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Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.

What makes filamentous phage ideal scaffold for bioconjugation?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What happens in in asthmatic epithelium in IFV infection?

MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The

What is the transmission of MERS-CoV is defined as?

Backcalculating the Incidence of Infection with COVID-19 on the Diamond Princess https://doi.org/10.3390/jcm9030657 SHA: 0938d2fb07611897abf38cea727ddbeea77b73d9 Authors: Nishiura, Hiroshi Date: 2020 DOI: 10.3390/jcm9030657 License: cc-by Abstract: To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy where E(.) represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] : where is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest.

What was the time period of peak infection of COVID-19 on the Diamond Princess cruise ship?

Backcalculating the Incidence of Infection with COVID-19 on the Diamond Princess https://doi.org/10.3390/jcm9030657 SHA: 0938d2fb07611897abf38cea727ddbeea77b73d9 Authors: Nishiura, Hiroshi Date: 2020 DOI: 10.3390/jcm9030657 License: cc-by Abstract: To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy where E(.) represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] : where is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest.

What is the estimated mean incubation period for COVID-19 infection on the Diamond Princess cruise ship?

Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/ SHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b Authors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier Date: 2011-09-29 DOI: 10.1371/journal.pone.0025738 License: cc-by Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent. Viral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab. Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source). Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (P,0.0001). The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001). To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671). We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001). We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus. Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

What are the results of the study?

MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The

When does the WHO recommend samlinf from the LRT?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What alternatives to classical vectored vaccines are needed?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What happens when the 3 cytokines are expressed?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What were linked to the exacerbation of the airway inflammation disease?

Port d’Entrée for Respiratory Infections – Does the Influenza A Virus Pave the Way for Bacteria? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742597/ SHA: ee0050c6fb81a4067d134010d0c80d21edb5df0b Authors: Siemens, Nikolai; Oehmcke-Hecht, Sonja; Mettenleiter, Thomas C.; Kreikemeyer, Bernd; Valentin-Weigand, Peter; Hammerschmidt, Sven Date: 2017-12-21 DOI: 10.3389/fmicb.2017.02602 License: cc-by Abstract: Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases (Weinstock, 2012) . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx (Hilty et al., 2012) are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide (Prina et al., 2016) . The composition of the nasopharyngeal microbiome is highly dynamic (Biesbroek et al., 2014a,b,c) and many factors, including environmental and host factors, can affect microbial colonization (Koppen et al., 2015) . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age (Bosch et al., 2016a) . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae (pneumococci) as a predominant pathobiont emerges (Miller et al., 2011; Bosch et al., 2016a,b) . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions (Blaser and Falkow, 2009 ). Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both (Pettigrew et al., 2008; Murphy et al., 2009 ). However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system (Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009) . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues (Ichinohe et al., 2011) . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection (IAV), oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice (Ichinohe et al., 2011) . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls (Leung et al., 2013) . However, another study published in the same year contradicted these results (Chaban et al., 2013) . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed (Chaban et al., 2013) . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin (HA) and neuraminidase (NA) they are classified into 16 classical HA and 9 classical NA subtypes (Neumann et al., 2009) . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge (Neumann et al., 2009 ). In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease (van der Meer et al., 2010) . Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually (Tjon-Kon-Fat et al., 2016) . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people (age > 65), pregnant women, and children under the age of one (Rothberg et al., 2008) . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic (Spanish Flu), often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death (Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006) . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years (Brundage and Shanks, 2007; Chien et al., 2009) . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae (Morens et al., 2008) . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus (GAS) and Haemophilus influenzae (Brundage and Shanks, 2008) . During the next two pandemics (H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu (Giles and Shuttleworth, 1957; Trotter et al., 1959) . Still, pneumonia accounted for about 44% of deaths during the Asian Flu (Giles and Shuttleworth, 1957) . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension (Giles and Shuttleworth, 1957) . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases (Hers et al., 1957 (Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959) , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu (Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971) . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic (Dawood et al., 2009 (Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions (Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012) . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia (Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 ). Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species (Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012) . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others (Brundage and Shanks, 2008; Ampofo et al., 2010) . When it does appear, it is typically third in incidence (Chaussee et al., 2011) . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population (Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013) . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections (Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b) . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections (Tregoning and Schwarze, 2010) . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects (Figure 1) . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin (Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009) . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus (MRSA) is now a major cause of hospital acquired infections (Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005) . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections (Rasigade et al., 2010) . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases (McCaskill et al., 2007; Sicot et al., 2013) . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized (McCullers, 2006) . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis (Bogaert et al., 2004; Valles et al., 2016) . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly (Black et al., 2010; Valles et al., 2016) . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population (Okumura and Nizet, 2014) . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year (Carapetis et al., 2005) . Listed as number nine in the list of global killers with around 500,000 deaths annually (Carapetis et al., 2005) , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections (Cunningham, 2000; Carapetis et al., 2005) . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age (O'Brien et al., 2009) , in the elderly (Marrie et al., 2017) , and in immuno-compromised individuals (Baxter et al., 2016) . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract (Webster et al., 1992) . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells (Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines (Durbin et al., 2013; Iwasaki and Pillai, 2014) . In addition, inflammasome activation leads to the release of IL-1β and IL-18 (Pothlichet et al., 2013; Iwasaki and Pillai, 2014) . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium (Korteweg and Gu, 2008) . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response (Yang and Tang, 2016) . All these events contribute to lung tissue injury (Imai et al., 2008; Davidson et al., 2014) . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment (Plotkowski et al., 1993) . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA (Figure 2 ) (Bottcher-Friebertshauser et al., 2013) or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus (Scheiblauer et al., 1992; Tse and Whittaker, 2015) . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. (MSCRAMMs), such as PspC, PspA, and PsaA in pneumococci (Hammerschmidt, 2006) , SPA, FnbA, ClfA, and ClfB in S. aureus (Bartlett and Hulten, 2010; Otto, 2010) , and M-protein, PrtF1, and PrtF2 in GAS (Cunningham, 2000) , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK (Fulde et al., 2013) , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen (McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012) . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci (Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015) , α-hemolysin and leukocidins of S. aureus (Mairpady Shambat et al., 2015) , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes (Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence (McCullers and Bartmess, 2003) . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion (Cundell and Tuomanen, 1994) . As the patient begins to recover from viral infection, secondary bacterial infections might occur (Louria et al., 1959) due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs (Louria et al., 1959; Puchelle et al., 2006) . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses (Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013) . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages (Figure 2 ) (Ghoneim et al., 2013) . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections (Ghoneim et al., 2013) . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs (Figure 2 ) (Sun and Metzger, 2008; Tian et al., 2012) . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors (TLR) 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state (Shahangian et al., 2009) . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages (Nakamura Frontiers in Microbiology | www.frontiersin.org FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial (Bact.) attachment. IAV is able to induce suppression and killing of resident alveolar macrophages (AM), which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function (NK), including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils (PMN), leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein (HBP). et al., 2011). Another study by Shahangian et al. (2009) revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer (NK) cell responses in the airway to subsequent S. aureus infection (Small et al., 2010) . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages (Figure 2 ) (Small et al., 2010) . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology (Li et al., 2015) . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons (Yamamoto et al., 2004; Spelmink et al., 2016) . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary (Sharma-Chawla et al., 2016) . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years (Snelgrove et al., 2008; Hussell and Cavanagh, 2009 ). These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production (van der Sluijs et al., 2004; Metzger and Sun, 2013) . The anti-inflammatory state suppresses the expression of pattern recognition receptors (PRR) on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology (Sun and Metzger, 2008; Goulding et al., 2011) . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis (Morton et al., 2014) . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm (Figure 2 ) (Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016) . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection (van der Poll and Herwald, 2014). Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016) . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor (Domon et al., 2016) . Staphylococcal cytotoxins (α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL) are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs (Mairpady Shambat et al., 2015) . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis (Snall et al., 2016; Uhlmann et al., 2016) . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production (Ramos and Fernandez-Sesma, 2012) . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality (Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013) . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals (Hraiech et al., 2015) . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans (Hackam and Redelmeier, 2006) . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies (Hackam and Redelmeier, 2006) . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested (Hackam and Redelmeier, 2006) . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials (Hackam and Redelmeier, 2006) , whereas about 44% replication rate was reported for highly cited human studies (Ioannidis, 2005) . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans (Hraiech et al., 2015) . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice (Mizgerd and Skerrett, 2008) . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes (Mizgerd and Skerrett, 2008) . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including (i) the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection (Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011) , (ii) pathogen synergism (Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015) , (iii) enhanced inflammatory response at the onset of infection (Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014) leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms (Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and (iv) host receptor availability for sustained bacterial infection (Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008) . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection (Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains (Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets (Diavatopoulos et al., 2010; McCullers et al., 2010) . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV (Diavatopoulos et al., 2010) . et al., 2010) . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine (Thangavel and Bouvier, 2014) . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection (Tripp and Tompkins, 2009) . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection (Sanford and Ramsay, 1987) . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV (Smith and McCullers, 2014) . The biggest advantages of using ferrets as a model include (i) their susceptibility to nonadapted human pathogens, (ii) efficiency in transmitting IAV and bacteria from one individual to another, and (iii) presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform (Bouvier and Lowen, 2010) . In recent years, the guinea pig (Cavia porcellus) was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease (Canning and Chou, 2008) . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model (Bouvier and Lowen, 2010) . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms (Lowen et al., 2006; Gabbard et al., 2014) . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection (Gabbard et al., 2014) . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus (Saito et al., 1988) . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts (Saito et al., 1988) . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus (Ponte et al., 1996) . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h (Ponte et al., 1996) . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat (Sigmodon hispidus) was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation (Ottolini et al., 2005) . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks (Ottolini et al., 2005) . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes (Oliveira et al., 2016) , metabolic syndromes (Feng et al., 2015) , cirrhosis , pharmaco-kinetics and dynamics (Antonopoulou et al., 2015; Hoover et al., 2015) , intoxication (Davis et al., 1991) , immunization (Iinuma and Okinaga, 1989) , and general bacterial virulence factors (Shanley et al., 1996) on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 (Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome (Harford et al., 1946) . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages (Carsillo et al., 2009) , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits (Oryctolagus cuniculus) are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz (1913) . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci (Kline and Winternitz, 1913) . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits (Stillman and Branch, 1926) . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals (Stillman and Branch, 1926) . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis (Diep et al., 2010 (Diep et al., , 2017 , as well as drug efficiency and efficacy studies (Cabellos et al., 1992; Croisier-Bertin et al., 2011) . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations (Loza-Tulimowska et al., 1977) . Also, rabbits are used for safety investigations of vaccines (e.g., CoVaccine HT or Aflunov) (Heldens et al., 2010; Gasparini et al., 2012) . In recent years the shedding of avian IAV by cottontails (Sylvilagus spp.) was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA (Root et al., 2014) . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails (Root et al., 2017) . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fasciluraris). Although it was shown early that macaques were susceptible to IAV (Saslaw et al., 1946) , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain (H5N1) with a conventional H1N1 strain (Rimmelzwaan et al., 2001) . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia (Rimmelzwaan et al., 2001) . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome (Kobasa et al., 2007) . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques (Itoh et al., 2009 ). The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques (Marriott et al., 2016) . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis (Virtaneva et al., 2005) and lower respiratory tract infection in cynomolgus macaques (Olsen et al., 2010a) . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans (Olsen et al., 2010a) . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection (Olsen et al., 2010b) . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected (Olsen et al., 2010b) . The same research group has developed a non-lethal IAV (H3N2)-S. aureus co-infection model in cynomolgus macaques (Kobayashi et al., 2013) . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone (Kobayashi et al., 2013) . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires (Jia et al., 2017) , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx (Philipp et al., 2012) . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% (Philipp et al., 2012) . The same group tested detoxified pneumolysin (dPly) and pneumococcal histidine triad protein D (PhtD) as potential vaccine candidates to prevent pneumonia (Denoel et al., 2011) . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity (i) to greatly reduce bacterial load within the first week post-challenge and (ii) the levels of PhtD-and Ply-specific antibodies (Denoel et al., 2011) . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia (Figure 1) . The composition and size of the porcine genome is comparable to that of humans (Hart et al., 2007) . In addition, human and porcine organs have many common features and functions (Swindle et al., 2012) . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice (Horter et al., 2003) . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans (Dawson et al., 2016) . Most of the immune cell compartments identified in humans are also present in pigs (Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011) . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells (Fairbairn et al., 2011) . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs (Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011) . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice (Fairbairn et al., 2011) . In contrast to human monocytes, which can be divided in three subclasses (classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + ), porcine monocytes consist of four subclasses (Chamorro et al., 2005; Fairbairn et al., 2013) . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 (Chamorro et al., 2005) . The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989) , osteomyelitis (Jensen et al., 2010) , and sepsis (Nielsen et al., 2009) . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs (De Greeff et al., 2016) . In addition, research on respiratory infections of pigs by human pathogens including S. aureus (Luna et al., 2009) , Mycobacterium tuberculosis (Gil et al., 2010) , Bordetella pertussis (Elahi et al., 2007) , Pseudomonas aeruginosa (Luna et al., 2009) , and IAV (Khatri et al., 2010) , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV (H1N1, H3N2), and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections (Van Reeth et al., 1998) . Especially IAV infections are already well established in swine (Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011) . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs (Staats et al., 1997) . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs (Staats et al., 1997) . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome (Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007) . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism (Seitz et al., 2012; Auger et al., 2016) , several other studies have shown the advantage of using swine as a natural host for S. suis (Bi et al., 2014; Ferrando et al., 2015) . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens (Lin et al., 2015) . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens (Lin et al., 2015) . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group (Lin et al., 2015) . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting (Figure 1) . Tissue engineering approaches were originally focused on regenerative medicine (Langer and Vacanti, 1993) . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids (Lancaster et al., 2013; Dang et al., 2016) , Helicobacter pylori infections of gastric epithelial organoids (McCracken et al., 2014; Schlaermann et al., 2016) , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids (Saxena et al., 2015; VanDussen et al., 2015) , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue (Petropolis et al., 2014 (Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016) , and staphylococcal and Andes hantavirus infections of human lung tissue (Mairpady Shambat et al., 2015; Sundstrom et al., 2016) . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top (Nguyen Hoang et al., 2012) . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells (Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015) . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia (Mairpady Shambat et al., 2015) . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils (Mairpady Shambat et al., 2015) . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation (Russell, 1995) . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci (Szymanski et al., 2012; Fatykhova et al., 2015) , Bacillus anthracis (Chakrabarty et al., 2007) , Haemophilus influenzae (Zhang et al., 2016) , and IAV (Nicholls et al., 2007; Chan et al., 2009) , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus (SIV) and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage (Meng et al., 2015) . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia.

What factors make bacterial and viral co-infections so lethal?

SARS to novel coronavirus – old lessons and new lessons https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/ SHA: 5d254ed178c092d3639ce70ae9653593acc471f9 Authors: McCloskey, Brian; Heymann, David L. Date: 2020-02-05 DOI: 10.1017/s0950268820000254 License: cc-by Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future. Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days. By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal. This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable? According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date. This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR). One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified. Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014. On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO. Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic. While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety. To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems. The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations. Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk. A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] . The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China. The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response. The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact. At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners. There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event. The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less. The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared. The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful. A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic. Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged. Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future. However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure? These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event. This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available. In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves. Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult. In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce.

How big was the temporary hospital built in Wuhan City for treatment of COVID-19 patients?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What follows in the event of a viral infection such as RV infection?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

Why are adenovirus vectors most attractive?

1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger" and David M. Morens1- The “Spanish" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population (or z500 million persons) were infected and had clinical- ly apparent illnesses (1,2) during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics (3,4). Total deaths were estimated at z50 million (577) and were arguably as high as 100 mil- lion (7). The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide (except- ing human infections from avian Viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses (now known to be H1N1 Viruses) were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic (8). Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 (“Asian flu”), the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer (9). They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage (so-called classic swine flu), and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains (prevalent from 1968 until the present). H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus (10). These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies (11717) and a number of reviews covering different aspects of the 1918 pandemic have recently been published ([8720) and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus (19) and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America (the first wave was best described in the United States in March 1918). Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus (21), and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context ([9). Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 (22). Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively (Figure 1). Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps (23), but a counter argument is that if a strain with a new hemagglutinin (HA) was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous (or nearly simultaneous) infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 (12,20). Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase (NA) surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before (12,15, 24). More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 (20). Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date (19). In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 (21). In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months (Figure 1). Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 (21). Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures (bene- ficial to thermolabile Viruses such as influenza), optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter (of the Northern Hemisphere), respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 (more than a year after the first pandemic appearance) and the third in early 1892 (21 ). As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients ([6), nothing can yet be said about whether the first (spring) wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source (17,19), as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods (24,25). For example, the 1918 nucleoprotein (NP) gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains (13,19). One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses (26); both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 (273) linked sugars (27729). Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 (2%) link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change (30), and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it (16). This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice (31). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (32). These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA (and possibly the NA) contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped (Figure 2), exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third (middle) distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years (35). Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic (33). A sharper perspective emerges when 1918 age-specific influenza morbidity rates (21) are used to adj ust the W- shaped mortality curve (Figure 3, panels, A, B, and C [35,37]). Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence (Figure 3, panel A). But even after adjusting age-specific deaths by age-specif— ic clinical attack rates (Figure 3, panel B), a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 (Figure 3, panel C). Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior Virus exposures [38]) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough (>35 years) to have been infected during that prior era (35). But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 (21). Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 (dashed line) and for the pandemic year 1918 (solid line) (33,34). Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia (P&l) (combined) age-specific incidence rates per 1,000 persons per age group (panel A), death rates per 1,000 persons, ill and well combined (panel B), and case-fatality rates (panel C, solid line), US Public Health Service house-to-house surveys, 8 states, 1918 (36). A more typical curve of age-specific influenza case-fatality (panel C, dotted line) is taken from US Public Health Service surveys during 1928—1929 (37). the 5 to 15-year-old group jumped to 25% of influenza cases (compatible with exposure to an antigenically novel Virus strain), while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum (Figure 2), a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 (>95% in most locales in industrialized nations) were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time (Figure 1). Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus (39), though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza (HPAI) Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation (e.g., receptor binding) are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the (alleged) pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second (fall) and third (winter) waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge (UK): Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. 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Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

Are the modern descendant influenza viruses as dangerous as the 1918 parent swine flu (Spanish Influenza) H1N1 virus?

A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.

What compartments were the bats divided into?

Which Kind of Provider’s Operation Volumes Matters? Associations between CABG Surgical Site Infection Risk and Hospital and Surgeon Operation Volumes among Medical Centers in Taiwan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459823/ SHA: f3cbc0503581249a834895fc94cd3bae24714a0d Authors: Yu, Tsung-Hsien; Tung, Yu-Chi; Chung, Kuo-Piao Date: 2015-06-08 DOI: 10.1371/journal.pone.0129178 License: cc-by Abstract: BACKGROUND: Volume-infection relationships have been examined for high-risk surgical procedures, but the conclusions remain controversial. The inconsistency might be due to inaccurate identification of cases of infection and different methods of categorizing service volumes. This study takes coronary artery bypass graft (CABG) surgical site infections (SSIs) as an example to examine whether a relationship exists between operation volumes and SSIs, when different SSIs case identification, definitions and categorization methods of operation volumes were implemented. METHODS: A population-based cross-sectional multilevel study was conducted. A total of 7,007 patients who received CABG surgery between 2006 and 2008 from19 medical centers in Taiwan were recruited. SSIs associated with CABG surgery were identified using International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9 CM) codes and a Classification and Regression Trees (CART) model. Two definitions of surgeon and hospital operation volumes were used: (1) the cumulative CABG operation volumes within the study period; and (2) the cumulative CABG operation volumes in the previous one year before each CABG surgery. Operation volumes were further treated in three different ways: (1) a continuous variable; (2) a categorical variable based on the quartile; and (3) a data-driven categorical variable based on k-means clustering algorithm. Furthermore, subgroup analysis for comorbidities was also conducted. RESULTS: This study showed that hospital volumes were not significantly associated with SSIs, no matter which definitions or categorization methods of operation volume, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon’s volumes varied. Most of the models demonstrated that the low-volume surgeons had higher risk than high-volume surgeons. CONCLUSION: Surgeon volumes were more important than hospital volumes in exploring the relationship between CABG operation volumes and SSIs in Taiwan. However, the relationships were not robust. Definitions and categorization methods of operation volume and correct identification of SSIs are important issues for future research. Text: data, which should use hierarchical models, may result in biased estimation of the variation and also lead to incorrect conclusions. SSIs following coronary artery bypass graft (CABG) procedures place a heavy burden on patients and healthcare systems. The total length of stay and expenditure for patients with SSIs after CABG surgery is significantly longer and higher than those without SSIs. [20, 21] In 2008, the Centers for Medicare & Medicaid of the United States of America implemented the "Never Event" policy, where hospitals would no longer receive higher payments for the additional costs associated with treating patients for certain healthcare-acquired infections, including those related to CABG. In view of the accuracy of SSIs identification and the heterogeneity of definition and categorization methods, no existing studies have used different infection case identification nor definitions and categorization methods of operation volume simultaneously to explore the relationship between operation volumes and infection. The current study takes CABG SSIs as an example to examine whether a relationship exists between operation volumes and SSIs, given different SSI cases identification, operation volume definitions and categorization methods. This retrospective and cross-sectional study adopted a multilevel design to examine the relationships between provider volumes and SSIs after adjusting for patient-, surgeon-, and hospital-level covariates. We used data from the Taiwan National Health Insurance Research Database (NHIRD) from 2005 and 2008. The NHIRD, published by the Taiwan National Health Research Institute, includes all the original claims data and registration files for beneficiaries enrolled under the National Health Insurance (NHI) program. The database covers the 23 million Taiwanese enrollees (approximately 98% of the population) in the NHI program. It is a de-identified secondary database containing patient-level demographic and administrative information; however, treatment items are aggregated and without time-related and clinical information. The data is released for research purposes. The protocol for the study was approved by the Institutional Review Board of the National Taiwan University Hospital (protocol #201001027R). The dataset we used in this study was secondary data; all information was de-identified by data owners. In this study, we adopted the ICD-9-CM SSI codes (hereafter referred to as the ICD-9-CM based model) and the Classification and Regression Trees (CART) model, which was developed in our previous work [11] to identify SSI cases. As we mentioned above, the ICD-9-CM SSI codes were the most popular tool to identify the SSI cases in claims data. In the ICD-9-CM based model, SSI cases were divided into two categories: index hospitalization events and post-discharge events (i.e., SSIs that occurred within 1 year after discharge and required readmission to a hospital and/ or the use of ambulatory services). Following Wu et al [13] , this study adopted the secondary ICD-9-CM diagnosis codes for index hospitalization events (ICD-9-CM code: 996.03, 996.61, 996.72, and 998.5), and the primary and secondary diagnosis codes for post-discharge events (ICD-9-CM code: 038.0-038. 4 ) as the criteria for SSI identification, in order to avoid cases in which infection existed prior to hospitalization. If a case had an index hospitalization event or a post-discharge event, then he/ she will be identified as SSIs by the ICD-9-CM based model. In the CART model, we adopted the type of antibiotics, dose of cefazolin, length of stay, and number of vessels obstructed (as a proxy indicator of duration of operation) as the parameters to identify the SSIs, according to our previous findings. [11] In our previous work, we used the 2005-2008 National Health Insurance claims data and healthcare-associated infection surveillance data from two medical centers for model development and model verification. Infection cases based on surveillance were identified by infection control personnel if the patient met the Taiwan CDC's criteria, which are the same as those adopted in the U.S. CDC. They manually review medical records of all patients at risk for the specified healthcare-associated infection. The classification algorithms, the multivariable regression model, and the data mining model were adopted to develop alternative models based on surrogate indicators to identify cases of CABG SSIs and to compare the performance among these models and the ICD-9-CMbased model. For the classification algorithms, researchers build up several criteria, and if a case satisfies (or exceeds) a specific number of criteria, then it will be identified as a case of infection. For the multivariable regression model, researchers usually calculated a risk score by the logistic regression model, and the optimal cutoff point was determined according to the resulting receiver operating characteristic curve. Concerning the data mining approach, which is widely used for predicting and classifying objects, the characteristics are: automatic discovery of patterns, prediction of likely outcomes, creation of actionable information, and focus on large data sets and databases. The classification and regression tree (CART) model, which is the most popular approach as applied in our work, and the growing, stopping, and pruning of the tree were determined by Gini improvement measures. [22, 23] After referring to the literature and conferring with infectious disease specialists, we adopted the following seven parameters: type of antibiotic, doses of antibiotic, doses of cefazolin, use of second-line antibiotics, length of stay, and number of vessels obstructed. Additionally, cross-validation was also employed, where data from one medical center was used for model development, and another one was used for model validation. The results of our previous work revealed that the CART model offered better performance than that of the other identification models or the ICD-9-CM based model, especially in the positive predictive value (>70%), which was only found to be 20% in the ICD-9-CM based model. (Table 1 ) The findings also implied that the CART was a decidedly better tool for identifying cases of SSI in the Taiwan National Health Insurance database. Therefore, this study also adopted the CART model for identifying CABG SSIs. To ensure homogeneity, current study analyzed 7,007 patients from 19 medical centers in Taiwan who underwent CABG surgery (ICD-9-CM procedure codes 36.1x-36.2x) between 2006 and 2008. CABG patients under the age of 18 years or over 85 years were excluded in this study. A total of 302 cases were identified as SSIs by ICD-9-CM based model, and a total of 107 cases were identified as SSIs by CART model. In this study, we used the following two definitions to define operation volumes: (1) the cumulative operation volumes by each surgeon and hospital within the study period, which was the most common definition in the literature; and (2) following Yasunaga et al.'s study, [24] cumulative operation volumes by each surgeon and hospital in the previous one year for each surgery. However, our data was skewed, which did not follow a normal distribution. Therefore, we conducted the log transformations on operation volumes. The current work treated operation volumes in three different ways: (1) a continuous variable; (2) a categorical variable based on the first and the third quartile as cutoff points (the most common method to categorize service/ operation volumes) [25] [26] [27] [28] ; and (3) a data-driven categorical variable based on k-means clustering algorithm. This study categorized surgeon and hospital volumes into low, medium, and high volume groups by quartile method and kmeans clustering algorithm. In the quartile method, the cut-off value (transformed by logarithm) of the first quartile (<25%) for hospital volumes was 5.65, and the third quartile (>75%) was 6.43. In terms of surgeon volumes, the first quartile was 4.38, and the third was 5.35, when we used the cumulative operation volumes within the study period as the definition. While the definition changed, first quartile (<25%) for hospital volumes was 4.66, and the third quartile (>75%) was 5.31. In terms of surgeon volumes, the first quartile was 3.40, and the third was 4.32. K-means clustering is an unsupervised machine-learning algorithm introduced by MacQueen in 1960s. This method is not only a simple and very reliable method in categorization/ classification, but is also recognized as one of the top 10 algorithms in data mining. [29] This method has often been applied in many fields. [30] [31] [32] Yu and his colleagues even applied it to define the quality of CABG care, and to explore the relationship among patient's income status, the level of quality of care, and inpatient mortality. [33] The main idea of this method is to partition observed data points into k non-overlapping clusters by minimizing the within-group sum of squares. Each point is assigned to the mean of its cluster using the Euclidian distance. Firstly, k cluster centers were randomly generated. Previous studies usually divided surgeons and hospitals into low-, medium-, and high-volume groups; therefore, we also predetermined the surgeon and hospital service volumes into 3 groups (k = 3). Then, participants were assigned to the cluster with the shortest distance to these cluster centers. Finally, the cluster centers were recomputed using the new cluster assignment and these steps would be iterated until convergence was achieved. [34] The cut-off values of hospital volumes were 5.21 and 5.69, and for surgeon's volumes were 2.40 and 4.38 respectively, when cumulative operation volumes within the study period was used as the definition. Likewise, when cumulative operation volumes before each surgery was used as definition, the cut-off values were 4.11 and 4.89 for hospital volumes, and 2.64 and 3.91 for surgeon's volumes. All cutoff values were transformed by logarithm. The results of k-means clustering are demonstrated in Figs 1-4. As the results show, the operation volumes were divided into three groups separately. In addition to surgeon and hospital volumes and SSI, we collected patient-, surgeon-, and hospital-level data. Firstly, patient-level variables included age, gender, length of ICU stay, number of vessels obstructed that were involved in the surgical operation, and the presence of important underlying diseases (e.g. diabetes mellitus, chronic obstructive pulmonary disease (COPD), heart failure, renal failure and renal insufficiency, which were associated with SSI). [13] Secondly, the surgeon-level variables included age and gender. Thirdly, the hospital-level variables included hospital ownership and geographic location. All statistical analyses of volume-infection relationship were performed using SAS (version 9.2, SAS Institution Inc., Cary, NC, USA). In statistical testing, a two-sided p value 0.05 was considered statistically significant. The distributional properties of continuous variables were expressed by mean ± standard deviation (SD), whereas categorical variables were presented by frequency and percentage. In univariate analysis, the potential three-level predictors of SSI were examined using chi-square test or two-sample t-test as appropriate. Next, to account for the correlations within surgeon (level-2) and hospital (level-3), multivariate analysis was conducted by fitting mixed-effects logistic regression models to each patient's data for estimating the effects of three-level predictors on the probability of post-operational SSI. Furthermore, subgroup analysis for comorbidities was also conducted. Table 2 shows that there were 7,007 patients with CABG performed by 199 surgeons in 19 hospitals during 2006-2008 in Taiwan. The majority of patients were male (77.5%), and the mean age of patients was 65.3 years. The average ICU stay was 6.05 days, the mean level of number of vessels obstructed was around 1.6, while 51.8% of patients had diabetes mellitus, 33.3% had heart failure, 14.1% had renal failure and renal insufficiency, and 22.0% had COPD. Three hundred and two patients (4.31%) were identified as having the ICD-9-CM SSI codes. However, identification by the CART model only revealed 107 infection cases, and 94 cases were identified in both models. Most cases received CABG surgery by male surgeons, with a mean age of 45.0 years, and the surgeon's average operation volumes within the study period was 151.64, while the average operation volumes before surgery was 52.18. More than half of the cases were performed with CABG in not-for-profit hospitals, and the hospitals' average operation volumes within the study period was 473.60, while the average operation volumes before each surgery was 158.79. Moreover, most of patients received their surgeries by high-volume surgeons and hospitals, when k-means algorithm was used for categorization, regardless of which definition of operation volumes were used. Table 3 shows the results of multilevel mixed-effect models, with the SSIs being identified by ICD-9-CM codes, and the operation volumes defined as the cumulative volumes within the study period. The results of Model 1 (continuous) reveal that the surgeon's volumes were negatively associated with SSIs, while hospital's volumes were not associated with surgical site infection SSIs. Model 2 (quartile) suggests that low-volume surgeons had higher SSI risk (OR = 2.220, p-value = 0.022) than high-volume surgeons. There were also no associations between hospital's operation volumes and SSIs. Model 3 (k-means) shows that the association did not exist between hospital's/ surgeon's volumes and SSIs. Table 4 displays the results of multilevel mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volumes within the study period. Model 1 again indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results revealed low-volume surgeons had higher risk (OR = 1.691, p = 0.002) than high-volume surgeons. Table 5 displays the results of multilevel mixed-effect models, in which the SSIs were identified by ICD-9-CM codes, but the operation volumes were defined as the cumulative volume in the previous one year for each surgery. Model 1 also indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.642, p = 0.040) than high-volume surgeons. Table 6 displays the results of multilevel mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volume in previous one year for each surgery. In Model 1, different to the above findings, there was no association between hospital's/ surgeon's volumes and SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.163, p = 0.020) than high-volume surgeons. We further examined the associations of surgeon and hospital volumes with SSIs in stratification analyses by underlying diseases. When the operation volumes were defined as the cumulative operation volume within the study period, no relationships existed between hospital/ surgeon operation volumes and SSIs. (Table 7 ) However, when the operation volumes were defined as the cumulative operation volumes in the previous one year for each surgery, the results suggested that there was a negative association between surgeon volumes and SSIs in the diabetes group, except that the volumes were treated as continuous variable and the infection cases were identified by ICD-9 codes. In terms of hospital operation volumes, the association did not exist. (Table 8 ) No studies have evaluated how different service/ operation volumes definitions and categorization methods affect volume-infection relationships. Moreover, several studies have pointed out the inappropriateness of identifying infection cases using the ICD-9-CM codes in claims data. Given these reasons, this study adopted two approaches to identifying SSIs, two definitions of operation volumes, and three methods for categorizing operation volumes to examine the relationships between operation volumes and SSIs. Our findings showed that the relationships between hospital volumes and SSIs did not exist, no matter which definitions, categorization mehods, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon volumes and SSIs were not robust in our data. It might be affected by different definitions and categorization methods of operation volumes, and also by different SSI cases identification approaches. In summary, most of the models demonstrated that the low-volume surgeons had higher risk than high-volume surgeons, and they also showed the risks were similar between medium-volume and high-volume surgeons. However, why did surgeon volume relate to SSIs, but hospital volume did not? Except for those issues we were concerned about in this study, there are some disagreements in the literature. Such as "Does provider volume really represent quality of care?" [12, 35] Or "Is provider volume the only one predictor for outcome of care?" [35, 36] These issues are worthy of further discussion, but are out of the scope of this study. Service/ operation volumes are treated as a proxy indicator for experiences; previous studies used it to examine whether practice makes perfect or not. But, except for provider's experiences, SSIs are also impacted by many factors, such as environmental and clinical factors. Wu et al once used Taiwan 2001 NHI claims data to explore the relationship between provider CABG operation volumes and SSIs. [13] They found that hospital volumes had a greater effect than surgeon volumes and claimed that this may imply that hospital teamwork is more important than individual surgeon. However, our findings demonstrated that there was no relationship between hospital volumes and SSIs. Wu et al. adopted the cumulative operation volumes within the study period as the definition, and identified SSIs by ICD-9-CM codes. Except, there were two differences between our work and Wu et al., which were the length and year of the data; our data was longer and more updated than theirs. Moreover, it is worth noting that there was an outbreak of severe acute respiratory syndrome (SARS) in Taiwan in 2003, after which the hospital infection control system in Taiwan was reviewed and re-designed. Wu et al data was before SARS, so these efforts may also have improved the level of SSIs control in hospitals, leading to different findings in this study. In addition, although most models revealed that there were negative relationships between surgeon's volumes and surgical site infection, the relationships were not robust. The results varied between different definitions and categorization method of operation volumes, and between SSIs identification approaches. Researchers need to consider how to identify SSIs correctly, how to choose optimal cut-off values, and how to decide on which definition is appropriate. Finally, the results of stratification analyses showed that low-volume surgeon had higher risk than high-volume surgeon in the diabetes mellitus group, when the cumulative operation in the previous one year before surgery was used as definition. A large number of studies have indicated diabetes mellitus is associated with a higher risk of SSIs, [37] [38] [39] and the findings of this study suggest that CABG patients with diabetes mellitus should be cared for by experienced surgeons. A multilevel analysis was applied to manage the nested factors, and two definitions of operation volume along with three different operation volume categorization methods were adopted to examine the relationship between volume and SSIs under two kinds of SSIs identification approaches. Nevertheless, the study suffered from several major limitations. First, the accuracy of SSIs identification was still an issue. Although the performance of the CART model to identify CABG SSIs was better than ICD-9-CM codes in Taiwan NHI claims data, it did not reach the perfect scenario. The accuracy of SSIs identification was still a challenge in our work. The second limitation relates to unmeasured variables, such as length of stay before operation, infection condition, hair removal, clinical information (e.g. blood glucose level, causative microorganism), time-related information (e.g. the duration of operation), the environment, surgical skills, use of post-operative drains, number of operations involved, and surgical site and wound care, etc. [40] Furthermore, information about type (elective or urgent) and incision site for surgery was not available in the Taiwan NHI claims data. In conclusion, the findings of this study suggest that different definitions and categorization methods of operation volumes, and different SSIs identification approaches might lead to different findings, although surgeon volumes were more important than hospital volumes in exploring the relationships between CABG operation volumes and SSIs in Taiwan, but they were still not robust. Definitions and categorization methods of operation volumes, and correct identification of SSIs are important issues for future research.

Patients from how many medical centers were studied?

The human viral challenge model: accelerating the evaluation of respiratory antivirals, vaccines and novel diagnostics https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013893/ SHA: f13c88733ea45be9e923a282dfd42f8c277c187c Authors: Lambkin-Williams, Rob; Noulin, Nicolas; Mann, Alex; Catchpole, Andrew; Gilbert, Anthony S. Date: 2018-06-22 DOI: 10.1186/s12931-018-0784-1 License: cc-by Abstract: The Human Viral Challenge (HVC) model has, for many decades, helped in the understanding of respiratory viruses and their role in disease pathogenesis. In a controlled setting using small numbers of volunteers removed from community exposure to other infections, this experimental model enables proof of concept work to be undertaken on novel therapeutics, including vaccines, immunomodulators and antivirals, as well as new diagnostics. Crucially, unlike conventional phase 1 studies, challenge studies include evaluable efficacy endpoints that then guide decisions on how to optimise subsequent field studies, as recommended by the FDA and thus licensing studies that follow. Such a strategy optimises the benefit of the studies and identifies possible threats early on, minimising the risk to subsequent volunteers but also maximising the benefit of scarce resources available to the research group investing in the research. Inspired by the principles of the 3Rs (Replacement, Reduction and Refinement) now commonly applied in the preclinical phase, HVC studies allow refinement and reduction of the subsequent development phase, accelerating progress towards further statistically powered phase 2b studies. The breadth of data generated from challenge studies allows for exploration of a wide range of variables and endpoints that can then be taken through to pivotal phase 3 studies. We describe the disease burden for acute respiratory viral infections for which current conventional development strategies have failed to produce therapeutics that meet clinical need. The Authors describe the HVC model’s utility in increasing scientific understanding and in progressing promising therapeutics through development. The contribution of the model to the elucidation of the virus-host interaction, both regarding viral pathogenicity and the body’s immunological response is discussed, along with its utility to assist in the development of novel diagnostics. Future applications of the model are also explored. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12931-018-0784-1) contains supplementary material, which is available to authorized users. Text: Acute respiratory infections (ARIs) manifest as Upper (URI) or Lower (LRI) respiratory tract infections and may move between the two compartments; ARIs represent the most common infectious diseases and are predominantly of viral aetiology. The global burden of ARI is substantial with significant morbidity and mortality occurring in children, the elderly and immunocompromised [1] . In the UK alone during the period 2014-2015, respiratory disease caused an estimated 15,800 excess winter deaths [2] . In the USA, influenza and respiratory syncytial virus (RSV) cause substantial mortality especially among people aged 65 and older [3] . However, although deaths in the industrialised world are widely reported, developing countries feel the burden particularly; out of an estimated 1.9 million child deaths from ARIs in 2000, 70% of those deaths occurred in Africa and south-east Asia [4] . The Millennium Summit at the United Nations in 2000 led to the setting up of the Millennium Development Goals. A study reported the progress made in meeting those goals in 40 developing countries; it concluded that the prevalence of ARI was 13%, health expenditure and per capita gross domestic product is directly associated with the prevalence of the disease [5] . Viral heterogeneity associated with ARIs is well established [6] . In the past, human rhinovirus (HRV) has been identified as the virus most frequently associated with respiratory illness with 30-50% of infections annually on average, and up to 80% of upper respiratory infections during the autumn outbreaks [7] . After HRVs, coronaviruses (CoV), influenza, respiratory syncytial virus (RSV) and parainfluenza viruses (PIV) are the next most frequent. More recently an evaluation of illness in 6,266 children under ten years of age in Australia, South East Asia and Latin America emphasised both the viral heterogeneity and the impact of ARI. Of the 2,421 children who experienced 3,717 individual influenza-like Illness (ILI) episodes, rhinovirus/enterovirus was most prevalent (41. 5%). Influenza followed this (15.8%), adenovirus (ADV) (9.8%), PIV and RSV (both 9.7%), CoV (5.6%), human metapneumovirus (HMPV) (5.5%) and human bocavirus (HBoV) (2.0%). The percentage of children missing school or childcare was between 21.4% for HBoV and 52.1% for influenza [8] . We have compared the data from the two reports one from 2003 [7] and the other in 2017 [8] and found that the reports, despite being separated by 14 years, were similar, with the single exception of HBoV, discovered in 2005 (Table 1) , which we discuss later. Feng et al. [9] described in detail the distribution of ARIs causing hospitalisation by age group: they observed that RSV was predominantly observed in the young and elderly, and influenza although significant in the young was noticeably more predominant in the elderly. Interestingly they observed that co-detection of viruses tended to occur more commonly in the younger age groups, particularly those under the age of five. Rhinovirus (the "common" cold) HRV infections, often considered trivial can significantly contribute to missed days from work and school, though infections are typically self-limiting [7] . HRV infections throughout the year and in many cases, manifest with symptoms such as nasal congestion, rhinorrhoea, sneezing, sore throat, and cough. HRV is known to be the primary cause of ARI and a severe contributing factor in exacerbations of atopic disease, e.g., asthma as well other conditions such as chronic obstructive pulmonary disease (COPD) [10] [11] [12] [13] . HRV infections are associated with significant economic implications as well as being an important contributor to sinusitis, otitis media, bronchitis and primary pneumonia [14] [15] [16] . HRV is a considerable cause of morbidity in specific at-risk groups such as infants, the elderly, immunocompromised, and, as already mentioned, chronic respiratory diseases such as asthma, COPD and cystic fibrosis. At present, HRV is considered the number one cause of asthma exacerbations [15] [16] [17] [18] [19] . Asthma is a complex disease, characterised by chronic airway inflammation, and a history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough. Over time these symptoms can vary in their intensity [20] . Each year over 300 million people worldwide are affected by asthma: approximately 250,000 people die as a result. Many deaths are due to suboptimal long-term medical care and delay in obtaining help during severe exacerbations of the disease [21] . Treatments to prevent worsening of symptoms and other therapies for mild to moderate asthma that avert relapse, i.e., the symptoms worsen again when the treatment stops, are significant unmet medical needs. The human challenge model has been used to investigate the viral pathogenicity [22] [23] [24] [25] [26] and recent publications on the asthma challenge model have focused on how the asthmatic host responds to HRV infection. Work is ongoing as to susceptibility to viral induced asthma worsening [27, 28] innate immune dysregulation [29] and induction of innate, and type 2 responses in nasal and bronchial epithelial secretions [30] . The pathogenesis of rhinoviral infection, along with other ARIs, in exacerbations of airway disease, has been investigated extensively. Impaired host responses to virus infection, a better understanding of the mechanisms of abnormal immune responses and the potential to develop novel therapeutic targets for virus-induced exacerbations have all used the HVC model [12, [31] [32] [33] [34] . Despite previous research work on multiple small molecule antivirals, such as pleconaril which have been tested using both the experimental challenge model and field studies [35] [36] [37] , there is currently no licensed treatment for HRV infections Other compounds have been tested against HRV, such as Vapendavir (BTA798) which prevented the release of viral RNA into the target cell and demonstrated a reduction in peak viral load in the HVC model [38] . A subsequent study in asthmatics was completed and although not published the compound did have a limited effect [39] . Pirodavir an intranasal capsid-binding molecule reached phase 3 clinical trials for HRV prevention and treatment in the 1990s. Although the compound decreased viral replication and shedding, it failed to show a significant reduction in the duration or severity of symptoms [40, 41] . A Protease inhibitor, rupintrivir thats prevents cleavage of viral proteins required for replication was tested in an HRV challenge trial. Rupintrivir was well tolerated and reduced viral loads and respiratory symptoms [36] . However, in studies of natural infection, it did not significantly affect viral loads or symptom severity [42] . Treatments such as zinc-containing products are now widely discredited as demonstrated by the withdrawal of a Cochrane report and JAMA editorial [43] [44] [45] . Current treatment of HRV infections primarily consists of over-the-counter (OTC) medicines to manage symptoms. There is also no licensed vaccine, and while there has been some progress on developing multivalent vaccines [46] , development in this area is hampered by the sheer number of serotypes that need to be covered (at present over 160). Despite HRV being associated with up to 50% of adult asthma exacerbations and up to 80% of childhood exacerbations, there are no HRV-specific asthma therapies [34] . As we better understand the interaction between the virus and the host, new therapies such as the monoclonal antibodies (anti-IgE [omalizumab] and anti-IL-5 [mepolizumab]) along with small molecules carefully targeting specific immune signalling pathways, HRV-specific prophylactic treatment may become practical [47] [48] [49] [50] . In order to prevent exacerbations, the design of new therapeutics could potentially improve efficacy by both directly acting to inhibit viral replication and alleviate the symptoms of asthma and COPD [51] . Influenza virus is a well-known human pathogen and can cause severe morbidity and mortality, particularly in older patients, those with co-morbidities and in the immunocompromised. In 2009, the first pandemic virus of the 21 st century hospitalised 195,000 to 403,000 in the US alone resulting in 8,870 to 18,300 deaths by mid-2010 [52] . A World Health Organization (WHO) global pooled analysis of 70,000 laboratory-confirmed hospitalised H1N1 pandemic patients from 19 countries revealed that of the 9,700 patients admitted to intensive care units, 2,500 died, and that morbid obesity might be a risk factor for hospitalisation and/or death [52] . Obesity was confirmed as a factor associated with a higher likelihood of admission to hospital in influenzainfected patients [53] . The 2009 pandemic was considered mild. However, the classic W shaped age distribution curve of infection for a pandemic virus was observed. That is high mortality in the very young and the old, but an additional spike in death amongst the "young and healthy". The pandemic, as did previous outbreaks, occurred in successive waves, but despite national policies favouring the use of antiviral drugs, few patients received these before admission to hospital, and many were given antibiotics [54] . The lack of real, or perceived, "real world" efficacy of currently available antivirals leads to the overuse of antibiotics and the subsequent problems that may arise [55] [56] [57] . The yearly seasonal morbidity and mortality of influenza results in hospitalisation and death mainly among the high-risk groups. Each year epidemics of seasonal influenza are estimated to result in about 3 to 5 million cases of severe illness, and about 290,000 to 650,000 deaths worldwide [58] . In first world / industrialised countries, most deaths associated with influenza occur among people age 65 or older [59] . Clinics and hospitals, in many countries, can be overwhelmed during peak illness periods, and there can be substantial economic cost [60] . The virus itself has been well characterised, and the two surface proteins, the haemagglutinin (HA) and the neuraminidase (NA) are important in both vaccine and antiviral development [61] . The effects of seasonal influenza epidemics in developing countries are not fully known, but research estimates that 99% of deaths in children under five years of age with influenza-related lower respiratory tract infections are found in developing countries [59, 62] . Currently, vaccines and antivirals exist for the prevention and treatment of influenza, but both have limitations in efficacy due to the rapid evolution of the virus as it mutates on a yearly basis and the sudden unexpected emergence of pandemic influenza strains. The effectiveness of recent annual influenza vaccines (to date mostly based on the HA, and rarely the NA surface glycoproteins) has languished between 37% and 70% over successive influenza seasons. In particular, the failure of the vaccine across the winter season of 2014-2015, where the overall adjusted effectiveness was 23% [95% confidence interval 14, 31] [63] is memorable. In a mismatched year, the mortality rate is increased in the most at-risk populations [64, 65] . The problem of ensuring that the seasonal vaccine is correctly matched to the upcoming circulating strain highlights the need for rapid development of inter-seasonal/universal vaccines and also the need for a way of testing their efficiency rapidly and accurately before the lengthy and expensive mass production is engaged which takes many months [66, 67] . Antiviral drugs exist of which currently the NA inhibitor oseltamivir is most commonly used. This is active against all known NA subtypes of influenza, and one would, therefore, assume against all influenza strains. They may have decreasing effect with the emergence of resistant influenza strains in which NA protein changes preventing efficient oseltamivir binding and thus its ability to inhibit the essential activity of the viral NA. For example, one genetic mutation known as 'H275Y'a substitution of histidine for tyrosine at NA position 275 -confers an evolutionary advantage to the virus including the 2009 H1N1 influenza [68] . During the 2013-2014 influenza season, 59 (1.2%) of 1,811 influenza A(H1N1) pdm09 virus isolates in 20 of 50 US states had the H275Y oseltamivir resistance substitution. No isolates were resistant to zanamivir [69] . Although animal studies have demonstrated limited transmission of mutant viruses [70, 71] , it is thought that the rise of oseltamivir resistance may be due to community transmission [72, 73] rather than the H275Y mutation becoming fixed in the viral genome. Asystematic systematic review and meta-analysis of published data from 2000 onwards concluded that most RSV-associated child deaths occur particularly in preterm infants and in infants up to 1-year of age [62, 74] . An effective maternal RSV vaccine or monoclonal antibody could have a substantial effect on disease burden in this age group [75] . The RSV-specific monoclonal antibody palivizumab is approved for prevention of serious LRI caused by RSV in susceptible infants. Economic benefit in a UK health setting has not been shown due to the high cost and lack of benefit on serious outcomes [76] . A single-centre cohort study of 22 infants showed no difference in treatment outcomes for patients receiving palivizumab when compared to patients only receiving "standard of care" treatment [77] . Despite the lack of evidence for clinical benefit, post-licensure data supports the use of palivizumab for reducing RSV-associated hospitalisations in premature infants under 33 weeks and in children with chronic lung and heart diseases [78] . Importantly, palivizumab resistant mutant virus has rarely been isolated in clinical specimens [79] . The RSV treatment ribavirin is limited due to difficulty with aerosol delivery, cost and potential harm to healthcare workers, despite off-label treatment of immunocompromised patients being reasonably successful. In the immunocompromised, therapy with a concomitant immunoglobulin or palivizumab has had mixed results, probably due to the difficulty of knowing when to initiate treatment [80] . Despite the call for the accelerated development of prevention and treatment strategies for an effective RSV vaccine for children [81] , research has stalled for decades since the death in the 1960s of two subjects in a clinical study. These subjects were infected with a communityacquired RSV infection after receiving the US National Institutes for Health (NIH's) formalin-inactivated, alumprecipitated RSV candidate vaccine. In contrast to influenza for which vaccines to date have shown themselves to be moderately effective but in need of improvement, RSV vaccines require substantially more research. There is currently no licensed vaccine for RSV; the most advanced candidate vaccine recently failed to show efficacy in a field study [82] . Effective treatments are urgently required. RSV is, even amongst healthcare professionals, considered a childhood disease and other confounders have obscured the understanding of the consequences of RSV in adults. RSV is poorly understood as a disease in the elderly [83] , and while the morbidity and mortality in children are of importance, it has been clearly shown that RSV has a comparable health burden to influenza in the elderly [84] . As an example, a recent study was conducted on adult (≥18 years) patients admitted to an emergency department with suspected ARI during 2013-2015 (N = 3743). Multiplex PCR was used to diagnose the cause of the respiratory infection. Eighty-seven patients were identified with RSV. A comparator group with influenza (n=312) was utilised. Based on a 20-day all-cause mortality endpoint, adult patients were less likely to be diagnosed with RSV than with flu (2.3 vs 8.3%, respectively), also they were older, often diagnosed with pneumonia, COPD, hypoxemia, and bacterial co-infection. RSV infection in the elderly was significantly associated with a greater risk of death than seasonal influenza, adjusted for potential confounders and comorbidities. [85] The clinical significance of viral/bacterial co-infections has long been a controversial topic. While severe bacterial pneumonia following influenza infection has been well described, associations are less clear among infections caused by viruses common in young children; secondary infections due to other viruses are less well understood and has been reviewed by others [86] . Although assessing the overall contribution of bacteria to disease severity is complicated by the presence of many confounding factors in clinical studies, understanding the role of viral/bacterial co-infections in defining the outcome of paediatric ARI may potentially reveal novel treatment and prevention strategies, improving patient outcomes [33, [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] . A recent (2017) publication considered the role of bacterial colonisation with Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis during symptomatic and asymptomatic viral upper respiratory infection in the nasopharynx of 4 to 7-year-old children during URI and when well. Using a multiplex PCR, virus was detected in about 80% of upper respiratory tract infections (URIs) in children and is also detectable in the nasopharynx of 30% of asymptomatic children. All three bacteria "levels" were higher during acute URI visits compared to asymptomatic surveillance visits by the children. Of note, however, is that even during asymptomatic follow-up visits, if the virus was present, all bacteria were detected at higher levels [96] . It is worth noting that the presence of confounding infections, can mask the importance of the primary aetiology. Taylor et al. [8] report the incidence of HBoV following its identification in 2005 from the respiratory tract samples of children, as an important respiratory pathogen in children. However, the role of this virus on its own as a pathogen of significance was initially unclear, co-infection with other viruses or bacteria was common and confounding. Moesker et al. [97] studied whether HBoV alone could cause acute respiratory infections in children. Using Next Generation Sequencing (NGS), they were able to exclude co-infections amongst those admitted to intensive care unit and studied HBoV viral loads. Of the 990 children who tested positive for a respiratory virus by RT-PCR, HBoV and RSV were detected in 178 and 366 of the children respectively. Forty-nine HBoV-positive patients and 72 RSV-positive patients were admitted to the intensive care. Seven HBoV-infected cases with severe ARI had no other co-infection (7/49, 14%). Importantly, these children did not have another detectable virus as determined by highly sensitive NGS. Also, they had much higher HBoV loads than other patients positive for HBoV, i.e., those with a co-infection. Although small, this study provides strong support that HBoV can cause serious ARI in children with no viral and bacterial co-infections. The history of the human viral challenge model Since Sir Edward Jenner performed the first documented HVC study with smallpox on the 14 th of May 1796 the usefulness of such studies has been apparent [98] . More than a century later, Sir Christopher Andrews returned from the US in 1931 he had observed the use of chimpanzees in the study of influenza. The funding for similar work in the UK was insufficient, and therefore Sir Christopher enrolled students from St Bartholomew's Hospital in London. He explained the next best thing would be a "Bart's" student as "they were cheaper than chimpanzees". Over 100 students immediately enrolled, but continued their studies and were not isolated in the same way the chimpanzees had been in the USA [99] . Unfortunately the investigators believed that the symptoms observed may not have been due to the challenge virus, but other respiratory infections acquired in the community, thus confounding the studies. A year later the UK's Medical Research Council (MRC) terminated the work. After the conclusion of World War II, the withdrawal of the US troops from the UK left the American Red Cross 'Harvard Hospital' Field Unit on Salisbury plain. The hospital became the Common Cold Unit (CCU) led by Dr David Tyrell, from 1946, volunteers were inoculated by instilling small quantities of the virus into their noses [100] . The CCU housed healthy volunteers in relative isolation from other people, thereby reducing the risk of contact with community-acquired sources of infection or from them passing on the virus to members of the public. The unit was eventually closed in 1989; during four decades of research, it attracted 20,000 volunteers. Its research contributed to a better understanding of respiratory viruses, viral lifecycle, possible vaccines [101] as well as the first licensed antiinfluenza compound amantadine [102] . The use of healthy volunteers in the HVC model provided, and still offers, a unique opportunity to describe the viral lifecycle. Investigators know with certainty the time of infection, nasal virus shedding can be measured, symptoms recorded prospectively, and participants are selected with low pre-existing immunity to the challenge virus to ensure a statistically significant infection rate with a small number of volunteers. Thus, such studies can maximise the safety and efficacy data obtained while minimising the risk to study volunteers and limited research funding. Although serum IgG, for influenza virus, was traditionally measured via the HAI assay, as the entry criteria for volunteers into studies, micro neutralisation assays are used for RSV and HRV. Other work does suggest screening for antibodies to the NA influenza surface protein should be considered [103] or T-cell responses to internal proteins [104] should be considered. After the closure of the CCU experimental infection studies continued in the USA using small motels and hotels replacing the huts on Salisbury Plain. These studies contributed to the significant development of the new NA inhibitors during the 1990s, including the inhaled drug zanamivir and the orally available drug oseltamivir [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] . Studies however also continued in the UK, specifically the University of Southampton who performed important work in atopic volunteers, demonstrating they had more severe colds when experimentally challenged with rhinovirus, than non-atopic controls [115] . The experimental A/Texas H1N1 influenza virus that was used successfully during the 1990s was implicated in the development of myocarditis in an experimentally infected subject, although a causal link was never demonstrated [116] . However, this incident halted work in the USA for a substantial period. Most, if not all, challenge viruses are manufactured according to Good Manufacturing Practice (GMP) standard. Although controlled nasal inoculation differs from naturally occurring infectionin which exposure to variable quantities of the virus may occur at various mucosal sites -the developed HVC model used in challenge studies mimics natural disease as far as possible [25, 117, 118] . We have described the production of a new GMP stock of virus using an HRV-16 isolate from an 18-year-old experimentally infected healthy female volunteer, provided by colleagues from University of Virginia Children's Hospital, USA. Importantly, the clinical sample was provided with the appropriate medical history and consent of the donor. We manufactured this new HRV-16 stock by minimal passage in a WI-38 cell line, to reduce the risk of mutations during the Good Manufacturing Practice process. Having first subjected the stock to rigorous adventitious agent testing and determining the virus suitability for human use, we conducted an initial "safety and pathogenicity" clinical study in adult volunteers in a dedicated clinical quarantine facility in London [118] . Our group started HVC studies in the UK in 2001, and since then we have conducted multiple studies with over 2,500 volunteers inoculated with influenza, respiratory syncytial virus (RSV) or human rhinovirus (HRV), and provided numerous proofs of concept [119] [120] [121] . The human viral challenge model: shortening the drug development pathway for ARIs Influenza, RSV and HRV infection have similar symptomatology, but this differs in severity and predominance of upper, lower or systemic symptoms as has been described by the Center for Disease Control [122] . However, it is not easy to diagnose between the different aetiologies of ARIs, and better diagnostics are needed [123] . Symptoms are common to each infection and manifest on a gradient. Generally, but far from always, influenza infection is more likely to result in a patient feeling so unwell as to take to their bed and have a fever, than RSV, an HRV, CoV or other common cold virus infection, during which daily life is usually less impacted. A variety of animal models exist to research respiratory viruses such as influenza [124] [125] [126] , RSV [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] , HRV [22, [138] [139] [140] . No single animal offers a platform for all respiratory viruses that infect humans, and different animal models exist for the same virus, which can give different, often conflicting results. In addition, the principles of the 3Rs (Replacement, Reduction and Refinement) were developed over 50 years ago to provide guidance and ensure humane animal research. Over time they have become national and international legislation/regulations. The policies of organisations that fund or conduct animal research include these principles as part of the condition of funding [141] . The shared symptomatology of respiratory viruses requires a single standard research platform that can be used to evaluate respiratory disease pathogenesis and the efficacy of candidate therapeutics. The use of a dedicated, purpose-built 24 en-suite bedroom isolation facility in which carefully screened volunteers can be safely inoculated with challenge viruses and intensively monitored may help reduce the use of animals while providing a single consistent research platform with standardised evaluable endpoints for respiratory virus research. Also, we have used a standardised diary card across our studies, which allows for comparison of the symptoms that each virus causes and the efficacy of the therapeutic being tested. We have included a copy of the diary card in the Additional file 1. It is difficult to evaluate the efficacy of a specific antiviral therapeutic "in the field" due to the presence of circulating community co-infections of differing microbial aetiology. The HVC model allows the opportunity to study a virus in isolation. HVC studies and field studies are complementary research stratagems necessary for the development of effective ARI therapeutics. In contemporary HVC trials, (Fig. 1 ) healthy volunteers are administered an investigational therapeutic either before (prophylaxis trials) or after (treatment trials) inoculation with the specific challenge strain of the virus. The viruses used in the HVC model are not attenuated and produce symptoms consistent with clinically observed ARI [25, 117, 118] . Each virus is propagated under GMP conditions, with a minimal number of passages from the isolates to the challenge stocks [118, 142] . The few mutations that occur within the virus are rapidly selected out due to a genetic bottleneck, with the consequence that the virus in the human host is considered wild-type [143] . The similarity between virus recovered from the inoculated host and the originator reference virus strain provides assurance that the model disease process is closely aligned with the reference virus strain and is not altered nor attenuated. There are limited licensed therapeutic options against respiratory viruses, highlighting a significant unmet medical need. A model such as the HVC allows the rapid evaluation of novel therapeutics. The model shortens both preclinical and early clinical development phases by providing a better understanding of the host and pathogen's initial interaction and has the potential to make the necessary vaccines and medicines more rapidly available than traditional development approaches otherwise might. Shortening the traditional development pathway through the early use of a Proof of Concept (PoC) study that incorporates the HVC model (Fig. 2) provides essential evaluable endpoints. Unlike conventional phase 1 studies which rarely include any assessment of efficacy, almost all HVC studies include evaluable efficacy endpoints such as reduction in AUC viral load (mainly recovered from upper respiratory tract samples such as nasal wash or nasopharyngeal swab), volunteer self-reported symptoms, peak symptom score, total symptom score amongst others. Small numbers of subjectsoften in the order of 30-45 per treatment group-are typically included in these rapid to execute short duration studies. The resulting safety and pharmacokinetic (PK) and pharmacodynamic (PD) data in controlled conditions, guide decisions on whether or not to progress to field studies, providing a most valuable set of data immediately after, or even as part of, the conventional phase 1 safety study. The HVC model also opens a different development route alongside traditional phase 1 allowing rapid progress to statistically powered phase 2b studies that will generate the efficacy data needed to support licensing, while still providing suitable safety data. The FDA guidance on developing influenza therapeutics [144] states that challenge trials cannot take the place of efficacy (phase 2) trials. The guidance states; "…Challenge trials can provide useful exposure-response and safety information, as well as an opportunity to demonstrate pharmacological antiviral activity in humans under controlled conditions outside the influenza season. Specifically, data from challenge trials can contribute to dose selection for phase 2b and phase 3 trials, and provide the opportunity to explore the effects of different times of drug initiation relative to virus exposure...". Challenge trial refinements are closing the gap between the experimental infection model and the natural infection setting. The HVC study duration of several weeks is shorter than a field-based phase 2 study that waits for a natural outbreak of the virus and the duration of which can be several months/years. These studies save Fig. 1 The Human Viral Challenge Model. The study typically consists of inputs, such as the volunteers, their selection criteria, isolation in quarantine and exposure to a GMP virus. There are two treatment options; a vaccination/prophylaxis with an antiviral or b treatment with an antiviral. Outputs from the study, summarised on the right, such as virus symptoms, virus shedding etc. X is the number of days before virus exposure vaccination may occur. Y is the number of days post virus exposure that a volunteer may be followed for development time when the transition between phases is fully optimised. Importantly, unlike traditional phase 1b/phase 2 studies, HVC studies are not dependent on a natural outbreak of infection, which can occur at random, and for which the exact time of infection may not be apparent. They provide evaluable endpoints, comparative PD and PK data, along with additional biomarker data on product performance in humans. It must, however, be stated that most often such studies enrol otherwise healthy young adults which imply that the outcome of the infection in the placebo group may be seen as mild to moderate, to some extent. The safety of volunteers has to remain the priority of investigators. The HRV/HVC model can be a potent tool, not just to study HRV infection and disease, but also to investigate the mechanisms of exacerbation in patients with chronic respiratory disease and to conduct efficacy studies for new therapies. Human challenge studies with HRV have been shown to produce infection in over 90% of serologically susceptible subjects and result in a clinical syndrome that is comparable to that reported with natural colds [145, 146] . Symptoms usually appear within 24 hours and peak at 48-72 hours after inoculation. Virus shedding follows a pattern similar to that of their symptoms. In recent times, several hundred inoculations of adult subjects have been reported and have established this as a safe and effective method in which to study HRV-related disease in both healthy and asthmatic subjects [145] . These studies have provided a knowledge base to further develop the HRV experimental model and provide a controlled and useful tool to develop new therapies for the disease areas associated with HRV infection. New treatments for asthma and COPD are urgently needed, and small animal models of asthma are poorly predictive of efficacy. Most drugs that are effective in these animal models are not found to be effective in later stages of development in humans. Models that more closely follow clinical features of human asthma and COPD are needed [32, [147] [148] [149] [150] [151] ]. We have already described current influenza antiviral drugs that can shorten disease and reduce the severity of symptoms if taken early enough after infection, and their prophylactic use can decrease the risk of infection; their utility has been debated however [152] . The two main classes of currently effective antiinfluenza drugs are the NA inhibitors, such as zanamivir (Relenza™), oseltamivir (Tamiflu™), peramivir (Rapivab™) [153] and M2 inhibitors, although drug resistance makes this class unusable [154] . The HVC model has recently been used extensively to evaluate new classes of antiviral compounds against influenza, including those such as experimental monoclonal antibodies targeting epitopes within the highly conserved and exposed part of the M2 viral surface Fig. 2 The role of the HVC model in the clinical development pathway. Short duration proof of concept studies, which incorporate the HVC model, typically include small numbers of subjects. The resulting safety and, particularly, efficacy data can more accurately guide decisions on whether to expose a larger number of subjects to promising candidate therapeutics in field studies than conventional phase 1 safety data alone otherwise might protein [155, 156] the conserved stalk of the HA [157] and small molecule antiviral drugs that target the viral polymerase, e.g. favipiravir [158] . The HVC model allows for the rapid evaluation of novel therapeutic compounds which may be difficult to evaluate in the field, due to the nature of "at risk" groups, e.g. paediatrics. Specifically, and given the described historical experience with RSV vaccines, it is important that benefit can first be demonstrated in a healthy population. In the past, unlike influenza and HRV, the HVC model has not been routinely used with RSV. Recently, however, there are several antiviral therapeutics that have reached an advanced stage of development using the model. We had for some time wished to restart the HVC/RSV studies at the University of London, the two significant challenges that had stalled antiviral development for RSV presented a considerable research need. In association with the DeVincenzo lab at the University of Tenessee and the biotech company Alnylam, we set about designing possibly the first HVC/RSV study. Alnylam pioneered the use of RNA interference (RNAi) which is a natural mechanism that regulates protein expression and is mediated by small interfering RNAs (siRNA). Working with both groups, we manufactured an RSV Type A virus to GMP standard and titrated it in 35 human volunteers who we divided into five groups, each which was intranasally inoculated with increasing titre (3.0-5.4 log plaque-forming units/person) of the challenge virus. Intranasally. Overall, in this new model, 77% of volunteers consistently shed virus. Infection rate, viral loads, disease severity, and safety were similar between cohorts and were unrelated to the quantity of RSV received. Symptoms began soon after initial viral detection, peaked in severity near when viral load peaked and subsided as viral loads slowly declined. We concluded that regardless of the titre administered once infections were established the viral load drove illness. We saw no adverse events linked to the virus [25] . Using this new model we conducted an HVC clinical study and demonstrated for the first time that an RNAi had significant antiviral activity against human RSV infection -this established the first-ever proof of concept for an RNAi therapeutic in humans adults [159] . An editorial in the American Journal of Respiratory and Critical Care Medicine, described the utility of the HVC/RSV model saying; "This model permits the relatively quick and efficient study of new therapeutics in humans and assists in making critical decisions whether to advance a product into costly human trials in populations at highest risk for disease; children, elderly or immunocompromised patients. This constitutes a major and welcome advance in the field of RSV." [81] It is notable that two compounds that have distinct modes of action have recently been evaluated using the HVC model. First-in-class nucleoside analogue ALS-008176, the efficacy of which was first demonstrated in the HVC model, is currently under evaluation in hospitalised infants [160, 161] . The HVC trial was of randomised, double-blind design, and studied healthy adults inoculated with RSV Memphis 37B [25] . A total of 62 participants received ALS-008176 or placebo for five days after confirmation of RSV infection by PCR (tested twice daily post inoculation). The primary endpoint was the area under the curve (AUC) for viral load post infection. More rapid RSV clearance and a greater reduction in viral load, with accompanying improvements in the severity of clinical disease, were demonstrated in the groups treated with ALS-008176 when compared to the placebo group [160] . Intensive sampling allowed for any potential mutations associated with resistance to be rapidly identified. No such resistant mutations were observed [160] . An RSV-entry inhibitor, GS-5806, a second molecule, first-in-[its]-class was also evaluated. Among the 54 subjects that received active treatment, lower viral load, lower total mucus weight and a lower AUC symptom score were highly significant when compared to placebo [119] . Based on these challenge study data, this therapeutic is now also progressing into potentially pivotal field studies [162] . An essential element of design in both studies was the timing of the first administration of therapeutic postexperimental virus inoculation; the timing was dependent on the detection of virus in nasal wash samples post inoculation of challenge virus by a rapid PCR assay [163] , rather than at an arbitrary time point. Subsequently the therapeutic was administered every 12 hours. Careful dose timing, at a clinically relevant point of detection, contributed to the positive outcomes of both studies. It is also believed that by using this "triggered dosing" model, it better mimicked what would happen in a clinical setting as symptoms are known to appear soon after the onset of virus shedding. The HVC model is not limited to novel antiviral compounds but is also important for the evaluation of novel vaccines. Influenza vaccine performance in recent years raises questions about the most appropriate correlates of protection. Unlike field studies, HVC studies are useful tools for assessing the correlates of protection, vital for vaccine development [103, 104, 164] . Specifically, the importance of the humoral and cellular responses has been highlighted along with the pre-existing T-cell immunity for other respiratory viruses [104] . A recent publication describes the use of the HVC model to demonstrate the efficacy of a novel intranasal proteosome-adjuvanted trivalent inactivated influenza vaccine (P-TIV). In two separate studies, selected subjects who were naïve to A/Panama/2007/1999 (H3N2) virus, were dosed via nasal spray with one of three regimens of P-TIV or placebo. Together, the studies evaluated one or two doses, 15 μg or 30 μg, either once only or twice 14 days apart (1 x 30 μg, 2 x 30 μg, 2 x 15 μg) and subjects were challenged with A/Panama/2007/1999 (H3N2) virus. Immune responses to the vaccine antigens were measured by haemagglutination inhibition (HAI) assay and nasal wash secretory IgA (sIgA) antibodies. Vaccine efficacy was observed ranging from 58% to 82%, comparable to traditional vaccines. The studies also demonstrate that protection against illness associated with evidence of influenza infection significantly correlated with pre-challenge HAI (serum IgG) titres (p = 0.0003) and mucosal IgA (p≤0.0001) individually, and HAI (p = 0.028) and sIgA (p = 0.0014) together. HAI and sIgA levels were inversely related to rates of illness. These studies demonstrated the efficacy of this novel intranasal vaccine and answered some important questions concerning true correlates of protection against influenza infection which will help drive future vaccine design. As well as achieving its primary aims, it revealed valuable insights into the correlates of protection and will, we hope, aid future vaccine design [164] . An inter-seasonal or universal influenza vaccine is desperately needed; it will save many lives, whether in those unexpected years when the recommended composition is not matched, or when a pandemic occurs, as it did in 2009. The significance of the 1918 pandemic [165, 166] makes it very clear; up to 100 million people died. A universal vaccine is one that can be prepared for the unexpected, a virus that occurs due to the reassortment of viral genes from different host species. The HVC model is possibly the only way to initially test such a universal vaccine. A universal candidate could generate an immune response against the highly conserved virus ion channel protein M2, [167] [168] [169] [170] , although no vaccine has been shown to be effective in this regard; monoclonal antibodies alone have, the HVC model showed their efficacy [156] . Alternatively, a vaccine may target the conserved stalk of the HA protein [104, 171] , or elicit a T-cell response to the internal proteins [172] [173] [174] [175] . All are possibilities that have been and can be explored more efficiently using the HVC model. Although HVC studies provide PoC, researchers, as we have shown, have employed regulatory design standards typical of later phase efficacy studies. With the development of molecular technology, it is now possible to refine the statistical analysis by stratifying the subjects based on their immune profile. For instance, it is now possible to assess whether a subject is carrying other known respiratory pathogens (bacteria, viruses etc.) and if there is a possible impact on the set of results from the volunteer. Subjects often consent for further analysis of their samples, which allows a valuable biobank of samples to be built for further testing. Moving forward, such samples will allow the use of the HVC model to understand further what happens when a virus infects a person. It is worth noting that the HVC model is not limited to PoC work on potential therapeutic agents; it is also extensively being used for research purposes, upon which improved treatments for respiratory viruses can be built. In recent years it has been used to demonstrate "gene switching signatures" that could form part of a diagnostic that would reveal infected individuals before they become symptomatic, in the early stages of infection; this could be vitally important in the event of a pandemic [176, 177] . Also, the HVC model has been used to allow a comparison of the relative disease dynamics of different respiratory viruses [24] and to provide a better understanding of the interaction of the virus and the human host [26, 178, 179] . The HVC model has increased our understanding of the viral life cycle and disease pathogenesis in a tightly controlled setting using small numbers of volunteers. Each volunteer is isolated from each other, and the wider community, ensuring that the disease under consideration is the only one of interest. The applicability of the virus used to challenge volunteers in the HVC model to a virus that an individual might become exposed to in the "real world" is significant. Whether challenge trials are feasible is dependent on the availability of adequately safety-tested challenge virus strains that are of know providence. The HVC model provides certain knowledge of the character of the virus; the exact time point of infection; measurability of nasal virus shedding; prospective recording of symptoms and pre-selection of participants for viral challenge who are sero-suitable. This ensures that a statistically significant rate of infection is achieved with the minimal number of volunteers, thus optimising the risk-benefit ratio that supports the determination of therapeutic efficacy. Crucial to HVC study design is the timing of administration of the first dose of product under investigation to determine optimal effectiveness, not just in the challenge study itself, but in both later stage clinical studies and final clinical use. The HVC model is an important tool in drug development, in particular with regard to acute respiratory infections. It can accelerate the development of therapeutics that address multiple unmet medical needs. It helps in the understanding of the relationship between a virus and its human host and offers the potential for the development of early-stage diagnostics. It contributes towards identifying new areas for therapeutic intervention. Possibly, and arguably, more importantly, it can ensure that scarce medical resources are directed towards later stage clinical development in an evidence-based manner, and promising therapeutic opportunities are prioritised. A careful and targeted study design process is a crucial step towards the successful outcome of a challenge trial, because almost all parameters, can be either controlled or at least known (either pre-or post-hoc). Furthermore, results from such trials can be used to make commercial decisions and can lead to major publications, expanding the collective understanding of the scientific community. Samples from such experiments are of immense value to researchers for the understanding of host interaction mechanisms and the development and validation of therapeutics. Utilisation of consistently collected historical data from HVC studies informs the accurate design and powering of subsequent studies. HVC studies have been successful in providing proof of concept for DNA vaccines, T-cell vaccines, intranasal vaccines, monoclonal antibodies and small molecules against a range of important respiratory viruses. It is also encouraging to see that the HVC model is now expanding into further patient populations such as the elderly, asthmatics and those with other conditions such as chronic obstructive pulmonary disease. An expanding archive of data from preceding studies is an invaluable asset to assist in the selection of volunteers, decide on appropriate endpoints and refine future field study designs. This allows for safer, statistically sound and more rapidly delivered research. drafted the initial version of this manuscript with author RLW. hVIVO was responsible for overall management of this work and verified the accuracy of the data presented. Other non-author contributors included Ben Murdoch of hVivo who provided figures. hVivo would like to thank the volunteers without whose altruism the human viral challenge studies conducted at hVivo over many years would not have been possible. The work, including professional medical writing services for preparing this manuscript, was wholly funded by hVivo Services Limited, the employer of all authors. Author RLW conceived the strategy for this paper. Author RLW and professional medical writer Samina Hamilton drafted the article (see 'Acknowledgements'). Authors RLW and AG critically reviewed the complete article for important intellectual content. Authors RLW and AG had full authority over the choice of the journal and approved the final article. Author RLW is a guarantor for the paper and takes overall responsibility for this publication. All other authors contributed to the writing and review of this manuscript. Ethics approval and consent to participate All clinical studies were described received appropriate Ethical Committee approval, including informed consent of volunteers. All authors declare that they are employees of hVivo and as such, have provided or do provide ethical professional clinical research services to academic, biotechnology, or pharmaceutical clients. A patent (patent applications 14/366602 (US) 12813946.6 (EP) application is in progress regarding specific utilisation of the HVC model. This does not alter the authors' adherence to International Society for Medical Publication Professionals (ISMPP) 'Good Publication Practice for Communicating Company-Sponsored Medical Research: GPP3'.

Why is ribavirin treatment limited?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What features can be created for creating vectored vaccines?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What happens upon viral infection in the airway?

Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.

What do applications of filamentous phage depend on?

C. difficile 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485338/ SHA: f0fb3bbd96dad4c907c7fd456cd5783ed8fa7bd6 Authors: Rosenbusch, Katharina E.; Bakker, Dennis; Kuijper, Ed J.; Smits, Wiep Klaas Date: 2012-10-31 DOI: 10.1371/journal.pone.0048608 License: cc-by Abstract: Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds. Spo0A is the key regulator for sporulation [1, 2] . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA [3] [4] [5] . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay [6] . The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. The two functional domains, the N-terminal phosphorylation and dimerization domain (receiver domain), and the C-terminal DNA binding (effector) domain are separated by a hinge region that is relatively poorly conserved [7] . Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization [8, 9] , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation [10] . Extensive characterization of Spo0A targets has revealed a motif that represents a high affinity Spo0A binding site, the 0A box [10, 11] . The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence [12, 13] . It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis [14] [15] [16] , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis [17] [18] [19] [20] [21] , and solvent production in Clostridium acetobutylicum [22, 23] . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection (CDI) (for recent reviews see [24, 25] ). Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed [26] . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 (BI/ NAP1) and 078 [27, 28] . Its main virulence factors are the major clostridial toxins A and B [29, 30] . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin [31, 32] . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow [24, 25] . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores [33] and the gene is required for persistence and transmission in mice [34] . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A [2] . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A [35] . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum [36] . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter [37] . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes. Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta(DE3) pLysS (Novagen). C. difficile strains were grown in a glucose-free trypton-yeast based medium (TTY; 3% w/v bactotrypton (BD), 2% yeast extract (Fluka), 0.1% w/v thioglycollate (Sigma) pH 7.4), supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates (Biomerieux). All plasmids are listed in Table 1 . Primers (obtained from Sigma Aldrich) are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase (Fermentas) according to the instructions of the manufacturer. Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO (Invitrogen), yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE (20 mM Tris Acetate, 0.5 mM EDTA) gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation (using a GeneJET Gel Extraction kit, Fermentas) and cloned into similarly digested pMF14 [10] that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 (see below). Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 (see below). Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit (Macherey Nagel) according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry (Invitrogen) on an ABI3130 sequencer (Perkin Elmer), according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix (Invitrogen) in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid (Invitrogen) at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 (SciEd) and Geneious version 5.6.2 (Biomatters Ltd). Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta(DE3) pLysS (Novagen). Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described [10] . In short, cells were disrupted in 4 mL lysis buffer (2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry (Clontech) in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column (BioRad) and washed with 2 mL wash buffer (20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column (identical to wash buffer but with 50, 100, 250 or 500 mM imidazole). The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT) using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa (Pierce). Proteins were stored at 280uC in storage buffer (identical to dialysis buffer but containing 20% glycerol). Protein concentrations were determined using Bradford reagent (BioRad), according to the manufacturer's instructions. DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase (Promega) and chromosomal DNA from B. subtilis JH642 (Bacillus Genetic Stock Center 1A96; http://www.bgsc.org), plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm [38] as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS) and a QIAExII kit (Qiagen), according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase (Invitrogen) according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter (Beckman). EMSA conditions were based on previous studies [10] . In short, binding reactions were carried out in binding buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol) in the presence of 200 mg/mL bovine serum albumin (NEB) and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer. Electrophoresis was carried out for 120 min at 85 V. After vacuum drying the gels onto filter paper, they were imaged after overnight exposure on Phosphorimager screens on a Typhoon instrument (GE Healthcare). The toxic effects of C. difficile culture supernatants on Vero cells (a kind gift of Eric Snijder [39] ) were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium (Dulbecco modified Eagle medium (Lonza) supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum), before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin (Techlab) was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB (Techlab) was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects (cell rounding) were observed. Statistical significance was evaluated with an independent sample t-test. Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute (Hinxton, UK). Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer (10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC (Roche)). After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer (0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB) was added, and samples were heated to 96uC for 5 mins. Total cell lysates (amounts corrected for OD 600 ) were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in PBST buffer (phosphate buffered saline with 0.1% v/v Tween-20) containing 5% membrane blocking reagent (Amersham Biosciences). To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection (Amersham Bioscience), or a goatanti-mouse-biotin-conjugated secondary antibody (Dako) followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody (Jackson). Detection was done using on a Typhoon instrument (GE Healthcare). Background corrected peak volumes were quantified using ImageQuant TL (Amersham Biosciences). Alignments of B. subtilis and C. difficile spo0A were made using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) on the basis of the published genome sequences, Genbank accession numbers AL009126 and AM180355, respectively, and the 630Derm spo0A sequence as determined in this study. The sequence for spo0A of C. difficile strain 630Derm was deposited in Genbank (accession no JX050222). Consensus Spo0A boxes were identified using a Single string Search command in Genome2D [40] , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel. The results were manually inspected for those boxes within 500 bp upstream of a gene on the same strand. Figures for publication were prepared using ImageQuant TL (Amersham Biosciences), Adobe Photoshop CS3 (Adobe Systems Inc) and Corel Graphics Suite X5 (Corel Corporation). In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain (DBD) were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli (Fig. 1A) and purified to near homogeneity using metal affinity chromatography ( Fig. 1A ; lanes P). Full length protein was used to raise antibodies to detect Spo0A in total lysates of C. difficile strains, and the purified DNA binding domain was used in subsequent in vitro binding assays (see below). We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells. We performed immunoblotting using polyclonal antibodies against C. difficile Spo0A on total lysates of wild type and spo0A mutant cells grown in a trypton-yeast based medium (TTY). We found a clear signal of the size expected for full length Spo0A (,31 kDa) as early as 3 hours post inoculation (exponential growth phase), through transition phase (8 h) as well as 24 and 48 hours post inoculation (stationary growth phase) ( Figure 1B; 630Derm) . The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant (Fig. 1B , CT::spo0A). We obtained similar results in other media, such as the commonly used supplemented brain heart infusion broth (BHIS; data not shown). To determine relative levels of Spo0A throughout growth, we performed an immunoblot experiment using fluorescent antibodies, which gives more quantitative information compared to the use of horseradish peroxidase conjugated antibodies in our hands. We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation ( Figure 1C ). Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase. Spo0A of C. difficile Strain 630Derm Contains a 6aminoacid Duplication BLAST homology searches readily identify a homolog of the well-characterized B. subtilis Spo0A protein in C. difficile 630 (CD1214) and previous work demonstrated that a spo0A mutant (an insertional inactivation of cd1214) -as expected -no longer forms spores [41] . In silico analyses suggest a similar secondary structure for both proteins ( Fig. 2A) , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region [7, 12] . We compared the sequence of CD1214 obtained from our lab strain 630Derm [38] to that of the published C. difficile 630 genome [42] . Strain 630Derm is a spontaneous erythromycin sensitive strain, which is commonly used in mutagenesis studies and was obtained by serial passaging of strain 630 [33, 38] . The 630Derm spo0A sequence (Genbank accession no JX050222) was derived from the expression plasmids constructed for this study, and confirmed in a whole genome sequence of strain 630Derm generated in our lab (data not shown). We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid (NVGNIE) duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation (hinge), flanking the highly conserved DNA binding domain ( Fig. 2A and B) . We verified the absence of this duplication in strain 630 by PCR (Fig. 2C ) as well as sequencing from the chromosomal DNA of C. difficile 630 (data not shown), to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 (to which 630 and 630Derm belong) by PCR, but the duplication was found to be unique to 630Derm among the isolates tested (data not shown). C. difficile Spo0A-DBD Shows Similar Specificity as B. subtilis Spo0A-DBD Next, we examined the conservation of the DNA binding domain of Spo0A (Spo0A-DBD) between B. subtilis and C. difficile. In B. subtilis amino acid residues contacting the backbone of the DNA and interacting with specific residues of the Spo0A binding sequence have been defined [13] . We found that all these residues were conserved in the C. difficile protein sequence (Fig. 2B) , indicating that the protein likely recognizes a similar motif. DNA binding by full length Spo0A in B. subtilis requires phosphorylation dependent dimerization [8, 9] . However, it was shown that the isolated DBD is capable of binding to legitimate targets of the full length protein [10] . Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene (PabrB) of B. subtilis. PabrB is commonly used as a high-affinity control in binding assays with the B. subtilis Spo0A or Spo0A-DBD protein [43, 44] . It is noteworthy that we failed to identify a homolog of abrB in C. difficile using BLAST, indicating that potential indirect regulation by Spo0A cannot occur through abrB in C. difficile as it does in B. subtilis. We found that C. difficile Spo0A-DBD bound with high affinity to PabrB (Fig. 2D and E) . We performed electrophoretic mobility shift assays (EMSAs) using radiolabeled PabrB and increasing amounts of purified C. difficile Spo0A-DBD that was purified using a C-terminal 66His-tag. The addition of protein leads to a dose-dependent retardation of the DNA fragment with an apparent K D of ,50 nM. In the same range of protein concentrations, no binding was observed for a negative control (a DNA fragment of B. subtilis citG [45] ) (Fig. 2E) , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence (0A box), that is characterized by a 7 bp core motif (TGTCGAA) [10, 11] . Structural studies have revealed that the protein makes specific contacts with the G at position 2 (G2), and the C at position 4 (C4) and 5 (G5) of this motif [13] . We introduced G2A, C4A, G5A, G2A/C4A and C4A/G5A mutations in the perfect consensus core 0A-box present in PabrB. We found that the affinity of C. difficile Spo0A for these mutated PabrB fragments was highly reduced (Fig. 2E) . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence. For the single point mutations in the DNA, the affinity decreased ,10-fold. There did not seem to be an additive effect of a second point mutation for the two combinations tested. None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other (non-consensus) 0A boxes in the abrB promoter [44] . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD. Value for Binding by C. difficile Spo0A-DBD Above, we have established that the Spo0A-DBD of C. difficile is highly homologous to that of the B. subtilis Spo0A protein, and that the proteins recognize a similar consensus sequence (Fig. 2 ). Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D [40] . Such an analysis revealed the presence of 102 matching motifs, of which 45 were located within 500 bp of the initiating ATG of an open reading frame on the same strand (see Table S1 ). Our attention was drawn to spo0A and sigH, as these two genes were previously found to be regulated by Spo0A in B. subtilis and/or play important roles in sporulation [3, [46] [47] [48] . We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames. We found that the addition of Spo0A-DBD to the reactions caused retardation of the spo0A and sigH DNA fragments (Fig. 3A) , but not of a spoVG fragment which did not contain a consensus 0A box (Fig. 3B) . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA. Moreover, the presence of multiple shifted species could indicate the presence of more than one strong binding site. These results establish that spo0A and sigH are likely legitimate targets of Spo0A in C. difficile, and confirm that spoVG is not, in line with results obtained in B. subtilis [10] . We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon. This positioning is similar to that observed for spo0A (275) and sigH (278). We confirmed in vitro binding of the C. difficile Spo0A-DBD to the promoter regions of lplA and ssuA. We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene (CD1654; box at 267) and the ssuA gene (CD1484; box at 282) (Fig. 3A) . The lplA gene encodes a predicted lipoate-protein ligase, and ssuA is annotated as an aliphatic sulfonates ABC transporter; to our knowledge, neither of these have been directly implicated in sporulation or have found to be targets for Spo0A in other organisms. Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway [33] . However, the in silico identification of upstream regions with a consensus Spo0A binding site did not point to any of the early sporulation genes (downstream of spo0A itself) as direct targets of Spo0A. This is likely the result of variations in the 0A-box in these promoters that were disregarded in the box search. In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A (such as spoIIAA and spoIIE) do not contain a 100% match to the core motif, but rather one or more near-consensus boxes [5, 49] . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above (Fig. 3B ). For spoIIAA (encoding an antianti sigma-factor) and spoIIE (encoding a serine phosphatase), we observed a low intensity shifted species at concentrations as low as 150 nM. For spoIIGA (encoding a sporulation specific protease) we observed the shifted species only at higher concentrations of protein (.200 nM). The negative control (spoVG) did not demonstrate binding of Spo0A-DBD at these concentrations. Moreover, the shift we observed was reversible using unlabeled DNA containing a high affinity binding site, but not using unlabeled DNA that lacked such a site ( Figure S1B-D) . Therefore, we consider the binding to spoIIAA, spoIIE and spoIIGA genes to be specific, despite the fact that increasing the amount of protein did not seem to cause a significant increase in the amount of DNA in the complex. Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells [35] . Considering the absence of a homolog of the abrB repressor, direct binding of Spo0A and concomitant activation of toxin gene transcription is a likely mechanism through which this could occur. We found evidence for direct binding of Spo0A-DBD to the region upstream of tcdB, encoding one of the major clostridial toxin genes, and possibly tcdC, but this did not seem to result in lower toxin levels in our hands. We performed EMSAs using DNA upstream of tcdR (encoding a sigma factor responsible for the activation of toxin gene transcription), tcdB (encoding toxin B), tcdA (encoding toxin A). In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE (encoding a holin-like protein [50, 51] ) and tcdC (encoding a putative negative regulator of toxin production [52] [53] [54] ), even though this regulator does not have a significant effect on toxin levels under the conditions we used [55, 56] . Of the regions tested, we only observed a clear shifted species, indicative of Spo0A binding, for tcdB ( Figure 4A ); the shifted species in our EMSA assay was reversed by the addition of unlabeled DNA containing a high affinity binding site, but not by DNA lacking such a site ( Figure S1E ). For tcdC, some smearing was observed at all concentrations of proteins tested ( Figure 4A ), and there did not seem to be a clear effect of the addition of unlabeled DNA fragments ( Figure S1F ). The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested. We found no lower toxicity towards Vero cells of culture supernatants derived from spo0A mutant cells compared to wild type. We grew three independent biological replicates of a wild type (630Derm) or Clostron-generated spo0A mutant (CT::spo0A -a kind gift of the Minton lab) in glucose-free TTY medium. We harvested culture supernatant at late-exponential phase (approximately 7 hours post inoculation), the transition phase between exponential and stationary growth phase (approximately 9 hours post inoculation), as well as two time points in stationary phase (24 and 48 hours post inoculation) and determined the toxin endpoint titres (see Materials and Methods). In contrast to previous findings, we observed a small (#4-fold) increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant (p.0.05, independent sample t-test). In other medium (BHIS), we observed no differences at all (data not shown). We conclude that Spo0A does not positively affect toxin production in C. difficile 630Derm and the in vivo relevance of the binding to regions upstream of tcdB and/or tcdC is therefore limited under our experimental conditions. The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box [5, 10, 11, 45] . Depending on the analysis, this motif is flanked by one or more adenine or thymine residues [10, 11] . Interestingly, many target genes do not harbor a perfect match to this consensus sequence, but rather contain one or more degenerate motifs. The differences in these motifs may reflect different promoter architectures (e.g. AT content), modes of action (e.g. activation or repression) or levels of regulation. Spo0A genes in B. subtilis can be divided in different classes that respond to different levels of phosphorylated Spo0A [43, 57] . For C. difficile, we conclude that the Spo0A protein likely recognizes a motif that is similar to the B. subtilis Spo0A box on the basis of four lines of evidence; 1. All DNA binding/contacting residues are conserved (Fig. 2B) , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A (Fig. 2D) , 3. Mutagenesis of key residues in the B. subtilis Spo0A box reduces affinity of C. difficile Spo0A for DNA (Fig. 2E ) and 4. A B. subtilis Spo0A box has predictive value for DNA binding by C. difficile Spo0A (Fig. 3A) . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein. It is likely that differences do exist between the preferred binding sites for both proteins that will be evident when a comprehensive analysis is performed of in vivo DNA binding of C. difficile Spo0A; based on the limited data set of this study, a MEME analysis [58] already suggests possible differences in the extended Spo0A motif (W.K. Smits, unpublished observations). These differences may relate to the much higher AT content of C. difficile compared to B. subtilis (71 vs. 56.5%, respectively), or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation (for review see ref [1, 59] ). The activation of Spo0A is controlled by a multi-component phosphorelay that can integrate environmental cues [60] and ensures a gradual increase in the level of phosphorylated Spo0A in the cell [57] . In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter [46] , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter [48] . In C. difficile, there are some interesting differences and similarities in the regulatory pathways. Most notably, there seems to be no phosphorelay [2] and the phosphorylation state of Spo0A is supposedly controlled by orphan histidine kinases [35] . The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H [37] , as it is in B. subtilis [61] . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile (Fig. 3A) , raising the possibility of auto-regulation of spo0A. The putative direct regulation of sigH by Spo0A may reflect that the C. difficile genome does not harbor a homolog of the pleiotropic regulator AbrB, which is responsible for the Spo0A-dependent regulation of sigH in B. subtilis [48] . Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase ( Figure 1C) . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA (Fig. 3B ). All these observations are consistent with direct regulation of these genes by Spo0A in other organisms [5, 45, 49, 62] , and the conservation of the sporulation pathway [2] . Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology [10, [14] [15] [16] . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB. Additionally, transcription of abrB responds already to low levels of Spo0A,P [43] . As a result these effects are detectable in late-exponential and early stationary phase, as some Spo0A is present throughout growth in B. subtilis cells. Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect. In Clostridium acetobutylicum and C. beijerinckii, Spo0A is a direct regulator of solvent formation, as well as sporulation [22, 23] . It seems therefore conceivable that Spo0A in C. difficile also affects aspects of metabolism. In this respect, it is important to note that also in C. difficile Spo0A is detectable from early exponential growth phase on ( Figure 1B) . We observed direct binding of C. difficile Spo0A to the promoter region of sigH (Fig. 3A) . This gene encodes the key sigma factor for the transition phase, and regulates processes outside sporulation as well [37] . Moreover, we found significant levels of Spo0A from early stationary phase on ( Fig. 1B and unpublished observations) , indicating the regulatory actions of Spo0A need not be limited to stationary phase in C. difficile. In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA (Fig. 3A) . The presence of a putative Spo0A binding site upstream of these genes, as well as the spacing compared to the start codon, is conserved in the problematic Stoke-Mandeville strain (R20291), a member of PCR ribotype 27. This could indicate that these aspects of regulation by Spo0A are conserved in multiple strains of C. difficile. It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed. In order to distinguish direct from indirect effects, in vivo binding profiles of Spo0A should be performed. The antibodies generated for this study should prove to be useful for this type of experiments. Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control [17] . Similarly, the production of the emetic toxin cereulide in B. cereus is greatly repressed in a spo0A mutant, in an AbrB-dependent manner [63] . In contrast, Spo0A directly represses the expression of the cry toxin genes in B. thuringiensis and a spo0A mutant is therefore a hyper-producer of the insecticidal crystal protein [18, 21] . In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A [64] and also the production of enterotoxin in this organism seems to be (indirectly) dependent on sporulation [65, 66] . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A (TcdA), both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B (TcdB) [35] . Our in vitro binding data indicate a potential binding site for Spo0A upstream of tcdB and possibly tcdC (Fig. 4A) . However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant (a kind gift of the Minton lab; [33] ) did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type (#2fold in exponential phase cells up to 4-fold in late-stationary phase cells). The small, and not significant, differences in toxin levels in our experiments might be attributed to differences in the susceptibility of cells for lysis rather than the production of toxin, but could also indicate a negative regulatory effect of Spo0A on toxin production. In support of the latter hypothesis, it was recently reported that a spo0A mutant of C. difficile strain R20291 (a PCR ribotypes 027/BI/NAP1 epidemic strain) demonstrates ,10fold higher toxin levels than its isogenic wild type 30 h post inoculation, and is significantly more virulent in a mouse model of disease [34] . The differences between Underwood et al [35] on the one hand and our study as well as the study of Deakin and coworkers [34] on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously (data not shown). Alternatively, the differences could indicate integration of the group II intron at more than one location in the chromosome in the strain used in Underwood et al [35] . In the absence of a complementation experiment and/or Southern blot data, this remains to be established. In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays. In the present study we have for the first time demonstrated direct binding of the DNA binding domain of C. difficile Spo0A to putative target DNA. This work has revealed that aspects of Spo0A binding are conserved between Bacillus and C. difficile (0A box, possible auto-regulation and binding to early sporulation promoters), whereas others are not (the absence of abrB as a direct target in C. difficile, binding to DNA upstream of lplA, ssuA). The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis [18, 21] . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo. The identification of genes affected by Spo0A in C. difficile may shed light on the role of the protein in virulence and pathogenesis of this organism. Figure S1 Specificity controls for binding by Spo0A-DBD-his6. Arrows indicate the position of shifted species (DNA:protein complexes). Titrations with PCR fragments of PabrB (containing a high affinity binding site) and PtcdA (lacking such a site) correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A. Comparison of binding of Spo0A-DBD-his6, Spo0A-his6 and CD2195-his6 binding to the upstream region of spoIIAA. B. Binding of Spo0A-DBD-his6 to the upstream region of spoIIAA is reversed by the addition of PabrB, but not by the addition of PtcdA). C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA. E. Binding of Spo0A-DBD-his6 to the upstream region of tcdB is reversed by the addition of PabrB, but not by the addition of PtcdA. F. Binding of Spo0A-DBD-his6 to the upstream region of tcdC is not or moderately affected by the addition of PabrB and/or PtcdA. (TIF) Text S1 Oligonucleotides used in this study and PCR cycling conditions for the EMSA probes. (PDF)

What is sporulation?

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

How long did the patient breath into the RTube?

MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The

How many clades have become apparent in genome of MERS-COV from humans and DCs?

A Global Champion for Health—WHO’s Next? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924837/ SHA: f2f9088055600d4160e36db5cb6ea000916390a3 Authors: nan Date: 2016-06-28 DOI: 10.1371/journal.pmed.1002059 License: cc-by Abstract: In this month’s editorial, the PLOS Medicine Editors propose ideal qualities for the World Health Organization's next Director General, for whom the selection process is now underway. Text: response to the Ebola outbreak [1] . Reformation of WHO to ready it to lead responses to future health emergencies is one area of active debate. Chan will step down from WHO on June 30, 2017 after more than a decade in the post. The process for choosing WHO's next leader has begun, promising to be protracted and rigorous as befits the importance of the role. Factoring in the many influential stakeholders in the process of appointing Chan's successor, however, transparency of the selection process may be one area unlikely to attract plaudits. Although too soon to speculate about the identity of WHO's next Director-General, it is worth reflecting on what qualities an incoming leader should bring to WHO and how that person might need to conceive changes in the structure and behavior of the organization against a landscape of important and evolving threats to the health of the fastgrowing global population. Instead of electing a new Director-General, Lorenz Von Seidlein of Mahidol University, Thailand, argued that "the problems. . .are now so deeply ingrained that replacing the WHO with new, more appropriate organizations is the logical solution. . .at a fraction of current cost, free of cumbersome, archaic obligations and entitlements and [with] an ability to respond to new problems." This viewpoint is indicative of the strength of feeling that WHO's deficiencies have come to evoke in some of those committed to the cause of improving the health of people in low-income and middle-income countries. But this perception acknowledges that an accountable global body will always be needed to promote, set standards in, and evaluate progress toward better health for people in all countries. The next Director-General will need to heed critics of the organization and craft a process of streamlining and restructuring to produce a new WHO that is demonstrably effective in leading responses to threats to health, and efficient in doing so. As Gostin commented to PLOS Medicine, "WHO urgently needs a bold reform agenda to fix long-standing problems recognized by every independent group that has evaluated the Organization." Political machinations and the enemy within, bureaucracy, are likely to impede reform. For example, WHO's regional and country offices are seen by some as unaccountable, yet the agency of the future will need to be connected and responsive to the resources and needs of all constituent countries. As Gostin also noted, "[WHO] has failed to include civil society in its governance, unlike. . .newer organizations." WHO's next Director-General should be a proven leader and advocate, perhaps from a lowincome or middle-income country. The new recruit will be greeted by a full in-tray, and featuring prominently are likely to be the constraints imposed by WHO's current funding mechanisms. A substantial proportion of WHO's existing budget is earmarked for specific projects, leaving the organization with little financial flexibility to respond to unanticipated demands. However, any improved funding mechanism is likely to follow, and be dependent on, organizational reform. According to Kruk, "WHO is both essential and hamstrung. . .the election of the Director-General should be a moment for member countries and other funders to reflect on whether they want an implementation agency for their favored health agenda, or an independent institution with the intelligence, agility, and operational capacity to tackle the coming global health challenges." Above all, the incoming leader of WHO will need to be open-minded and creative. More than one of the experts we contacted emphasized the fluid nature of the threats to human health to which WHO should shape the world's response. WHO must be able to lead responses in some areas of global health, but, in other areas, working together with more nimble and focused organizations will be pragmatic. Large-scale infectious disease outbreaks are continuing, and noncommunicable diseases, including cancer, dementia, and mental illnesses, are growing in prevalence and increasing demand for treatment and care. The resources and ingenuity of researchers and clinicians will need to be harnessed, and interventions adapted to new settings, with much greater dynamism. The secular issues of population ageing, conflict, climate change, migration, and others will produce health problems that only an organization with a global reach, responsible to all, can hope to meet. We look forward to welcoming a new leader for WHO with the energy and vision to remold the organization to meet the health needs of the world's people and societies for the 21st century.

When did the last Director General of the WHO resign?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What is the advantage of the AAV vector?

Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review https://doi.org/10.3390/jcm9030623 SHA: 9b0c87f808b1b66f2937d7a7acb524a756b6113b Authors: Pang, Junxiong; Wang, Min Xian; Ang, Ian Yi Han; Tan, Sharon Hui Xuan; Lewis, Ruth Frances; Chen, Jacinta I. Pei; Gutierrez, Ramona A.; Gwee, Sylvia Xiao Wei; Chua, Pearleen Ee Yong; Yang, Qian; Ng, Xian Yi; Yap, Rowena K. S.; Tan, Hao Yi; Teo, Yik Ying; Tan, Chorh Chuan; Cook, Alex R.; Yap, Jason Chin-Huat; Hsu, Li Yang Date: 2020 DOI: 10.3390/jcm9030623 License: cc-by Abstract: Rapid diagnostics, vaccines and therapeutics are important interventions for the management of the 2019 novel coronavirus (2019-nCoV) outbreak. It is timely to systematically review the potential of these interventions, including those for Middle East respiratory syndrome-Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS)-CoV, to guide policymakers globally on their prioritization of resources for research and development. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Supplementary strategies through Google Search and personal communications were used. A total of 27 studies fulfilled the criteria for review. Several laboratory protocols for confirmation of suspected 2019-nCoV cases using real-time reverse transcription polymerase chain reaction (RT-PCR) have been published. A commercial RT-PCR kit developed by the Beijing Genomic Institute is currently widely used in China and likely in Asia. However, serological assays as well as point-of-care testing kits have not been developed but are likely in the near future. Several vaccine candidates are in the pipeline. The likely earliest Phase 1 vaccine trial is a synthetic DNA-based candidate. A number of novel compounds as well as therapeutics licensed for other conditions appear to have in vitro efficacy against the 2019-nCoV. Some are being tested in clinical trials against MERS-CoV and SARS-CoV, while others have been listed for clinical trials against 2019-nCoV. However, there are currently no effective specific antivirals or drug combinations supported by high-level evidence. Text: Since mid-December 2019 and as of early February 2020, the 2019 novel coronavirus (2019-nCoV) originating from Wuhan (Hubei Province, China) has infected over 25,000 laboratory-confirmed cases across 28 countries with about 500 deaths (a case-fatality rate of about 2%). More than 90% of the cases and deaths were in China [1] . Based on the initial reported surge of cases in Wuhan, the majority were males with a median age of 55 years and linked to the Huanan Seafood Wholesale Market [2] . Most of the reported cases had similar symptoms at the onset of illness such as fever, cough, and myalgia or fatigue. Most cases developed pneumonia and some severe and even fatal respiratory diseases such as acute respiratory distress syndrome [3] . The 2019 novel coronavirus (2019-nCoV), a betacoronavirus, forms a clade within the subgenus sarbecovirus of the Orthocoronavirinae subfamily [4] . The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also betacoronaviruses that are zoonotic in origin and have been linked to potential fatal illness during the outbreaks in 2003 and 2012, respectively [5, 6] . Based on current evidence, pathogenicity for 2019-nCoV is about 3%, which is significantly lower than SARS-CoV (10%) and MERS-CoV (40%) [7] . However, 2019-nCoV has potentially higher transmissibility (R0: 1.4-5.5) than both SARS-CoV (R0: [2] [3] [4] [5] and MERS-CoV (R0: <1) [7] . With the possible expansion of 2019-nCoV globally [8] and the declaration of the 2019-nCoV outbreak as a Public Health Emergency of International Concern by the World Health Organization, there is an urgent need for rapid diagnostics, vaccines and therapeutics to detect, prevent and contain 2019-nCoV promptly. There is however currently a lack of understanding of what is available in the early phase of 2019-nCoV outbreak. The systematic review describes and assesses the potential rapid diagnostics, vaccines and therapeutics for 2019-nCoV, based in part on the developments for MERS-CoV and SARS-CoV. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies examining the diagnosis, therapeutic drugs and vaccines for Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the 2019 novel coronavirus (2019-nCoV), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There were two independent reviewers each focusing on SARS, MERS, and 2019-nCoV, respectively. A third independent reviewer was engaged to resolve any conflicting article of interest. We used the key words "SARS", "coronavirus", "MERS", "2019 Novel coronavirus", "Wuhan virus" to identify the diseases in the search strategy. The systematic searches for diagnosis, therapeutic drugs and vaccines were carried out independently and the key words "drug", "therapy", "vaccine", "diagnosis", "point of care testing" and "rapid diagnostic test" were used in conjunction with the disease key words for the respective searches. Examples of search strings can be found in Table S1 . We searched for randomized controlled trials (RCTs) and validation trials (for diagnostics test) published in English, that measured (a) the sensitivity and/or specificity of a rapid diagnostic test or a point-of-care testing kit, (b) the impact of drug therapy or (c) vaccine efficacy against either of these diseases with no date restriction applied. For the 2019-nCoV, we searched for all in vitro, animal, or human studies published in English between 1 December 2019 and 6 February 2020, on the same outcomes of interest. In addition, we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial searches. Studies that examined the mechanisms of diagnostic tests, drug therapy or vaccine efficacy against SARS, MERS and 2019-nCoV were excluded. A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles. With the emergence of 2019-nCoV, real time RT-PCR remains the primary means for diagnosing the new virus strain among the many diagnostic platforms available ( [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ; Table S3 ). Among the 16 diagnostics studies selected, one study discussed the use of RT-PCR in diagnosing patients with 2019-nCoV [11] ( Table 1 ). The period and type of specimen collected for RT-PCR play an important role in the diagnosis of 2019-nCoV. It was found that the respiratory specimens were positive for the virus while serum was negative in the early period. It has also suggested that in the early days of illness, patients have high levels of virus despite the mild symptoms. Apart from the commonly used RT-PCR in diagnosing MERS-CoV, four studies identified various diagnostic methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), RT-insulated isothermal PCR (RT-iiPCR) and a one-step rRT-PCR assay based on specific TaqMan probes. RT-LAMP has similar sensitivity as real time RT-PCR. It is also highly specific and is used to detect MERS-CoV. It is comparable to the usual diagnostic tests and is rapid, simple and convenient. Likewise, RT-iiPCR and a one-step rRT-PCR assay have also shown similar sensitivity and high specificity for MER-CoV. Lastly, one study focused on the validation of the six commercial real RT-PCR kits, with high accuracy. Although real time RT-PCR is a primary method for diagnosing MERS-CoV, high levels of PCR inhibition may hinder PCR sensitivity (Table 1) . There are eleven studies that focus on SARS-CoV diagnostic testing (Table 1) . These papers described diagnostic methods to detect the virus with the majority of them using molecular testing for diagnosis. Comparison between the molecular test (i.e RT-PCR) and serological test (i.e., ELISA) showed that the molecular test has better sensitivity and specificity. Hence, enhancements to the current molecular test were conducted to improve the diagnosis. Studies looked at using nested PCR to include a pre-amplification step or incorporating N gene as an additional sensitive molecular marker to improve on the sensitivity (Table 1 ). In addition, there are seven potential rapid diagnostic kits (as of 24 January 2020; Table 2 ) available on the market for 2019-nCoV. Six of these are only for research purposes. Only one kit from Beijing Genome Institute (BGI) is approved for use in the clinical setting for rapid diagnosis. Most of the kits are for RT-PCR. There were two kits (BGI, China and Veredus, Singapore) with the capability to detect multiple pathogens using sequencing and microarray technologies, respectively. The limit of detection of the enhanced realtime PCR method was 10 2 -fold higher than the standard real-time PCR assay and 10 7fold higher than conventional PCR methods In the clinical aspect, the enhanced realtime PCR method was able to detect 6 cases of SARS-CoV positive samples that were not confirmed by any other assay [25] • The real time PCR has a threshold sensitivity of 10 genome equivalents per reaction and it has a good reproducibility with the inter-assay coefficients of variation of 1.73 to 2.72%. • 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. The real-time RT-PCR reaction was more sensitive than the nested PCR reaction, as the detection limit for the nested PCR reaction was about 10 3 genome equivalents in the standard cDNA control. [34] Real-time reverse-transcription PCR (rRT-PCR); RNA-dependent RNA polymerase (RdRp); open reading frame 1a (ORF1a); Loop-mediated isothermal amplification (LAMP); enzyme-linked immunosorbent assay (ELISA); immunofluorescent assay (IFA); immunochromatographic test (ICT); nasopharyngeal aspirate (NPA). With the emergence of 2019-nCoV, there are about 15 potential vaccine candidates in the pipeline globally (Table 3 ), in which a wide range of technology (such as messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle) was applied. It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening. The published clinical trials were mostly done in United States except for one on the SARS vaccine done in China [44] . All vaccine candidates for SARS and MERS were reported to be safe, well-tolerated and able to trigger the relevant and appropriate immune responses in the participants. In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60. [47] Molecules developed by the university scientists inhibit two coronavirus enzymes and prevent its replication. The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] . The trial conducted in the United States in 2017 demonstrated SAB-301 to be safe and well-tolerated at single doses. Another trial on MERS therapeutics was found on ClinicalTrials.gov-a phase 2/3 trial in the United States evaluating the safety, tolerability, pharmacokinetics (PK), and immunogenicity on coadministered MERS-CoV antibodies REGN3048 & REGN3051 [100]. Rapid diagnostics plays an important role in disease and outbreak management. The fast and accurate diagnosis of a specific viral infection enables prompt and accurate public health surveillance, prevention and control measures. Local transmission and clusters can be prevented or delayed by isolation of laboratory-confirmed cases and their close contacts quarantined and monitored at home. Rapid diagnostic also facilitates other specific public health interventions such as closure of high-risk facilities and areas associated with the confirmed cases for prompt infection control and environmental decontamination [11, 101] . Laboratory diagnosis can be performed by: (a) detecting the genetic material of the virus, (b) detecting the antibodies that neutralize the viral particles of interest, (c) detecting the viral epitopes of interest with antibodies (serological testing), or (d) culture and isolation of viable virus particles. The key limitations of genetic material detection are the lack of knowledge of the presence of viable virus, the potential cross-reactivity with non-specific genetic regions and the short timeframe for accurate detection during the acute infection phase. The key limitations of serological testing is the need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses. The limitation of virus culture and isolation is the long duration and the highly specialized skills required of the technicians to process the samples. All patients recovered. Significantly shorted time from the disease onset to the symptom improvement in treatment (5.10 ± 2.83 days) compared to control group (7.62 ± 2.27 days) (p < 0.05) No significant difference in blood routine improvement, pulmonary chest shadow in chest film improvement and corticosteroid usgae between the 2 groups. However, particularly in the respect of improving clinical symptoms, elevating quality of life, promoting immune function recovery, promoting absorption of pulmonary inflammation, reducing the dosage of cortisteroid and shortening the therapeutic course, treatment with integrative chinese and western medicine treatment had obvious superiority compared with using control treatment alone. Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well-tolerated in healthy participants. [46] Where the biological samples are taken from also play a role in the sensitivity of these tests. For SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates have higher and more prolonged levels of viral RNA because of the tropism of the virus. MERS-CoV viral loads are also higher for severe cases and have longer viral shedding compared to mild cases. Although upper respiratory tract specimens such as nasopharyngeal or oropharyngeal swabs can be used, they have potentially lower viral loads and may have higher risk of false-negatives among the mild MERS and SARS cases [102, 103] , and likely among the 2019-nCoV cases. The existing practices in detecting genetic material of coronaviruses such as SARS-CoV and MERS-CoV include (a) reverse transcription-polymerase chain reaction (RT-PCR), (b) real-time RT-PCR (rRT-PCR), (c) reverse transcription loop-mediated isothermal amplification (RT-LAMP) and (d) real-time RT-LAMP [104] . Nucleic amplification tests (NAAT) are usually preferred as in the case of MERS-CoV diagnosis as it has the highest sensitivity at the earliest time point in the acute phase of infection [102] . Chinese health authorities have recently posted the full genome of 2019-nCoV in the GenBank and in GISAID portal to facilitate in the detection of the virus [11] . Several laboratory assays have been developed to detect the novel coronavirus in Wuhan, as highlighted in WHO's interim guidance on nCoV laboratory testing of suspected cases. These include protocols from other countries such as Thailand, Japan and China [105] . The first validated diagnostic test was designed in Germany. Corman et al. had initially designed a candidate diagnostic RT-PCR assay based on the SARS or SARS-related coronavirus as it was suggested that circulating virus was SARS-like. Upon the release of the sequence, assays were selected based on the match against 2019-nCoV upon inspection of the sequence alignment. Two assays were used for the RNA dependent RNA polymerase (RdRP) gene and E gene where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing. All assays were highly sensitive and specific in that they did not cross-react with other coronavirus and also human clinical samples that contained respiratory viruses [11] . The Hong Kong University used two monoplex assays which were reactive with coronaviruses under the subgenus Sarbecovirus (consisting of 2019-nCoV, SARS-CoV and SARS-like coronavirus). Viral RNA extracted from SARS-CoV can be used as the positive control for the suggested protocol assuming that SARS has been eradicated. It is proposed that the N gene RT-PCR can be used as a screening assay while the Orf1b assay acts as a confirmatory test. However, this protocol has only been evaluated with a panel of controls with the only positive control SARS-CoV RNA. Synthetic oligonucleotide positive control or 2019-nCoV have yet to be tested [106] . The US CDC shared the protocol on the real time RT-PCR assay for the detection of the 2019-nCoV with the primers and probes designed for the universal detection of SARS-like coronavirus and the specific detection of 2019-nCoV. However, the protocol has not been validated on other platforms or chemistries apart from the protocol described. There are some limitations for the assay. Analysts engaged have to be trained and familiar with the testing procedure and result interpretation. False negative results may occur due to insufficient organisms in the specimen resulting from improper collection, transportation or handling. Also, RNA viruses may show substantial genetic variability. This could result in mismatch between the primer and probes with the target sequence which can diminish the assay performance or result in false negative results [107] . Point-of-care test kit can potentially minimize these limitations, which should be highly prioritized for research and development in the next few months. Serological testing such as ELISA, IIFT and neutralization tests are effective in determining the extent of infection, including estimating asymptomatic and attack rate. Compared to the detection of viral genome through molecular methods, serological testing detects antibodies and antigens. There would be a lag period as antibodies specifically targeting the virus would normally appear between 14 and 28 days after the illness onset [108] . Furthermore, studies suggest that low antibody titers in the second week or delayed antibody production could be associated with mortality with a high viral load. Hence, serological diagnoses are likely used when nucleic amplification tests (NAAT) are not available or accessible [102] . Vaccines can prevent and protect against infection and disease occurrence when exposed to the specific pathogen of interest, especially in vulnerable populations who are more prone to severe outcomes. In the context of the current 2019-nCoV outbreak, vaccines will help control and reduce disease transmission by creating herd immunity in addition to protecting healthy individuals from infection. This decreases the effective R0 value of the disease. Nonetheless, there are social, clinical and economic hurdles for vaccine and vaccination programmes, including (a) the willingness of the public to undergo vaccination with a novel vaccine, (b) the side effects and severe adverse reactions of vaccination, (c) the potential difference and/or low efficacy of the vaccine in populations different from the clinical trials' populations and (d) the accessibility of the vaccines to a given population (including the cost and availability of the vaccine). Vaccines against the 2019-nCoV are currently in development and none are in testing (at the time of writing). On 23 January 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) announced that they will fund vaccine development programmes with Inovio, The University of Queensland and Moderna, Inc respectively, with the aim to test the experimental vaccines clinically in 16 weeks (By June 2020). The vaccine candidates will be developed by the DNA, recombinant and mRNA vaccine platforms from these organizations [109] . Based on the most recent MERS-CoV outbreak, there are already a number of vaccine candidates being developed but most are still in the preclinical testing stage. The vaccines in development include viral vector-based vaccine, DNA vaccine, subunit vaccine, virus-like particles (VLPs)-based vaccine, inactivated whole-virus (IWV) vaccine and live attenuated vaccine. The latest findings for these vaccines arebased on the review by Yong et al. (2019) in August 2019 [110] . As of the date of reporting, there is only one published clinical study on the MERS-CoV vaccine by GeneOne Life Science & Inovio Pharmaceuticals [47] . There was one SARS vaccine trial conducted by the US National Institute of Allergy and Infectious Diseases. Both Phase I clinical trials reported positive results, but only one has announced plans to proceed to Phase 2 trial [111] . Due to the close genetic relatedness of SARS-CoV (79%) with 2019-nCoV [112] , there may be potential cross-protective effect of using a safe SARS-CoV vaccine while awaiting the 2019-nCoV vaccine. However, this would require small scale phase-by-phase implementation and close monitoring of vaccinees before any large scale implementation. Apart from the timely diagnosis of cases, the achievement of favorable clinical outcomes depends on the timely treatment administered. ACE2 has been reported to be the same cell entry receptor used by 2019-nCoV to infect humans as SARS-CoV [113] . Hence, clinical similarity between the two viruses is expected, particularly in severe cases. In addition, most of those who have died from MERS-CoV, SARS-CoV and 2019-nCoV were advance in age and had underlying health conditions such as hypertension, diabetes or cardiovascular disease that compromised their immune systems [114] . Coronaviruses have error-prone RNA-dependent RNA polymerases (RdRP), which result in frequent mutations and recombination events. This results in quasispecies diversity that is closely associated with adaptive evolution and the capacity to enhance viral-cell entry to cause disease over time in a specific population at-risk [115] . Since ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, coronaviruses are likely to infect the upper respiratory and gastrointestinal tract and this may influence the type of therapeutics against 2019-nCoV, similarly to SAR-CoV. However, in the years following two major coronavirus outbreaks SARS-CoV in 2003 and MERS-CoV in 2012, there remains no consensus on the optimal therapy for either disease [116, 117] . Well-designed clinical trials that provide the gold standard for assessing the therapeutic measures are scarce. No coronavirus protease inhibitors have successfully completed a preclinical development program despite large efforts exploring SARS-CoV inhibitors. The bulk of potential therapeutic strategies remain in the experimental phase, with only a handful crossing the in vitro hurdle. Stronger efforts are required in the research for treatment options for major coronaviruses given their pandemic potential. Effective treatment options are essential to maximize the restoration of affected populations to good health following infections. Clinical trials have commenced in China to identify effective treatments for 2019-nCoV based on the treatment evidence from SARS and MERS. There is currently no effective specific antiviral with high-level evidence; any specific antiviral therapy should be provided in the context of a clinical study/trial. Few treatments have shown real curative action against SARS and MERS and the literature generally describes isolated cases or small case series. Many interferons from the three classes have been tested for their antiviral activities against SARS-CoV both in vitro and in animal models. Interferon β has consistently been shown to be the most active, followed by interferon α. The use of corticosteroids with interferon alfacon-1 (synthetic interferon α) appeared to have improved oxygenation and faster resolution of chest radiograph abnormalities in observational studies with untreated controls. Interferon has been used in multiple observational studies to treat SARS-CoV and MERS-CoV patients [116, 117] . Interferons, with or without ribavirin, and lopinavir/ritonavir are most likely to be beneficial and are being trialed in China for 2019-nCoV. This drug treatment appears to be the most advanced. Timing of treatment is likely an important factor in effectiveness. A combination of ribavirin and lopinavir/ritonavir was used as a post-exposure prophylaxis in health care workers and may have reduced the risk of infection. Ribavirin alone is unlikely to have substantial antiviral activities at clinically used dosages. Hence, ribavirin with or without corticosteroids and with lopinavir and ritonavir are among the combinations employed. This was the most common agent reported in the available literature. Its efficacy has been assessed in observational studies, retrospective case series, retrospective cohort study, a prospective observational study, a prospective cohort study and randomized controlled trial ranging from seven to 229 participants [117] . Lopinavir/ritonavir (Kaletra) was the earliest protease inhibitor combination introduced for the treatment of SARS-CoV. Its efficacy was documented in several studies, causing notably lower incidence of adverse outcomes than with ribavirin alone. Combined usage with ribavirin was also associated with lower incidence of acute respiratory distress syndrome, nosocomial infection and death, amongst other favorable outcomes. Recent in vitro studies have shown another HIV protease inhibitor, nelfinavir, to have antiviral capacity against SARS-CoV, although it has yet to show favorable outcomes in animal studies [118] . Remdesivir (Gilead Sciences, GS-5734) nucleoside analogue in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several coronaviruses (CoVs), including highly pathogenic CoVs and potentially emergent BatCoVs. The use of remdesivir may be a good candidate as an investigational treatment. Improved mortality following receipt of convalescent plasma in various doses was consistently reported in several observational studies involving cases with severe acute respiratory infections (SARIs) of viral etiology. A significant reduction in the pooled odds of mortality following treatment of 0.25 compared to placebo or no therapy was observed [119] . Studies were however at moderate to high risk of bias given their small sample sizes, allocation of treatment based on the physician's discretion, and the availability of plasma. Factors like concomitant treatment may have also confounded the results. Associations between convalescent plasma and hospital length of stay, viral antibody levels, and viral load respectively were similarly inconsistent across available literature. Convalescent plasma, while promising, is likely not yet feasible, given the limited pool of potential donors and issues of scalability. Monoclonal antibody treatment is progressing. SARS-CoV enters host cells through the binding of their spike (S) protein to angiotensin converting enzyme 2 (ACE2) and CD209L [118] . Human monoclonal antibodies to the S protein have been shown to significantly reduce the severity of lung pathology in non-human primates following MERS-CoV infection [120] . Such neutralizing antibodies can be elicited by active or passive immunization using vaccines or convalescent plasma respectively. While such neutralizing antibodies can theoretically be harvested from individuals immunized with vaccines, there is uncertainty over the achievement of therapeutic levels of antibodies. Other therapeutic agents have also been reported. A known antimalarial agent, chloroquine, elicits antiviral effects against multiple viruses including HIV type 1, hepatitis B and HCoV-229E. Chloroquine is also immunomodulatory, capable of suppressing the production and release of factors which mediate the inflammatory complications of viral diseases (tumor necrosis factor and interleukin 6) [121] . It is postulated that chloroquine works by altering ACE2 glycosylation and endosomal pH. Its anti-inflammatory properties may be beneficial for the treatment of SARS. Niclosamide as a known drug used in antihelminthic treatment. The efficacy of niclosamide as an inhibitor of virus replication was proven in several assays. In both immunoblot analysis and immunofluorescence assays, niclosamide treatment was observed to completely inhibit viral antigen synthesis. Reduction of virus yield in infected cells was dose dependent. Niclosamide likely does not interfere in the early stages of virus attachment and entry into cells, nor does it function as a protease inhibitor. Mechanisms of niclosamide activity warrant further investigation [122] . Glycyrrhizin also reportedly inhibits virus adsorption and penetration in the early steps of virus replication. Glycyrrhizin was a significantly potent inhibitor with a low selectivity index when tested against several pathogenic flaviviruses. While preliminary results suggest production of nitrous oxide (which inhibits virus replication) through induction of nitrous oxide synthase, the mechanism of Glycyrrhizin against SARS-CoV remains unclear. The compound also has relatively lower toxicity compared to protease inhibitors like ribavirin [123] . Inhibitory activity was also detected in baicalin [124] , extracted from another herb used in the treatment of SARS in China and Hong Kong. Findings on these compounds are limited to in vitro studies [121] [122] [123] [124] . Due to the rapidly evolving situation of the 2019-nCoV, there will be potential limitations to the systematic review. The systematic review is likely to have publication bias as some developments have yet to be reported while for other developments there is no intention to report publicly (or in scientific platforms) due to confidentiality concerns. However, this may be limited to only a few developments for review as publicity does help in branding to some extent for the company and/or the funder. Furthermore, due to the rapid need to share the status of these developments, there may be reporting bias in some details provided by authors of the scientific articles or commentary articles in traditional media. Lastly, while it is not viable for any form of quality assessment and metaanalysis of the selected articles due to the limited data provided and the heterogeneous style of reporting by different articles, this paper has provided a comprehensive overview of the potential developments of these pharmaceutical interventions during the early phase of the outbreak. This systematic review would be useful for cross-check when the quality assessment and meta-analysis of these developments are performed as a follow-up study. Rapid diagnostics, vaccines and therapeutics are key pharmaceutical interventions to limit transmission of respiratory infectious diseases. Many potential developments on these pharmaceutical interventions for 2019-nCoV are ongoing in the containment phase of this outbreak, potentially due to better pandemic preparedness than before. However, lessons from MERS-CoV and SARS-CoV have shown that the journeys for these developments can still be challenging moving ahead. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1 : Example of full search strategy in Pubmed, Table S2 : Google Search: 2019-nCoV diagnostics, Table S3 : Summary of diagnostic assays developed for 2019-nCoV, Table S4

What was a characteristic of SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates?

A super-spreading ewe infects hundreds with Q fever at a farmers' market in Germany https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618839/ SHA: ee1b5a9618dcc4080ed100486cedd0969e80fa4d Authors: Porten, Klaudia; Rissland, Jürgen; Tigges, Almira; Broll, Susanne; Hopp, Wilfried; Lunemann, Mechthild; van Treeck, Ulrich; Kimmig, Peter; Brockmann, Stefan O; Wagner-Wiening, Christiane; Hellenbrand, Wiebke; Buchholz, Udo Date: 2006-10-06 DOI: 10.1186/1471-2334-6-147 License: cc-by Abstract: BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study (cases were Q fever patients, controls were randomly selected Soest citizens) and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii (C. burnetii). RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3(rd )trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii (C. burnetii), a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats [1] . High concentrations of C. burnetii can be found in birth products of infected mammals [2] . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool [1] . Because the infectious dose is very low [3] and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances [4] [5] [6] . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare [1] . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases [2] . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis [1] . Children develop clinical disease less frequently [7, 8] . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay (IFA) is considered to be the reference method, complement fixation (CF), ELISA and microagglutination (MA) can also be used [9] . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections [1] . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year [10] . In 2001 and 2002, 293 and 191 cases were notified, respectively [11, 12] . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory (NCL) for Coxiellae and the Robert Koch-Institute (RKI), the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens (ELISA or IFA), a 4-fold increase in anti-phase II IgG antibody titer (ELISA or IFA) or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection [13, 14] we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case (CCS1 case) as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 (AFE) as (OR-1)/OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context ("notified case") was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition (as used in the vendors' study (s.b.)) identified through random sampling of the Soest population (within CCS2 (s.b.)) as well as in two cohorts (vendors' study and the 9 sailor friends (see below)). The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market (see below "Estimation of the overall attack rate") and gave an estimate of the proportion of clinically ill cases that were hospitalized (s.a.). In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit (approximately 3 meters). Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose (360°) in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east ( Figure 1 ). For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen (plus minus 45°). Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen (in stand units or meters) and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition (as defined in the cohort study on vendors). The overall attack rate among adults was estimated based on the following sources: (1) Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * (2) Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; (3) Results of the small cohort of 9 sailor friends (s.a.); (4) Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever (s.a. "determination of outbreak size and descriptive epidemiology") interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in (1), (3) and (4) we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children (numerator). For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions (number of visitors, proportion of adult visitors and clinical attack rate among adults) we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks (70 animals). Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 (CDC, Atlanta, USA). Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 (65%) of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. (Figure 2 ). If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases (mean +/-2 standard deviations) had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 (53%) were male and 140 (47%) were female. Of the notified cases, 189 (63%) were from the county of Soest, 104 (35%) were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state (Northrhine Westphalia) and 6 (2%) were from five other federal states in Germany (Figure 3 ). Only eight (3%) cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively ( Figure 4 ). 75 (25%) of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 (21%; 95% CI = 6-46%; (1/5 (CCS2), 2/11 (vendors study) and 1/3 (sailor friends)) clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 (56%) outbreak cases; 66 (22%) were confirmed by an increase in anti-phase II antibody titer (CF), 89 (30%) had IgM antibodies against phase II antigens, 11 (4%) were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 (44%) cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two (11%) of 18 cardiological patients and four (36%) of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman (case 3; Table 1 ). Serological follow-up of the mothers detected chronic infection in the same woman (case 3) 12 weeks after delivery. One year follow-up of two newborn children (of cases 1 and 3) identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender (OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23). Seventeen (85%) of 20 cases indicated that they had seen the cow (that also was on display at the market next to the sheep) compared to 7 (32%) of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 (32%) of 25 controls (OR = 5.0; 95%CI = 1.2-22.3; p = 0.03). Touching the sheep was also significantly more common among cases (5/20 (25%) CCS2 cases vs. 0/22 (0%) controls; OR undefined; lower 95% CI = 1.1; p = 0.02). 17 (85%) of 20 CCS2 cases, but only 6 (25%) of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" (OR = 17.0; 95%CI = 3.0-112.5; p < 0.01). Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 (86%) of 87 vendors, and received second hand information about 7 more (overall response rate: 94%). Fourty-five (56%) were male and 35 (44%) were female. 13 (16%) met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 (55%) became cases compared to only 7 (10%) of 70 persons who worked in a stand at a greater distance (relative risk (RR) = 5.5 (95%CI = 2.3-13.2; p = 0.002); Figure 1 ). Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen (section 4, Figure 1 ), 4 (9%) of 44 vendors became cases, compared to 2 (13%) of 15 persons who worked in section 1 (p = 0.6). Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 (32%) became cases. (Table 3 ). In all scenarios the AR among adults was significantly higher than that among children ( Figure 5 ). In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births (57 (86%)) had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 (25%) tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% (8%; 12%; 12%; 16%; 24%). We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market [15] . More recently a large outbreak occurred in Jena (Thuringia) in 2005 with 322 reported cases [16] associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases (source 5) were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] . An additional interview with the two cases with early onset (2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" (see Table 3 ). Displayed are attack rates and 95% confidence intervals. respectively) could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high [17] . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to (within 6 meters) the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% (8/ 191) [7] , 5% [1] and 10% (4/39) [5] ), and there was at least one study with a much higher proportion (63% (10/ 16)) [18] . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep (n = 1000-2000) [5, 7] the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep [19] . In the most severely affected village, the clinical attack rate was 16% (estimated from the data provided) [19] . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic (50% (3/6)) is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease [7] . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness [7, 8] few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock [20] . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks [7, 20, 21] , was 40% compared to 1% in sheep flocks not linked to human outbreaks [20] . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that (in such a setting) can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred [20] , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of (farm) animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents [20, [22] [23] [24] . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations [25] which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author(s) declare that they have no competing interests.

What health regulations were changes due to the outbreak of C. burnetti?

Responding to the COVID-19 pandemic in complex humanitarian crises https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085188/ SHA: d013e42811c6442b184da3b9bbfd9e334031a975 Authors: Poole, Danielle N.; Escudero, Daniel J.; Gostin, Lawrence O.; Leblang, David; Talbot, Elizabeth A. Date: 2020-03-21 DOI: 10.1186/s12939-020-01162-y License: cc-by Abstract: nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 [1] . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale [2] . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene (WASH) tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations [3] . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing [4] . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% [5] . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production [6] . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance [7] . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines [5, 8] . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence [9] . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations [10] . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings [2] . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic.

What can undermine interventions?

A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.

What was the R0 of SARS?

Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026206/ SHA: eff8bed68ef6109e8f0c51a8b1ec4b6ca5b6329e Authors: Theuns, Sebastiaan; Vanmechelen, Bert; Bernaert, Quinten; Deboutte, Ward; Vandenhole, Marilou; Beller, Leen; Matthijnssens, Jelle; Maes, Piet; Nauwynck, Hans J. Date: 2018-06-29 DOI: 10.1038/s41598-018-28180-9 License: cc-by Abstract: Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5X) after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (10(6.42)–10(7.01) copies/swab) and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread (n = 25, 56.8% positive) of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. Text: metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. Performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. Third-generation sequencing using MinION (Oxford Nanopore Technologies, ONT) might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. This signal is converted into a nucleotide sequence by computational algorithms (basecalling). Since the release of MinION technology, major advances have been made in terms of the number and the quality of reads generated 15 . In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 . MinION has also been used as a diagnostic tool during recent Ebolavirus outbreaks in West Africa, allowing fast on-site characterization of circulating strains 17, 18 . Coupled to a laptop-based bioinformatics workflow, MinION was able to detect Chikungunya virus, Ebola virus and hepatitis C virus in less than 6 hours using earlier versions of the technology 19 . A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 . In veterinary virology, the use of nanopore sequencing is growing. A novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing 22 . The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 . To the author's knowledge, the present study is the first using MinION as an aid in porcine health management. This study was aimed to explore the possibilities of MinION as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect (novel) viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. No gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the MinION's sensitivity. A porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. Moreover, archival (2014) fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium. be performed for 243,313 reads with a mean length of 740 nucleotides. Reads with a q-score lower than 7 were filtered out, resulting in 179,015 remaining sequences (mean length 816 nt) for use in downstream analyses. Results of the sequencing run, including taxonomical classification and mapping of reads against PEDV and rotavirus A (RVA) reference genomes are shown in Fig. 1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database. Of these, 39.3% (n = 5,985) and 10.3% (n = 1,564) were assigned to viral families comprising Porcine epidemic diarrhea virus (family Coronaviridae) and Rotavirus (family Reoviridae, subfamily Sedoreovirinae), respectively. A fraction of the reads (29.3%, n = 4,468) were assigned to order Caudovirales. These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore. Most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing (Fig. 1B) . PEDV and RVA sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time (Fig. 1C) . After one hour, sequencing depths were higher for PEDV (43.0X) than for RVA (4.9 to 22.1X). High sequencing depths were acquired after three hours of sequencing for PEDV (103.5X) and for most RVA gene segments (19.2 to 48.2X). De novo assembly was executed on the quality-filtered reads prior to identification (tBLASTx) to recover viral genomes. This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes (Table 1) . Higher assembly accuracies (97 to 99%) were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly (Table 1) . However, execution of de novo assembly prior to taxonomical classification (tBLASTx) reduced the time to identify entire viral genomes in the dataset. Virome composition of a young diarrheic piglet using nanopore sequencing. A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads (q-score >7, mean read length 653 nt) were used for further analyses. Different methods were used to compare the reads against a viral database using the HPC cluster of Ghent University and results are shown in Fig. 2 . Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx. However, there was a very high difference between wall times on the HPC cluster, with only 26 seconds of analysis time for BLASTn, versus almost 24 hours for tBLASTx. The majority of sequences were assigned to bacteriophages within the order Caudovirales and families Siphoviridae (n = 3,213 to 4,163 reads), Podoviridae (n = 2,506 to 3,002 reads) and Myoviridae (n = 912 to 1,202 reads). A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure (n = 4; category 1), somewhat sure (=14; category 2) and not so sure (=1; category 3) to be phage-like contigs (Fig. 2B) . Comparison of these contigs against the GenBank database using BLAST allowed classification into four different groups. Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 (data not shown). The longest contig with a size of 39,069 nucleotides, together with four other contigs showed similarities (95% nt identity) to different Escherichia phages. As they also mapped nicely distributed across the reference genome of Escherichia phage vB_EcoP_PhAPEC7, it seems that they must also belong to one phage genome (data not shown). Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome. Interestingly, three contigs were identified for which no similarities were found with existing viruses in GenBank, but contig 0105 mapped to the reference genome of the Enterococcus phage vB_EfaS_IME196 (data not shown). These might be novel phages or divergent variants from existing phages present in GenBank. Three eukaryotic porcine viruses, porcine kobuvirus (n = 18 to 22 reads), enterovirus G (n = 5 to 9 reads) and astrovirus (n = 4 reads) were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae. Kobuvirus reads were mapped against a European reference strain S-1/HUN/2007/Hungary, as shown in Fig. 2C . However, full-genome coverage at high sequencing depth was not obtained. Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C (RVC) was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in Fig. 3A . All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets (A and D) the shedding was sustained and lasted for at least 2 weeks (above the limit of quantification). Peak shedding titers of the porcine kobuvirus varied between 6.42 and 7.01 log 10 copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and PEDV. Moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. Diarrheic signs were only noticed in two piglets (A and B). In piglet B, an association between high RVC shedding and diarrheic episodes was observed. In contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. Interestingly, a peak in kobuvirus shedding was observed in piglet C at day 11 post-farrowing. This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. Retrospective analysis of porcine kobuviruses shedding in Belgian diarrheic suckling pigs and phylogenetic analysis. A total of 44 diarrheic fecal samples collected in 2014 were screened for the presence of kobuvirus using the new RT-qPCR. Of these, 25 samples (56.8%) tested positive and 18 samples showed quantifiable viral loads (4.31 to 6.83 log 10 copies/swab). Seven samples were positive, but viral loads were too low to allow accurate quantification. The presence of RVA and RVC had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in Fig. 3B 25 . Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load (5.16 to 5.42 log 10 copies/g) was observed. A single RVC infection was found in seven samples and in four of these tested positive for kobuvirus at high loads (4.31 to 5.59 log 10 copies/g). A dual RVA/RVC infection was seen in two samples, but neither contained quantifiable kobuvirus loads. Many (n = 10) of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 (92.1 to 94.0% nucleotide sequence identity) and the Hungarian reference strain S-1/Hun/2017 (93.4%). Furthermore, there was a high level of genetic variability between the 2014 Belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between 90.1 and 97.2%. A phylogenetic analysis, using the 3D gene of 17V079 and twelve Belgian isolates from 2014 ( Fig. 3C) , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. Veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. Only the most likely cause(s) of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. However, most NGS platforms require large investments and processing of the reads can only start at the end of the sequencing run. Viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present 13 . Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. All these factors lead to a long turnover time between sample collection and diagnostic reporting. The third-generation sequencing device MinION (ONT), holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. Sequencing of this pooled sample with the MinION resulted in rapid identification of both viruses. Real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by Greninger and colleagues using the SURPI analysis pipeline for rapid identification of human viruses from different clinical matrices 19 . Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths (43.0X) were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments (19.2-48.2X). Overall, higher sequencing depths were generated for PEDV that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as PEDV has a genome size of approximately 28 kb, and RVA gene segments are shorter (0.6 to 3.3 kb). This bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. While the technology can be useful for giving fast readouts of viruses (<3 hours) present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices (e.g. feces) and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. Real-time PCR analyses were conducted for RVA, RVC, PEDV and TGEV. Enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs 26 . Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted. However, BLASTn search options also reached high sensitivity, but at much lower time cost: 26 seconds instead of almost 24 hours. For rapid read analysis and searching for closely related non-divergent viral sequences, BLASTn or another fast methodology should thus be preferentially used. However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079. Astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of Belgian pigs and in feces from pigs around the globe 9,10,27 . In a recent study from Thailand, the difference in prevalence of astrovirus in diarrheic (8.4%) versus non-diarrheic (4.6%) piglets less than 4-weeks-old was not statistically significant. Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 . A recent study in 5 European countries (Hungary, Spain, Germany, Austria and Sweden) have indicated the widespread of porcine astroviruses in the swine population. A one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from Austria and Spain. Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections. Enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well 11, 12, [32] [33] [34] . In a study from Vietnam, no significant correlation was found between diarrhea status and presence of enterovirus G in feces 35 . The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. However, reporting of a porcine kobuvirus in Belgian piglets with MinION is unique. In Belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in Belgium 36 . In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding (6.42 to 7.01 log 10 copies/swab) and diarrheic signs was not observed. In one pig, an association was made between diarrheic episodes and the peak of rotavirus C shedding, a well-known enteric pathogen 37, 38 . Very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as PEDV and rotavirus [39] [40] [41] . Similar viral loads for porcine kobuvirus were also found in case (4.60 ± 1.76 copies/qPCR reaction) and control pigs (4.79 ± 1.72 copies/qPCR reaction) during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 . The finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. Hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. Highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries [44] [45] [46] . In our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. Such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus 47 . Of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines (MA104, ST and SK), and peripheral blood mononuclear cells. There was no evidence of cytopathogenic effect after several days of incubation. Antibodies to visualize antigen expression were not available and therefore the possibility of replication without SCIenTIFIC REPORTS | (2018) 8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. To assess more broadly the prevalence of kobuvirus in the Belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. A high proportion (40.9%) of the samples (n = 44) contained quantifiable viral loads ranging between 4.31 to 6.83 log 10 copies/g feces. Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. However, the prevalence of kobuvirus has been widely described in pigs from several European countries (The Netherlands (16.7%), Slovakia (63.4%), Hungary (81.0%), Czech Republic (87.3%), Austria (46.2%), Italy (52.4%), Germany (54.5%) and Sweden (45.0%)), American countries (The United States (21.9%) and Brazil (53.0%)), African countries (Kenya (14.9%) and Uganda (15.5%)) and Asian countries (Thailand (99%), South Korea (52.1%) and Vietnam (29.3%)) [44] [45] [46] [48] [49] [50] [51] [52] [53] . In a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in Hungary (54.5% prevalence in healthy pigs vs 92.3% prevalence in diarrheic pigs), Spain (47.5% healthy vs 74.4% diarrheic), Brazil (41% vs 78.4%), Thailand (19.3% vs 84.5%) and Vietnam (27.6% to 40.9%) 35, 45, 46, 52 . Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%. Furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. Because most (99%) of the reads generated during sequencing of the fecal sample 17V079 matched bacteriophages upon analysis with BLAST, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 . Several contigs showed high similarities to the Bacteroides phage B124-14, found in municipal wastewater and human fecal samples. It was shown to be absent in 30 samples collected from different animal species, including pigs, and is therefore considered a human-specific phage 55, 56 . The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. Interestingly, several of the contigs found also showed similarities to Escherichia phages. Two of the contigs were similar to Escherichia phages PhAPEC5 and PhAPEC7, isolated from Belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic Escherichia coli. Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 . Finally, one contig showed limited similarity to an Enterococcus phage, isolated from hospital sewage in China, while a last contig showed moderate similaraties to the bacterial Enterococcus hirae genome. This region may be a prophage, inserted in the bacterial genome. The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. The new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case 17V079 and it may be that bacteriophages are involved in the pathogenesis of this syndrome. So far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. Complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. However, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. Viruses. Porcine rotavirus A (RVA) strain RVA/Pig-tc/BEL/12R046/2012/G9P [23] was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml. The nucleotide sequences of the 11 gene segments of this strain were resolved earlier using Sanger sequencing (GenBank accession numbers: KM82070 (VP1), KM820707 (VP2), KM827014 (VP3), KM820720 (VP4), KM820728 (VP6), KM820735 (VP7), KM820742 (NSP1), KM820672 (NSP2), KM820679 (NSP3), KM820686 (NSP4) and KM820693 (NSP5)) 59 . A porcine epidemic diarrhea virus strain (PEDV, CV777) isolated in Belgium in the 1970s was adapted for growth in Vero cells in the 1980s 60 . In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml (GenBank accession number: AF353511). Origin of a fecal sample from diarrheic suckling piglets. A diarrheic fecal sample was collected from a Belgian pig on a farm housing a total of 620 sows and using a 2-week batch-production system, with a weaning age of 23 days. Topigs Norsvin sows were crossed with Piétrain boars, producing 32. toxins (Suiseng, Hipra). Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine (Lactovac, Zoetis). Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages (6.2-7.1%) were observed. Since the spring of 2017, enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in 7-days-old suckling piglets. A diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory (Dialab, Belsele, Belgium) and labeled 17V079. No virological cause was found to explain the diarrheic problems on the farm. The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule (inactivated autovaccine). No other pathogens were found in this sample. As the clinical picture hinted at a viral cause for the disease, the sample was sent to the Laboratory of Virology at the Faculty of Veterinary Medicine (Ghent University) for further analysis. The sample tested negative for RVA, RVC, PEDV and TGEV using in-house RT-qPCR assays 25, 61, 62 . Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids. First, viral enrichment was done based on the NetoVIR protocol to obtain pure viral nucleic acids for sequencing library preparation 13 . MinION analyses of cell culture grown viruses RVA and PEDV were conducted at the Laboratory of Clinical Virology (Rega Institute, KU Leuven), whereas the diarrheic fecal sample was analyzed at the Laboratory of Virology (Faculty of Veterinary Medicine, Ghent University). RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses. This mixture was filtered using a 0.8 µm polyethersulphone filter for 1 min at 17,000 × g, followed by a nuclease treatment for 2 hours at 37 °C to digest free nucleic acids in the suspension: 250 µl of the sample was added to 14 µl of home-made buffer (1 M Tris, 100 mM CaCl 2 and 30 mM MgCl 2 , pH 8), 4 µl of Benzonase Nuclease (Millipore) and 2 µl Micrococcal Nuclease (NEB) as described earlier 13 . Fourteen microliters of EDTA were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the QIAamp Viral RNA Mini Kit (Qiagen). The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications. A 10% w/v suspension of the diarrhea was made in Minimum Essential Medium and centrifuged. The supernatant was filtered through a 0.45 µm syringe filter (Sarstedt) and treated with Benzonase Nuclease for 1 hour to speed up the diagnostic pipeline. Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl. cDNA and second strand synthesis for nanopore sequencing. Nucleic acids were heated at 95 °C for 2 min and chilled on ice to resolve secondary RNA structures and to denature double-stranded RNA. Superscript IV Reverse Transcriptase (ThermoScientific) was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers (Random Primer 6, New England Biolabs), 1 µl dNTP mix (NEB) and 2.5 µl nuclease-free water. Primer annealing was conducted at 65 °C for 5 min, after which 4 µl Superscript IV Reaction Buffer (ThermoScientific), 1 µl dithiothreitol (ThermoScientific) and 1 µl SuperScript IV Reverse Transcriptase (ThermoScientific) were added in a total reaction volume of 20 µl. The reaction conditions were as follows: 23 °C for 10 min, 50 °C for 10 min, 80 °C for 10 min and an infinite hold step at 10 °C. A second strand of DNA was generated from single stranded (c)DNA molecules using the NEBNext Second Strand Synthesis Kit (NEB). Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water (80 µl total reaction volume). Isothermal amplification was done at 16 °C for 1 h and double-stranded nucleic acids were purified using 144 µl of magnetic AMPure XP Beads (Beckman Coulter). Two washing steps with freshly prepared 70% ethanol were conducted before eluting in 52 µl nuclease-free water. Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. Fifty microliters of (un) amplified DNA were mixed with 7 µl Ultra II End-Prep Reaction Buffer (New England Biolabs) and 3 µl Ultra II End-prep enzyme mix (New England Biolabs), and incubated at 20 °C for 5 min and 65 °C for 5 min. Next, nucleic acids were purified using 60 µl AMPure XP Beads and eluted in 31 µl nuclease-free water. Sequencing adapters, provided with the Ligation Sequencing Kit 1D (R9.4) (SQK-LSK108, ONT), were ligated to the dA-tailed nucleic acids. End-prepped DNA (30 µl) was mixed with 20 µl adapter mix (AMX, ONT) and 50 µl Blunt/TA Ligation Master Mix (New England Biolabs) in a total reaction volume of 100 µl and incubated at room temperature for 10 min. The sequencing library, containing double-stranded DNA with adapters ligated to the 3′ ends, was then purified using 40 µl AMPure XP beads. Two washing steps were conducted using 140 µl Adapter Bead Binding Buffer (ABB, ONT) before eluting in 15 µl of Elution Buffer (ELB, ONT). (EXP-LLB001, ONT), 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software (ONT). Bio-informatics analyses. Raw reads were produced by MinKNOW. Live basecalling was enabled for the first experiment using MinKNOW version 1.5.5. In the second experiment, basecalling was done after the sequencing run using Albacore (version 1.2.5., ONT). Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted. Sequences were then analyzed using different BLAST methods including BLASTn and tBLASTx (BLAST version 2.6.0; e-value cut-off 1e −3 -1e −10 ) to compare sensitivity and run-times to detect viral sequences among the reads. A complete viral database was composed of all virus sequences in GenBank (taxonomy ID 10239, containing sequences up to 17th of September 2017). The best hit (lowest e-value) was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk (https://github.com/lh3/seqtk) and used in downstream analyses. GraphMap (version 0.5.2) and Samtools (version 1.6) were used for mapping of reads against reference sequences, while Canu 1.6 was used for de novo assembly of viral genomes [65] [66] [67] [68] . VirSorter was run using the 'Viromes' database to look for phages, with the Virome Decontamination Mode on to identify phage contigs 54 . Bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of Ghent University. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene. A porcine kobuvirus was discovered in the sample 17V079 using the MinION. The sequence of the 3D gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit (Qiagen) with the newly designed primers Kobu_6049Fw and Kobu_7524Rv (IDT DNA Technologies) ( Table 2 ). The RT-PCR reaction contained 5 µl 5 × Qiagen OneStep RT-PCR Buffer, 1 µl dNTPs, 3 µl of each primer (10 µm), 7 µl nuclease-free water, 1 µl OneStep RT-PCR enzyme mix and 5 µl template RNA or water (total reaction volume of 25 µl). RT-PCR conditions were as follows: 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles of amplification (94 °C for 30 s, 50 °C for 30 s and 72° for 90 s) and a final extension step at 72 °C for 1 min. Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light. The amplicon was sent to GATC (Constance, Germany) for Sanger sequencing using an ABI 3730xl DNA Analyzer system. Quality control of the raw chromatograms was done using 4Peaks (Nucleobytes BV, The Netherlands) and BLASTn (NCBI, United States). Specific RT-qPCR primers (Table 2) for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer (IDT DNA Technologies) to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye (Primerdesign, Southampton, United Kingdom), 0.4 µl of each primer (200 nM) and 6.2 µl nuclease-free water. Three microliters of template RNA or water were added to each tube containing 17 µl mastermix. A synthetic RNA positive control (175nt) was generated by RT-PCR using the primers Kobu3D_qPCR +T7_Fw and Kobu3D_qPCR_Rv, followed by in vitro transcription of this PCR product using a T7 RNA polymerase. The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range (LDR) from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation (95 °C for 10 s) and annealing (58 °C for 60 s). Detection of SYBR Green fluorescence was done at the end of each annealing phase. A melt curve analysis was executed to assess specificity of the amplicons generated. Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. Assays were valid if the efficiency over the LDR was between 90 and 110%, and R 2 of the standard curve replicates was >0.99. Quantification of the viral loads was possible if the Cq-values of two qPCR replicates fell within the LDR of the assay. Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. Longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. Upon characterization of the virome with the MinION, a longitudinal follow-up study was setup between August and September 2017. To warrant the health status of the pig stock, entrance to the farm was strictly regulated. Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer. Sample collection in the longitudinal field study was done in agreement with the European legislation on animal experiments. Sample collection was approved by and done in accordance to the requirements of the Local Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University. One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. A dry cotton rectal swab (Copan) was collected from each individual piglet at days 1, 5, 8, 11, 14, 17, 20 and 22 after birth. The swab was placed immediately in 2 ml of viral transport medium (phosphate buffered saline containing 1000 U/ml penicillin (Continental Pharma, Puurs, Belgium), 1 mg/ml streptomycin (Certa, Braine l′Alleud, Belgium), 1 mg/ml gentamicin (Life Technologies) and 0.01% v/v Fungizone (Bristol-Myers Squibb, Braine l′Alleud, Belgium)) in a sterile 15 ml falcon tube (Sarstedt) and stored at −20 °C. Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. Upon arrival in the Laboratory of Virology, the samples were thawed and placed on a shaker for 30 min at 4 °C to release viral particles in the transport medium. Samples were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions and purified nucleic acids were eluted in 100 µl of AVE and stored at −70 °C until RT-qPCR analysis. RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 . Belgian suckling pigs. Fecal samples (n = 44) of diarrheic suckling piglets less than 2 weeks old were sent to a private laboratory by veterinarians (Dialab, Belsele, Belgium) for etiological diagnosis, as described earlier. These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 . RNA extraction was conducted using the QIAamp Cador Pathogen Mini Kit (Qiagen) as described above and RT-qPCR was done to quantify the load of kobuvirus RNA copies. Samples with a quantifiable viral load were subjected to RT-PCR to amplify the 3D polymerase gene, after which Sanger sequencing was performed. The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 . A maximum-likelihood phylogenetic tree was constructed with RAxML using a general time reversible model with gamma distribution (20 cats, alpha: 0.121, LogLK = 14938.461) and heuristic branch swapping 70 . Tree editing was done using Affinity Designer (Serif). Pairwise distances were calculated using the p-distance model in Mega with bootstrap values set at 500 replicates.

What was investigated in this study?

Backcalculating the Incidence of Infection with COVID-19 on the Diamond Princess https://doi.org/10.3390/jcm9030657 SHA: 0938d2fb07611897abf38cea727ddbeea77b73d9 Authors: Nishiura, Hiroshi Date: 2020 DOI: 10.3390/jcm9030657 License: cc-by Abstract: To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy where E(.) represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] : where is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest.

When was the first passenger patient on the Diamond Princess cruise ship diagnosed with COVID-19?

A super-spreading ewe infects hundreds with Q fever at a farmers' market in Germany https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618839/ SHA: ee1b5a9618dcc4080ed100486cedd0969e80fa4d Authors: Porten, Klaudia; Rissland, Jürgen; Tigges, Almira; Broll, Susanne; Hopp, Wilfried; Lunemann, Mechthild; van Treeck, Ulrich; Kimmig, Peter; Brockmann, Stefan O; Wagner-Wiening, Christiane; Hellenbrand, Wiebke; Buchholz, Udo Date: 2006-10-06 DOI: 10.1186/1471-2334-6-147 License: cc-by Abstract: BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study (cases were Q fever patients, controls were randomly selected Soest citizens) and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii (C. burnetii). RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3(rd )trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii (C. burnetii), a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats [1] . High concentrations of C. burnetii can be found in birth products of infected mammals [2] . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool [1] . Because the infectious dose is very low [3] and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances [4] [5] [6] . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare [1] . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases [2] . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis [1] . Children develop clinical disease less frequently [7, 8] . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay (IFA) is considered to be the reference method, complement fixation (CF), ELISA and microagglutination (MA) can also be used [9] . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections [1] . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year [10] . In 2001 and 2002, 293 and 191 cases were notified, respectively [11, 12] . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory (NCL) for Coxiellae and the Robert Koch-Institute (RKI), the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens (ELISA or IFA), a 4-fold increase in anti-phase II IgG antibody titer (ELISA or IFA) or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection [13, 14] we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case (CCS1 case) as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 (AFE) as (OR-1)/OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context ("notified case") was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition (as used in the vendors' study (s.b.)) identified through random sampling of the Soest population (within CCS2 (s.b.)) as well as in two cohorts (vendors' study and the 9 sailor friends (see below)). The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market (see below "Estimation of the overall attack rate") and gave an estimate of the proportion of clinically ill cases that were hospitalized (s.a.). In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit (approximately 3 meters). Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose (360°) in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east ( Figure 1 ). For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen (plus minus 45°). Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen (in stand units or meters) and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition (as defined in the cohort study on vendors). The overall attack rate among adults was estimated based on the following sources: (1) Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * (2) Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; (3) Results of the small cohort of 9 sailor friends (s.a.); (4) Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever (s.a. "determination of outbreak size and descriptive epidemiology") interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in (1), (3) and (4) we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children (numerator). For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions (number of visitors, proportion of adult visitors and clinical attack rate among adults) we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks (70 animals). Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 (CDC, Atlanta, USA). Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 (65%) of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. (Figure 2 ). If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases (mean +/-2 standard deviations) had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 (53%) were male and 140 (47%) were female. Of the notified cases, 189 (63%) were from the county of Soest, 104 (35%) were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state (Northrhine Westphalia) and 6 (2%) were from five other federal states in Germany (Figure 3 ). Only eight (3%) cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively ( Figure 4 ). 75 (25%) of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 (21%; 95% CI = 6-46%; (1/5 (CCS2), 2/11 (vendors study) and 1/3 (sailor friends)) clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 (56%) outbreak cases; 66 (22%) were confirmed by an increase in anti-phase II antibody titer (CF), 89 (30%) had IgM antibodies against phase II antigens, 11 (4%) were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 (44%) cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two (11%) of 18 cardiological patients and four (36%) of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman (case 3; Table 1 ). Serological follow-up of the mothers detected chronic infection in the same woman (case 3) 12 weeks after delivery. One year follow-up of two newborn children (of cases 1 and 3) identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender (OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23). Seventeen (85%) of 20 cases indicated that they had seen the cow (that also was on display at the market next to the sheep) compared to 7 (32%) of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 (32%) of 25 controls (OR = 5.0; 95%CI = 1.2-22.3; p = 0.03). Touching the sheep was also significantly more common among cases (5/20 (25%) CCS2 cases vs. 0/22 (0%) controls; OR undefined; lower 95% CI = 1.1; p = 0.02). 17 (85%) of 20 CCS2 cases, but only 6 (25%) of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" (OR = 17.0; 95%CI = 3.0-112.5; p < 0.01). Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 (86%) of 87 vendors, and received second hand information about 7 more (overall response rate: 94%). Fourty-five (56%) were male and 35 (44%) were female. 13 (16%) met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 (55%) became cases compared to only 7 (10%) of 70 persons who worked in a stand at a greater distance (relative risk (RR) = 5.5 (95%CI = 2.3-13.2; p = 0.002); Figure 1 ). Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen (section 4, Figure 1 ), 4 (9%) of 44 vendors became cases, compared to 2 (13%) of 15 persons who worked in section 1 (p = 0.6). Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 (32%) became cases. (Table 3 ). In all scenarios the AR among adults was significantly higher than that among children ( Figure 5 ). In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births (57 (86%)) had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 (25%) tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% (8%; 12%; 12%; 16%; 24%). We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market [15] . More recently a large outbreak occurred in Jena (Thuringia) in 2005 with 322 reported cases [16] associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases (source 5) were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] . An additional interview with the two cases with early onset (2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" (see Table 3 ). Displayed are attack rates and 95% confidence intervals. respectively) could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high [17] . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to (within 6 meters) the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% (8/ 191) [7] , 5% [1] and 10% (4/39) [5] ), and there was at least one study with a much higher proportion (63% (10/ 16)) [18] . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep (n = 1000-2000) [5, 7] the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep [19] . In the most severely affected village, the clinical attack rate was 16% (estimated from the data provided) [19] . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic (50% (3/6)) is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease [7] . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness [7, 8] few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock [20] . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks [7, 20, 21] , was 40% compared to 1% in sheep flocks not linked to human outbreaks [20] . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that (in such a setting) can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred [20] , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of (farm) animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents [20, [22] [23] [24] . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations [25] which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author(s) declare that they have no competing interests.

What causes Q fever?

Pre-existing immunity against vaccine vectors – friend or foe? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/ SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M. Date: 2013-01-27 DOI: 10.1099/mic.0.049601-0 License: cc-by Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) . A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) . Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity.

What is the effect of host immune response to the delivery vector on the efficacy of vaccination?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What is the issue with each of these vaccines?

Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/ SHA: f2d842780b9928cc70f38a4458553f2431877603 Authors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian Date: 2011-08-03 DOI: 10.3390/molecules16086489 License: cc-by Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, "Asian Influenza (1957/H2N2)" and "Hong Kong Influenza (1968/H3N2)" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view.

What do neuroaminidase inhibitors target?

Interferon-Induced Transmembrane Protein 3 Inhibits Hantaan Virus Infection, and Its Single Nucleotide Polymorphism rs12252 Influences the Severity of Hemorrhagic Fever with Renal Syndrome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206578/ SHA: 4328e18bdf9b52875c87f3f5ddb1911636a192d2 Authors: Xu-yang, Zheng; Pei-yu, Bian; Chuan-tao, Ye; Wei, Ye; Hong-wei, Ma; Kang, Tang; Chun-mei, Zhang; Ying-feng, Lei; Xin, Wei; Ping-zhong, Wang; Chang-xing, Huang; Xue-fan, Bai; Ying, Zhang; Zhan-sheng, Jia Date: 2017-01-03 DOI: 10.3389/fimmu.2016.00535 License: cc-by Abstract: Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS). Previous studies have identified interferon-induced transmembrane proteins (IFITMs) as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms (SNP) rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response (NRIR). Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS (2) . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins (IFITMs) was discovered 25 years ago to consist of interferon-stimulated genes (ISGs) (3) . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity (4) . Different IFITM proteins have different antiviral spectrum (5) . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice (6, 7) , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus (7) (8) (9) (10) (11) . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells (4, 12) . Single nucleotide polymorphisms (SNPs) are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro (13, 14) . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus (13, 15) . HTNV has been shown to induce a type I interferon response (though in later time postinfection) (16, 17) . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection (18) , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity (19) . Among them, negative regulator of interferon response (NRIR) (lncRNA NRIR, also known as lncRNA-CMPK2) is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection (20) . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay (ELISA) in our department. The classification of HFRS severity and the exclusion criteria were described as follows (21) : white blood cells (WBC), platelets (PLT), blood urea nitrogen (BUN), serum creatinine (Scr), and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory (shown in Table 1 ). According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described (21): (1) mild patients were identified with mild renal failure without an obvious oliguric stage; (2) moderate patients were those with obvious symptoms of uremia, effusion (bulbar conjunctiva), hemorrhage (skin and mucous membrane), and renal failure with a typical oliguric stage; (3) severe patients had severe uremia, effusion (bulbar conjunctiva and either peritoneum or pleura), hemorrhage (skin and mucous membrane), and renal failure with oliguria (urine output, 50-500 ml/day) for ≤5 days or anuria (urine output, <50 ml/day) for ≤2 days; and (4) critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria (urine output, 50-500 ml/day) for >5 days, anuria (urine output, <50 ml/day) for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: (1) any other kidney disease, (2) diabetes mellitus, (3) autoimmune disease, (4) hematological disease, (5) cardiovascular disease, (6) viral hepatitis (types A, B, C, D, or E), or (7) any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period (21) . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit (Gentra Systems, Minneapolis, MN, USA). The region encompassing the human IFITM3 rs12252 were amplified by PCR (forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′). The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer (Thermo Scientific, Waltham, MA, USA). The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project (http:// www.1000genomes.org). The HTNV load in plasma samples (collected during the acute phase) from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods (2) . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits (Invitrogen, Carlsbad, CA, USA). The SuperScript III Platinum One-Step Quantitative RT-PCR System kit (Invitrogen, Carlsbad, CA, USA) was employed for the real-time RT-PCR assay. The primers and probe (provided by Sangon Biotech, Shanghai, China) were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′-(FAM) ATCCCTCACCTTCTGCCTGGCTATC (TAMRA)-3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler (Bio-Rad, Hercules, CA, USA) with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells (ScienCell Research Laboratories, Carlsbad, CA, USA) were grown in ECM BulletKit (ScienCell Research Laboratories, Carlsbad, CA, USA) in a 5% CO2 incubator. A549 cells (ATCC Cat# CRM-CCL-185, RRID:CVCL_0023) were grown in our laboratory in DMEM with 10% FBS (Thermo Scientific, Waltham, MA, USA) in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells (ATCC Cat# CRL-1586, RRID:CVCL_0574) in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein (NP) as previously described (22) . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source (Piscataway, NJ, USA) and dissolved in the buffer provided by the manufacturer (composition not disclosed). HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 (purchased from GENECHEM, Shanghai, China) at a moi of 10. Puromycin (2 μg/ ml for HUVEC and 6 μg/ml for A549 cells) was used to create cell lines stably expressing IFITMs. Cells were transfected with control (scrambled) short interfering RNA (siRNA), IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA (10 nM) using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). SiRNAs were purchased from Origene (Rockville, MD, USA), and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the K1622 kit (Thermo Scientific, Waltham, MA, USA). Quantitative realtime PCR (qPCR) was performed using SYBR Premix Ex Taq II (Takara Biotechnology Co., Dalian, China) with a Bio-Rad iQ5 cycler (Bio-Rad, Hercules, CA, USA). β-actin was used as the reference gene. The primers (Sangon Biotech, Shanghai, China) were as follows: IFITM1 (forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′); IFITM2 (forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′); IFITM3 (forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′); IFITM3 pre-mRNA (forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′); HTNV S segment (forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′); β-actin (forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′); NRIR (forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′); NRAV (forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′). For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit (Invitrogen, Carlsbad, CA, USA) with a specific primer in gene-specific TaqMan assay kit (000454, Invitrogen, Carlsbad, CA, USA). MiR-130a level was determined using the gene-specific TaqMan assay kit (000454, Invitrogen, Carlsbad, CA, USA). U6 (001973, Invitrogen, Carlsbad, CA, USA) was used as an endogenous control (23) . Because the pre-mRNA levels can represent the initial transcription rate (24) , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described (25) . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon (24) . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment (20 IU/ml for 12 h) after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein (20 μg protein/lane) were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 (Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405), IFITM2, IFITM3 (Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821), and β-actin (Proteintech, Wuhan, Hubei, China) or HTNV NP (provided by the Department of Microbiology, The Fourth Military Medical University) overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA) and visualized using X-ray film. The blot densities were analyzed using the Quantity One software (Bio-Rad, Hercules, CA, USA). In addition, the RIPA buffer contains 50mM Tris (pH = 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail (Roche, Basel, Switzerland) was added before use. The cells were cultured on glass coverslips (Millipore, Billerica, MA, USA) until they were semi-confluence and then incubated with HTNV for 60 min (moi = 1). At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag (Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546), IFITM3, lysosome-associated membrane glycoprotein 1 (LAMP1, Cell Signaling Technology, Danvers, MA, USA), or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 (Abcam, Cambridge, MA, USA) secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system (Olympus, Tokyo, Japan) were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K (0.1 mg/ml, Thermo Scientific, Waltham, MA, USA). To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV (moi = 1) was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium (20 mM sodium succinate, pH = 5.5) for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl (26) . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance (ANOVA) with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section "Material and Methods. " We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database (68.29 vs. 52.16%, P = 0.0076). The frequency of rs12252 C in severe patients was also higher than those mild patients (68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 ). These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio (95% CI) of 2.124 (1.067-4.230). For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database (26.92% CC genotype, P = 0.03) as well as mildly infected patients (14.29%, P = 0.02, Figures 1A,B ; Table 2 ). However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. (c) The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase ( Figure 1C) . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele (Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes) leads to an impaired anti-influenza activity (14) . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated (NΔ21) proteins (Figure 2A ) with c-myc-tag to HUVEC and A549 cell using lentivirus vectors ( Figure 2B) . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment ( Figure 2C ) and more positive of HTNV NP ( Figure S3 in Supplementary Material). Indeed, compared with the mock (empty vector)-infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells (Figures 2C,D ; Figure S3 in Supplementary Material). To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells (Figures 3A,B ; Figure S1 in Supplementary Material). While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs ( Figure S2 in Supplementary Material), and the effect of the best oligo against each IFITMs (IFITM1C, IFITM2A, IFITM3B) was tested by Western blot in A549 ( Figure 4A ) and HUVEC cells ( Figure 4B) . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment (20 IU/ml for another 12 h). The cells were then challenged with HTNV (moi = 1) for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells (Figures 4C,D) . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells (Figure 5A) , and the cells were then challenged with HTNV (moi = 1) for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells (Figures 5B-D) . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells (Figures 5B-D) . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP ( Figure S3 in Supplementary Material). These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV (moi = 1) at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section "Materials and Methods." Expression of IFITM3 did not affect HTNV binding ( Figure 6A ) but significantly suppressed HTNV entry in both HUVEC and A549 cells (Figure 6B ). iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy (Figure 6C) . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells (27) . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry (28) , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one (downregulated) after HTNV infection ( Figure 7A ; Figure S4 in Supplementary Material) in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector ( Figure 7B) . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells (Figures 7C-E) . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome (HPS) in humans (21) . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury (1, 21) , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS (2). However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response (16) . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication (29) . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals (15) . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families (including filoviruses, rhabdoviruses, and flaviviruses) (7, (9) (10) (11) 30) . For example, HIV-1 and HCV infection are inhibited by IFITM1 (31) (32) (33) (34) . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry (12) . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein (5) . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles (32) , whereas IFITM3 confines influenza virus in acidified endosomal compartments (27) . Notably, retrovirus subvirus particles (ISVPs), which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry (35) . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging (FLIM) suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes (36) . In the present study, we demonstrated that IFN-α2a (20 U/ ml) significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV (18) . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices (shown in Figure 2A as black boxes) in IFITM3 (14) . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids (deleted part, shown in Figure 2A as red dotted line). Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence (15, 29) . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells (15) . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed (37) . In Chinese patients infected with influenza A (H1N1) virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 (13) . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression (38) . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes (25) . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection (20) . Mir-130a was also reported as a regulator of IFITM1 (23) . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV (NR_038854), remained unchanged after HTNV infection ( Figures S4A,B in Supplementary Material). Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection ( Figures S4C,D in Supplementary Material). In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein (NΔ21) that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.

What is the structure of Hantaan virus?

Acute Hemorrhagic Encephalitis Responding to Combined Decompressive Craniectomy, Intravenous Immunoglobulin, and Corticosteroid Therapies: Association with Novel RANBP2 Variant https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857578/ SHA: ef6638accc1ef599ad1aafd47b3a86f2b904cc76 Authors: Alawadhi, Abdulla; Saint-Martin, Christine; Bhanji, Farhan; Srour, Myriam; Atkinson, Jeffrey; Sébire, Guillaume Date: 2018-03-12 DOI: 10.3389/fneur.2018.00130 License: cc-by Abstract: BACKGROUND: Acute hemorrhagic encephalomyelitis (AHEM) is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein (RANBP2) gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone (IVMP) in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease (SCD) presented an acquired demyelinating syndrome (ADS) with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP (30 mg/kg/day). Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins (IVIG). She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant (c.4993A>G, p.Lys1665Glu) was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis (AHEM) or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis (ADEM). AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal (1) (2) (3) . Many treatment options have been used including intravenous (IV) steroids, intravenous immunoglobulins (IVIG), and plasmapheresis (4) . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy (5) (6) (7) (8) (9) . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy (ANE) which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children (10) . We report a new case of AHEM associated to a Ran Binding Protein (RANBP)-2 variant and responsive to combined craniectomy, intravenous methylprednisolone (IVMP), and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease (SCD). A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia [day 0 (D0): refers to the start of the diplopia] 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging (MRI) performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal (Figure 1 ). Brain MR angiography (MRA) showed a normal appearing circle of Willis. The cerebrospinal fluid (CSF) obtained by lumber puncture was normal (WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands). The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate (tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus), and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive (1:1,280) reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies (ANA) were positive (1:160). B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP (30 mg/kg/day) followed by 9 days of oral prednisone (1 mg/kg/day). At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness (D30) with normal alertness. She had normal blood pressure (120/81 mmHg). She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine (D30) she deteriorated with vomiting and reduced level of consciousness (Glasgow Coma Scale dropped from 15 to 8 over 30 min). Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal (Figures 2A-F) . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain (EVD). Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere (Figures 2G-I) . She was started on IVMP (30 mg/kg/ day for 5 days) and IVIG (1 g/kg/day for 2 days). Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions (Figure 3) . Prednisolone was tapered course over 6 weeks. At discharge (D71), she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR (HSV1/2) were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens (SSA-RO, SSB-LA, smith, RNP) were negative. Serum autoimmune antibodies panel (NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI) were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel ( Table 1 ) targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation (NM_006267.4, c.4993A>G, p.Lys1665Glu). This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease (6/117,118 alleles). Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging (SIFT and Align GVGD). Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A (11) . SCD patients are prone to ischemic or hemorrhagic strokes (12) . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case (13) . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients (13) (14) (15) (16) . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient (17) . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis (18) . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia (19) . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes (20, 21) . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context (7, 19) . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy (5) (6) (7) (8) (9) (22) (23) (24) (25) . Autosomal dominant mutations (with incomplete penetrance) in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy (26, 27) . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum (10) . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools (a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid). It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy (25, 26) making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells (28) . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation (2016). This work was supported by grants from Heart and Stroke Foundation of Canada (grant number: G-14-0005756), and Foundation of Stars.

When did she present with rapidly progressive right-hand weakness?

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067204/ SHA: c097a8a9a543d69c34f10e5c3fd78019e560026a Authors: Chan, Jasper Fuk-Woo; Kok, Kin-Hang; Zhu, Zheng; Chu, Hin; To, Kelvin Kai-Wang; Yuan, Shuofeng; Yuen, Kwok-Yung Date: 2020-01-28 DOI: 10.1080/22221751.2020.1719902 License: cc-by Abstract: A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Deltacoronavirus (δCoV), and Gammacoronavirus (γCoV) [1] . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV (SARS-CoV) which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV (MERS-CoV) which has caused a persistent epidemic in the Arabian Peninsula since 2012 [2, 3] . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host [the Himalayan palm civet (Paguma larvata) for SARS-CoV and the dromedary camel (Camelus dromedarius) for MERS-CoV] before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV (HCoV-229E and HKU-NL63) and 4 βCoV (HCoV-OC43 [ HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly [4] . In contrast, SARS-CoV (lineage B βCoV) and MERS-CoV (lineage C βCoV) may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively [5, 6] . On 31 December 2019, the World Health Organization (WHO) was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China [7] . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan [8, 9] . The earliest date of symptom onset was 1 December 2019 [10] . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 (32%) required intensive care and 6 (15%) died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans [10] . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan [11] . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank (accession no. MN975262) ( Table 1 ). The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees [12] . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [13] . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site [14] . All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X [15] . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction (PSIPRED) [16] . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). Secondary structure prediction in the 5 ′ -untranslated region (UTR) and 3 ′ -UTR was performed using the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/ RNAWebSuite/RNAfold.cgi) with minimum free energy (MFE) and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other (Table 2 ). There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV (Table 3) . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain (NTD) and receptor-binding domain (RBD), while the S2 subunit contains conserved fusion peptide (FP), heptad repeat (HR) 1 and 2, transmembrane domain (TM), and cytoplasmic domain (CP). We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs (SL-CoV ZXC21 and ZC45) and human SARS-CoV (Figure 2 ). Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials [18] . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV (Figure 3(A) ), the core domain of RBD (excluding the external subdomain) are highly conserved (Figure 3(B) ). Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain (RBD) when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b ( Figure 4 ). It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types [19] , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points [20] . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling [21] . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV [22] . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 [23] . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients [24] . In addition, we have previously identified two bat SARS-related-CoV (Bat-CoV YNLF_31C and YNLF_34C) and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV [25] . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 (human SARS-CoV GZ02), the mid-and late-phase SARS-CoV that includes the split orf8b (human SARS-CoV Tor2), civet SARS-CoV (paguma SARS-CoV), two bat SARS-related-CoV containing full-length orf8 (bat-CoV YNLF_31C and YNLF_34C), 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45), and bat SARS-related-CoV HKU3-1 ( Figure 5(A) ). As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5(B) ) which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes [26] , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet(s) containing six strands ( Figure 5(C) ). The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 (MG772934.1), and 82% with human SARS-CoV BJ01 2003 (AY278488) and human SARS-CoV Tor2 (AY274119). The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes (S, E, M, and N) were shown (Figure 6(A-E) ). For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 (A-C), the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif [27] . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b (marked in red), which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR (Figure 7(D-F) ). The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs [27] . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV.

How do most bat SARSr-COV differ from 2019-nCOV and human SARS-COV?

Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review https://doi.org/10.3390/jcm9030623 SHA: 9b0c87f808b1b66f2937d7a7acb524a756b6113b Authors: Pang, Junxiong; Wang, Min Xian; Ang, Ian Yi Han; Tan, Sharon Hui Xuan; Lewis, Ruth Frances; Chen, Jacinta I. Pei; Gutierrez, Ramona A.; Gwee, Sylvia Xiao Wei; Chua, Pearleen Ee Yong; Yang, Qian; Ng, Xian Yi; Yap, Rowena K. S.; Tan, Hao Yi; Teo, Yik Ying; Tan, Chorh Chuan; Cook, Alex R.; Yap, Jason Chin-Huat; Hsu, Li Yang Date: 2020 DOI: 10.3390/jcm9030623 License: cc-by Abstract: Rapid diagnostics, vaccines and therapeutics are important interventions for the management of the 2019 novel coronavirus (2019-nCoV) outbreak. It is timely to systematically review the potential of these interventions, including those for Middle East respiratory syndrome-Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS)-CoV, to guide policymakers globally on their prioritization of resources for research and development. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Supplementary strategies through Google Search and personal communications were used. A total of 27 studies fulfilled the criteria for review. Several laboratory protocols for confirmation of suspected 2019-nCoV cases using real-time reverse transcription polymerase chain reaction (RT-PCR) have been published. A commercial RT-PCR kit developed by the Beijing Genomic Institute is currently widely used in China and likely in Asia. However, serological assays as well as point-of-care testing kits have not been developed but are likely in the near future. Several vaccine candidates are in the pipeline. The likely earliest Phase 1 vaccine trial is a synthetic DNA-based candidate. A number of novel compounds as well as therapeutics licensed for other conditions appear to have in vitro efficacy against the 2019-nCoV. Some are being tested in clinical trials against MERS-CoV and SARS-CoV, while others have been listed for clinical trials against 2019-nCoV. However, there are currently no effective specific antivirals or drug combinations supported by high-level evidence. Text: Since mid-December 2019 and as of early February 2020, the 2019 novel coronavirus (2019-nCoV) originating from Wuhan (Hubei Province, China) has infected over 25,000 laboratory-confirmed cases across 28 countries with about 500 deaths (a case-fatality rate of about 2%). More than 90% of the cases and deaths were in China [1] . Based on the initial reported surge of cases in Wuhan, the majority were males with a median age of 55 years and linked to the Huanan Seafood Wholesale Market [2] . Most of the reported cases had similar symptoms at the onset of illness such as fever, cough, and myalgia or fatigue. Most cases developed pneumonia and some severe and even fatal respiratory diseases such as acute respiratory distress syndrome [3] . The 2019 novel coronavirus (2019-nCoV), a betacoronavirus, forms a clade within the subgenus sarbecovirus of the Orthocoronavirinae subfamily [4] . The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also betacoronaviruses that are zoonotic in origin and have been linked to potential fatal illness during the outbreaks in 2003 and 2012, respectively [5, 6] . Based on current evidence, pathogenicity for 2019-nCoV is about 3%, which is significantly lower than SARS-CoV (10%) and MERS-CoV (40%) [7] . However, 2019-nCoV has potentially higher transmissibility (R0: 1.4-5.5) than both SARS-CoV (R0: [2] [3] [4] [5] and MERS-CoV (R0: <1) [7] . With the possible expansion of 2019-nCoV globally [8] and the declaration of the 2019-nCoV outbreak as a Public Health Emergency of International Concern by the World Health Organization, there is an urgent need for rapid diagnostics, vaccines and therapeutics to detect, prevent and contain 2019-nCoV promptly. There is however currently a lack of understanding of what is available in the early phase of 2019-nCoV outbreak. The systematic review describes and assesses the potential rapid diagnostics, vaccines and therapeutics for 2019-nCoV, based in part on the developments for MERS-CoV and SARS-CoV. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies examining the diagnosis, therapeutic drugs and vaccines for Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the 2019 novel coronavirus (2019-nCoV), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There were two independent reviewers each focusing on SARS, MERS, and 2019-nCoV, respectively. A third independent reviewer was engaged to resolve any conflicting article of interest. We used the key words "SARS", "coronavirus", "MERS", "2019 Novel coronavirus", "Wuhan virus" to identify the diseases in the search strategy. The systematic searches for diagnosis, therapeutic drugs and vaccines were carried out independently and the key words "drug", "therapy", "vaccine", "diagnosis", "point of care testing" and "rapid diagnostic test" were used in conjunction with the disease key words for the respective searches. Examples of search strings can be found in Table S1 . We searched for randomized controlled trials (RCTs) and validation trials (for diagnostics test) published in English, that measured (a) the sensitivity and/or specificity of a rapid diagnostic test or a point-of-care testing kit, (b) the impact of drug therapy or (c) vaccine efficacy against either of these diseases with no date restriction applied. For the 2019-nCoV, we searched for all in vitro, animal, or human studies published in English between 1 December 2019 and 6 February 2020, on the same outcomes of interest. In addition, we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial searches. Studies that examined the mechanisms of diagnostic tests, drug therapy or vaccine efficacy against SARS, MERS and 2019-nCoV were excluded. A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles. With the emergence of 2019-nCoV, real time RT-PCR remains the primary means for diagnosing the new virus strain among the many diagnostic platforms available ( [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ; Table S3 ). Among the 16 diagnostics studies selected, one study discussed the use of RT-PCR in diagnosing patients with 2019-nCoV [11] ( Table 1 ). The period and type of specimen collected for RT-PCR play an important role in the diagnosis of 2019-nCoV. It was found that the respiratory specimens were positive for the virus while serum was negative in the early period. It has also suggested that in the early days of illness, patients have high levels of virus despite the mild symptoms. Apart from the commonly used RT-PCR in diagnosing MERS-CoV, four studies identified various diagnostic methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), RT-insulated isothermal PCR (RT-iiPCR) and a one-step rRT-PCR assay based on specific TaqMan probes. RT-LAMP has similar sensitivity as real time RT-PCR. It is also highly specific and is used to detect MERS-CoV. It is comparable to the usual diagnostic tests and is rapid, simple and convenient. Likewise, RT-iiPCR and a one-step rRT-PCR assay have also shown similar sensitivity and high specificity for MER-CoV. Lastly, one study focused on the validation of the six commercial real RT-PCR kits, with high accuracy. Although real time RT-PCR is a primary method for diagnosing MERS-CoV, high levels of PCR inhibition may hinder PCR sensitivity (Table 1) . There are eleven studies that focus on SARS-CoV diagnostic testing (Table 1) . These papers described diagnostic methods to detect the virus with the majority of them using molecular testing for diagnosis. Comparison between the molecular test (i.e RT-PCR) and serological test (i.e., ELISA) showed that the molecular test has better sensitivity and specificity. Hence, enhancements to the current molecular test were conducted to improve the diagnosis. Studies looked at using nested PCR to include a pre-amplification step or incorporating N gene as an additional sensitive molecular marker to improve on the sensitivity (Table 1 ). In addition, there are seven potential rapid diagnostic kits (as of 24 January 2020; Table 2 ) available on the market for 2019-nCoV. Six of these are only for research purposes. Only one kit from Beijing Genome Institute (BGI) is approved for use in the clinical setting for rapid diagnosis. Most of the kits are for RT-PCR. There were two kits (BGI, China and Veredus, Singapore) with the capability to detect multiple pathogens using sequencing and microarray technologies, respectively. The limit of detection of the enhanced realtime PCR method was 10 2 -fold higher than the standard real-time PCR assay and 10 7fold higher than conventional PCR methods In the clinical aspect, the enhanced realtime PCR method was able to detect 6 cases of SARS-CoV positive samples that were not confirmed by any other assay [25] • The real time PCR has a threshold sensitivity of 10 genome equivalents per reaction and it has a good reproducibility with the inter-assay coefficients of variation of 1.73 to 2.72%. • 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. The real-time RT-PCR reaction was more sensitive than the nested PCR reaction, as the detection limit for the nested PCR reaction was about 10 3 genome equivalents in the standard cDNA control. [34] Real-time reverse-transcription PCR (rRT-PCR); RNA-dependent RNA polymerase (RdRp); open reading frame 1a (ORF1a); Loop-mediated isothermal amplification (LAMP); enzyme-linked immunosorbent assay (ELISA); immunofluorescent assay (IFA); immunochromatographic test (ICT); nasopharyngeal aspirate (NPA). With the emergence of 2019-nCoV, there are about 15 potential vaccine candidates in the pipeline globally (Table 3 ), in which a wide range of technology (such as messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle) was applied. It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening. The published clinical trials were mostly done in United States except for one on the SARS vaccine done in China [44] . All vaccine candidates for SARS and MERS were reported to be safe, well-tolerated and able to trigger the relevant and appropriate immune responses in the participants. In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60. [47] Molecules developed by the university scientists inhibit two coronavirus enzymes and prevent its replication. The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] . The trial conducted in the United States in 2017 demonstrated SAB-301 to be safe and well-tolerated at single doses. Another trial on MERS therapeutics was found on ClinicalTrials.gov-a phase 2/3 trial in the United States evaluating the safety, tolerability, pharmacokinetics (PK), and immunogenicity on coadministered MERS-CoV antibodies REGN3048 & REGN3051 [100]. Rapid diagnostics plays an important role in disease and outbreak management. The fast and accurate diagnosis of a specific viral infection enables prompt and accurate public health surveillance, prevention and control measures. Local transmission and clusters can be prevented or delayed by isolation of laboratory-confirmed cases and their close contacts quarantined and monitored at home. Rapid diagnostic also facilitates other specific public health interventions such as closure of high-risk facilities and areas associated with the confirmed cases for prompt infection control and environmental decontamination [11, 101] . Laboratory diagnosis can be performed by: (a) detecting the genetic material of the virus, (b) detecting the antibodies that neutralize the viral particles of interest, (c) detecting the viral epitopes of interest with antibodies (serological testing), or (d) culture and isolation of viable virus particles. The key limitations of genetic material detection are the lack of knowledge of the presence of viable virus, the potential cross-reactivity with non-specific genetic regions and the short timeframe for accurate detection during the acute infection phase. The key limitations of serological testing is the need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses. The limitation of virus culture and isolation is the long duration and the highly specialized skills required of the technicians to process the samples. All patients recovered. Significantly shorted time from the disease onset to the symptom improvement in treatment (5.10 ± 2.83 days) compared to control group (7.62 ± 2.27 days) (p < 0.05) No significant difference in blood routine improvement, pulmonary chest shadow in chest film improvement and corticosteroid usgae between the 2 groups. However, particularly in the respect of improving clinical symptoms, elevating quality of life, promoting immune function recovery, promoting absorption of pulmonary inflammation, reducing the dosage of cortisteroid and shortening the therapeutic course, treatment with integrative chinese and western medicine treatment had obvious superiority compared with using control treatment alone. Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well-tolerated in healthy participants. [46] Where the biological samples are taken from also play a role in the sensitivity of these tests. For SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates have higher and more prolonged levels of viral RNA because of the tropism of the virus. MERS-CoV viral loads are also higher for severe cases and have longer viral shedding compared to mild cases. Although upper respiratory tract specimens such as nasopharyngeal or oropharyngeal swabs can be used, they have potentially lower viral loads and may have higher risk of false-negatives among the mild MERS and SARS cases [102, 103] , and likely among the 2019-nCoV cases. The existing practices in detecting genetic material of coronaviruses such as SARS-CoV and MERS-CoV include (a) reverse transcription-polymerase chain reaction (RT-PCR), (b) real-time RT-PCR (rRT-PCR), (c) reverse transcription loop-mediated isothermal amplification (RT-LAMP) and (d) real-time RT-LAMP [104] . Nucleic amplification tests (NAAT) are usually preferred as in the case of MERS-CoV diagnosis as it has the highest sensitivity at the earliest time point in the acute phase of infection [102] . Chinese health authorities have recently posted the full genome of 2019-nCoV in the GenBank and in GISAID portal to facilitate in the detection of the virus [11] . Several laboratory assays have been developed to detect the novel coronavirus in Wuhan, as highlighted in WHO's interim guidance on nCoV laboratory testing of suspected cases. These include protocols from other countries such as Thailand, Japan and China [105] . The first validated diagnostic test was designed in Germany. Corman et al. had initially designed a candidate diagnostic RT-PCR assay based on the SARS or SARS-related coronavirus as it was suggested that circulating virus was SARS-like. Upon the release of the sequence, assays were selected based on the match against 2019-nCoV upon inspection of the sequence alignment. Two assays were used for the RNA dependent RNA polymerase (RdRP) gene and E gene where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing. All assays were highly sensitive and specific in that they did not cross-react with other coronavirus and also human clinical samples that contained respiratory viruses [11] . The Hong Kong University used two monoplex assays which were reactive with coronaviruses under the subgenus Sarbecovirus (consisting of 2019-nCoV, SARS-CoV and SARS-like coronavirus). Viral RNA extracted from SARS-CoV can be used as the positive control for the suggested protocol assuming that SARS has been eradicated. It is proposed that the N gene RT-PCR can be used as a screening assay while the Orf1b assay acts as a confirmatory test. However, this protocol has only been evaluated with a panel of controls with the only positive control SARS-CoV RNA. Synthetic oligonucleotide positive control or 2019-nCoV have yet to be tested [106] . The US CDC shared the protocol on the real time RT-PCR assay for the detection of the 2019-nCoV with the primers and probes designed for the universal detection of SARS-like coronavirus and the specific detection of 2019-nCoV. However, the protocol has not been validated on other platforms or chemistries apart from the protocol described. There are some limitations for the assay. Analysts engaged have to be trained and familiar with the testing procedure and result interpretation. False negative results may occur due to insufficient organisms in the specimen resulting from improper collection, transportation or handling. Also, RNA viruses may show substantial genetic variability. This could result in mismatch between the primer and probes with the target sequence which can diminish the assay performance or result in false negative results [107] . Point-of-care test kit can potentially minimize these limitations, which should be highly prioritized for research and development in the next few months. Serological testing such as ELISA, IIFT and neutralization tests are effective in determining the extent of infection, including estimating asymptomatic and attack rate. Compared to the detection of viral genome through molecular methods, serological testing detects antibodies and antigens. There would be a lag period as antibodies specifically targeting the virus would normally appear between 14 and 28 days after the illness onset [108] . Furthermore, studies suggest that low antibody titers in the second week or delayed antibody production could be associated with mortality with a high viral load. Hence, serological diagnoses are likely used when nucleic amplification tests (NAAT) are not available or accessible [102] . Vaccines can prevent and protect against infection and disease occurrence when exposed to the specific pathogen of interest, especially in vulnerable populations who are more prone to severe outcomes. In the context of the current 2019-nCoV outbreak, vaccines will help control and reduce disease transmission by creating herd immunity in addition to protecting healthy individuals from infection. This decreases the effective R0 value of the disease. Nonetheless, there are social, clinical and economic hurdles for vaccine and vaccination programmes, including (a) the willingness of the public to undergo vaccination with a novel vaccine, (b) the side effects and severe adverse reactions of vaccination, (c) the potential difference and/or low efficacy of the vaccine in populations different from the clinical trials' populations and (d) the accessibility of the vaccines to a given population (including the cost and availability of the vaccine). Vaccines against the 2019-nCoV are currently in development and none are in testing (at the time of writing). On 23 January 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) announced that they will fund vaccine development programmes with Inovio, The University of Queensland and Moderna, Inc respectively, with the aim to test the experimental vaccines clinically in 16 weeks (By June 2020). The vaccine candidates will be developed by the DNA, recombinant and mRNA vaccine platforms from these organizations [109] . Based on the most recent MERS-CoV outbreak, there are already a number of vaccine candidates being developed but most are still in the preclinical testing stage. The vaccines in development include viral vector-based vaccine, DNA vaccine, subunit vaccine, virus-like particles (VLPs)-based vaccine, inactivated whole-virus (IWV) vaccine and live attenuated vaccine. The latest findings for these vaccines arebased on the review by Yong et al. (2019) in August 2019 [110] . As of the date of reporting, there is only one published clinical study on the MERS-CoV vaccine by GeneOne Life Science & Inovio Pharmaceuticals [47] . There was one SARS vaccine trial conducted by the US National Institute of Allergy and Infectious Diseases. Both Phase I clinical trials reported positive results, but only one has announced plans to proceed to Phase 2 trial [111] . Due to the close genetic relatedness of SARS-CoV (79%) with 2019-nCoV [112] , there may be potential cross-protective effect of using a safe SARS-CoV vaccine while awaiting the 2019-nCoV vaccine. However, this would require small scale phase-by-phase implementation and close monitoring of vaccinees before any large scale implementation. Apart from the timely diagnosis of cases, the achievement of favorable clinical outcomes depends on the timely treatment administered. ACE2 has been reported to be the same cell entry receptor used by 2019-nCoV to infect humans as SARS-CoV [113] . Hence, clinical similarity between the two viruses is expected, particularly in severe cases. In addition, most of those who have died from MERS-CoV, SARS-CoV and 2019-nCoV were advance in age and had underlying health conditions such as hypertension, diabetes or cardiovascular disease that compromised their immune systems [114] . Coronaviruses have error-prone RNA-dependent RNA polymerases (RdRP), which result in frequent mutations and recombination events. This results in quasispecies diversity that is closely associated with adaptive evolution and the capacity to enhance viral-cell entry to cause disease over time in a specific population at-risk [115] . Since ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, coronaviruses are likely to infect the upper respiratory and gastrointestinal tract and this may influence the type of therapeutics against 2019-nCoV, similarly to SAR-CoV. However, in the years following two major coronavirus outbreaks SARS-CoV in 2003 and MERS-CoV in 2012, there remains no consensus on the optimal therapy for either disease [116, 117] . Well-designed clinical trials that provide the gold standard for assessing the therapeutic measures are scarce. No coronavirus protease inhibitors have successfully completed a preclinical development program despite large efforts exploring SARS-CoV inhibitors. The bulk of potential therapeutic strategies remain in the experimental phase, with only a handful crossing the in vitro hurdle. Stronger efforts are required in the research for treatment options for major coronaviruses given their pandemic potential. Effective treatment options are essential to maximize the restoration of affected populations to good health following infections. Clinical trials have commenced in China to identify effective treatments for 2019-nCoV based on the treatment evidence from SARS and MERS. There is currently no effective specific antiviral with high-level evidence; any specific antiviral therapy should be provided in the context of a clinical study/trial. Few treatments have shown real curative action against SARS and MERS and the literature generally describes isolated cases or small case series. Many interferons from the three classes have been tested for their antiviral activities against SARS-CoV both in vitro and in animal models. Interferon β has consistently been shown to be the most active, followed by interferon α. The use of corticosteroids with interferon alfacon-1 (synthetic interferon α) appeared to have improved oxygenation and faster resolution of chest radiograph abnormalities in observational studies with untreated controls. Interferon has been used in multiple observational studies to treat SARS-CoV and MERS-CoV patients [116, 117] . Interferons, with or without ribavirin, and lopinavir/ritonavir are most likely to be beneficial and are being trialed in China for 2019-nCoV. This drug treatment appears to be the most advanced. Timing of treatment is likely an important factor in effectiveness. A combination of ribavirin and lopinavir/ritonavir was used as a post-exposure prophylaxis in health care workers and may have reduced the risk of infection. Ribavirin alone is unlikely to have substantial antiviral activities at clinically used dosages. Hence, ribavirin with or without corticosteroids and with lopinavir and ritonavir are among the combinations employed. This was the most common agent reported in the available literature. Its efficacy has been assessed in observational studies, retrospective case series, retrospective cohort study, a prospective observational study, a prospective cohort study and randomized controlled trial ranging from seven to 229 participants [117] . Lopinavir/ritonavir (Kaletra) was the earliest protease inhibitor combination introduced for the treatment of SARS-CoV. Its efficacy was documented in several studies, causing notably lower incidence of adverse outcomes than with ribavirin alone. Combined usage with ribavirin was also associated with lower incidence of acute respiratory distress syndrome, nosocomial infection and death, amongst other favorable outcomes. Recent in vitro studies have shown another HIV protease inhibitor, nelfinavir, to have antiviral capacity against SARS-CoV, although it has yet to show favorable outcomes in animal studies [118] . Remdesivir (Gilead Sciences, GS-5734) nucleoside analogue in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several coronaviruses (CoVs), including highly pathogenic CoVs and potentially emergent BatCoVs. The use of remdesivir may be a good candidate as an investigational treatment. Improved mortality following receipt of convalescent plasma in various doses was consistently reported in several observational studies involving cases with severe acute respiratory infections (SARIs) of viral etiology. A significant reduction in the pooled odds of mortality following treatment of 0.25 compared to placebo or no therapy was observed [119] . Studies were however at moderate to high risk of bias given their small sample sizes, allocation of treatment based on the physician's discretion, and the availability of plasma. Factors like concomitant treatment may have also confounded the results. Associations between convalescent plasma and hospital length of stay, viral antibody levels, and viral load respectively were similarly inconsistent across available literature. Convalescent plasma, while promising, is likely not yet feasible, given the limited pool of potential donors and issues of scalability. Monoclonal antibody treatment is progressing. SARS-CoV enters host cells through the binding of their spike (S) protein to angiotensin converting enzyme 2 (ACE2) and CD209L [118] . Human monoclonal antibodies to the S protein have been shown to significantly reduce the severity of lung pathology in non-human primates following MERS-CoV infection [120] . Such neutralizing antibodies can be elicited by active or passive immunization using vaccines or convalescent plasma respectively. While such neutralizing antibodies can theoretically be harvested from individuals immunized with vaccines, there is uncertainty over the achievement of therapeutic levels of antibodies. Other therapeutic agents have also been reported. A known antimalarial agent, chloroquine, elicits antiviral effects against multiple viruses including HIV type 1, hepatitis B and HCoV-229E. Chloroquine is also immunomodulatory, capable of suppressing the production and release of factors which mediate the inflammatory complications of viral diseases (tumor necrosis factor and interleukin 6) [121] . It is postulated that chloroquine works by altering ACE2 glycosylation and endosomal pH. Its anti-inflammatory properties may be beneficial for the treatment of SARS. Niclosamide as a known drug used in antihelminthic treatment. The efficacy of niclosamide as an inhibitor of virus replication was proven in several assays. In both immunoblot analysis and immunofluorescence assays, niclosamide treatment was observed to completely inhibit viral antigen synthesis. Reduction of virus yield in infected cells was dose dependent. Niclosamide likely does not interfere in the early stages of virus attachment and entry into cells, nor does it function as a protease inhibitor. Mechanisms of niclosamide activity warrant further investigation [122] . Glycyrrhizin also reportedly inhibits virus adsorption and penetration in the early steps of virus replication. Glycyrrhizin was a significantly potent inhibitor with a low selectivity index when tested against several pathogenic flaviviruses. While preliminary results suggest production of nitrous oxide (which inhibits virus replication) through induction of nitrous oxide synthase, the mechanism of Glycyrrhizin against SARS-CoV remains unclear. The compound also has relatively lower toxicity compared to protease inhibitors like ribavirin [123] . Inhibitory activity was also detected in baicalin [124] , extracted from another herb used in the treatment of SARS in China and Hong Kong. Findings on these compounds are limited to in vitro studies [121] [122] [123] [124] . Due to the rapidly evolving situation of the 2019-nCoV, there will be potential limitations to the systematic review. The systematic review is likely to have publication bias as some developments have yet to be reported while for other developments there is no intention to report publicly (or in scientific platforms) due to confidentiality concerns. However, this may be limited to only a few developments for review as publicity does help in branding to some extent for the company and/or the funder. Furthermore, due to the rapid need to share the status of these developments, there may be reporting bias in some details provided by authors of the scientific articles or commentary articles in traditional media. Lastly, while it is not viable for any form of quality assessment and metaanalysis of the selected articles due to the limited data provided and the heterogeneous style of reporting by different articles, this paper has provided a comprehensive overview of the potential developments of these pharmaceutical interventions during the early phase of the outbreak. This systematic review would be useful for cross-check when the quality assessment and meta-analysis of these developments are performed as a follow-up study. Rapid diagnostics, vaccines and therapeutics are key pharmaceutical interventions to limit transmission of respiratory infectious diseases. Many potential developments on these pharmaceutical interventions for 2019-nCoV are ongoing in the containment phase of this outbreak, potentially due to better pandemic preparedness than before. However, lessons from MERS-CoV and SARS-CoV have shown that the journeys for these developments can still be challenging moving ahead. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1 : Example of full search strategy in Pubmed, Table S2 : Google Search: 2019-nCoV diagnostics, Table S3 : Summary of diagnostic assays developed for 2019-nCoV, Table S4

How does RT-LAMP compare with other methods?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

When was the Middle East Respiratory Syndrome Coronavirus isolated first?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

How may the responses be different in a chronically inflamed airway?

MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The

What is the the proportion of deaths among those infected with MERS-CoV?

Etiology of respiratory tract infections in the community and clinic in Ilorin, Nigeria https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719735/ SHA: f2e835d2cde5f42054dbd0c20d4060721135c518 Authors: Kolawole, Olatunji; Oguntoye, Michael; Dam, Tina; Chunara, Rumi Date: 2017-12-07 DOI: 10.1186/s13104-017-3063-1 License: cc-by Abstract: OBJECTIVE: Recognizing increasing interest in community disease surveillance globally, the goal of this study was to investigate whether respiratory viruses circulating in the community may be represented through clinical (hospital) surveillance in Nigeria. RESULTS: Children were selected via convenience sampling from communities and a tertiary care center (n = 91) during spring 2017 in Ilorin, Nigeria. Nasal swabs were collected and tested using polymerase chain reaction. The majority (79.1%) of subjects were under 6 years old, of whom 46 were infected (63.9%). A total of 33 of the 91 subjects had one or more respiratory tract virus; there were 10 cases of triple infection and 5 of quadruple. Parainfluenza virus 4, respiratory syncytial virus B and enterovirus were the most common viruses in the clinical sample; present in 93.8% (15/16) of clinical subjects, and 6.7% (5/75) of community subjects (significant difference, p < 0.001). Coronavirus OC43 was the most common virus detected in community members (13.3%, 10/75). A different strain, Coronavirus OC 229 E/NL63 was detected among subjects from the clinic (2/16) and not detected in the community. This pilot study provides evidence that data from the community can potentially represent different information than that sourced clinically, suggesting the need for community surveillance to enhance public health efforts and scientific understanding of respiratory infections. Text: Acute Respiratory Infections (ARIs) (the cause of both upper respiratory tract infections (URIs) and lower respiratory tract infections (LRIs)) are a major cause of death among children under 5 years old particularly in developing countries where the burden of disease is 2-5 times higher than in developed countries [1] . While these viruses usually cause mild cold-like symptoms and can be self-limiting, in recent years novel coronaviruses such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have evolved and infected humans, causing severe illness, epidemics and pandemics [2] . Currently, the majority of all infectious disease outbreaks as recorded by the World Health Organization (WHO) occur in the continent of Africa where there is high transmission risk [3, 4] . Further, in developing areas (both rural and urban), there are increasing risk factors such as human-animal interfaces (due to residential-proximity to livestock). These changing epidemiological patterns have resulted in calls for improved ARI surveillance, especially in places of high transmission risk [5] . Nigeria is one such place with high prevalence of many of the risk factors implicated in ARI among children including; age, sex, overcrowding, nutritional status, socio-economic status, and where study of ARIs is currently limited [6] . These broad risk factors alongside limited resources have indicated the need for community-based initiatives for surveillance and interventions [6, 7] . For ARI surveillance in particular, infections in the community are those that do not get reported clinically. Clinical data generally represents the most severe cases, and those from locations with access to healthcare institutions. In Nigeria, hospitals are visited only when symptoms are very severe. Thus, it is hypothesized that viral information from clinical sampling is insufficient to either capture disease incidence in general populations or its predictability from symptoms [8] . Efforts worldwide including in East and Southern Africa have been focused on developing community-based participatory disease surveillance methods [9] [10] [11] [12] [13] . Community-based approaches have been shown useful for learning more about emerging respiratory infections such as assessing under-reporting [14] , types of viruses prevalent in communities [10] , and prediction of epidemics [15] . Concurrently, advancements in molecular identification methods have enabled studies regarding the emergence and epidemiology of ARI viruses in many locations (e.g. novel polyomaviruses in Australia [16, 17] , human coronavirus Erasmus Medical Center (HCoV-EMC) in the Middle East and United Kingdom [18, 19] , SARS in Canada and China [20] [21] [22] ), yet research regarding the molecular epidemiology of ARI viruses in Nigeria is limited. Diagnostic methods available and other constraints have limited studies there to serological surveys of only a few of these viruses and only in clinical populations [23, 24] . Thus, the utility of community-based surveillance may be appropriate in contexts such as in Nigeria, and the purpose of this pilot study was to investigate if clinical cases may describe the entire picture of ARI among children in Nigeria. We performed a cross-sectional study in three community centers and one clinical in Ilorin, Nigeria. Ilorin is in Kwara state and is the 6th largest city in Nigeria by population [25] . Three Local Government Areas (Ilorin East, Ilorin South and Ilorin West LGAs) were the community sites and Children's Specialist Hospital, Ilorin the clinical site. Convenience sampling was used for the purposes of this pilot study, and samples were obtained from March 28 to April 5 2017. Inclusion criteria were: children less than 14 years old who had visible symptoms of ARI within the communities or those confirmed at the hospital with ARI. Exclusion criteria were: children who were 14 and above, not showing signs of ARI and subjects whose parents did not give consent. Twenty-five children with symptoms were selected each from the three community locations while 16 symptomatic children were sampled from the hospital. The total sample size (n = 91) was arrived at based on materials and processing cost constraints, as well as to provide enough samples to enable descriptive understanding of viral circulation patterns estimated from other community-based studies [10] . Disease Surveillance and Notification Officers, who are employed by the State Ministry of Health and familiar with the communities in this study, performed specimen and data collection. Symptoms considered were derived in accordance with other ARI surveillance efforts: sore throat, fever, couch, running nose, vomiting, body ache, leg pain, nausea, chills, shortness of breath [10, 26] . Gender and age, type of residential area (rural/urban), education level, proximity of residence to livestock, proximity to an untarred road and number of people who sleep in same room, were all recorded. The general difference between the two settings was that those from the hospital had severe illnesses, while those from the community were generally "healthy" but exhibiting ARI symptoms (i.e. mild illness). Nasal swabs were collected from the subjects and stored in DNA/RNA shield (Zymo Research, Irvine, California). Collected samples were spinned and the swab removed. Residues containing the nasal samples were stored at -20 °C prior to molecular analysis. Viral RNA was isolated using ZR Viral RNA ™ Kit (Zymo Research, Irvine, California) per manufacturer instructions (http://www.zymoresearch.com/downloads/dl/file/ id/147/r1034i.pdf ). Real-time PCR (polymerase chain reaction), commonly used in ARI studies [10, 19, 27] , was then carried out using RV15 One Step ACE Detection Kit, catalogue numbers RV0716K01008007 and RV0717B01008001 (Seegene, Seoul, South Korea) for detection of 15 human viruses: parainfluenza virus 1, 2, 3 and 4 (PIV1-4), respiratory syncytial virus (RSV) A and B, influenza A and B (FLUA, FLUB), rhinovirus type A-C, adenovirus (ADV), coronavirus (OC 229 E/NL63, OC43), enterovirus (HEV), metapneumovirus (hMPV) and bocavirus (BoV). Reagents were validated in the experimental location using an inbuilt validation protocol to confirm issues of false negative and false positive results were not of concern. Amplification reaction was carried out as described by the manufacturer: reverse transcription 50 °C-30′, initial activation 94°-15′, 45 cycles: denaturation 94°-30″, annealing 60°-1′ 30″, extension 72°-1, final extension 72°-10′, hold 4°. Visualization was performed using electrophoresis on a 2% agarose gel in TBE 1X with EtBr, in presence of RV15 OneStep A/B/C Markers; molecular weight marker. Specimen processing was not blinded as there was no risk of experimental bias. Standardized procedures were used for community and clinic sampling. All statistical analyses were performed using R version 3.2.4. Univariate statistics [mean and 95% confidence interval (CI)] are described. Bivariate statistics (difference in proportions) were assessed using a two-proportion z-test. A p value < 0.001 was considered significant. No observations used in this study had any missing data for analyses in this study. Basic participant demographics are summarized in PCR results showed that ten different viruses (influenza A, coronavirus OC 229 E/NL63, RSVA, RSV B, parainfluenza 1-4) were detected. Figure 1 shows how these infections were distributed across virus types as well as in the community versus clinic samples. In sum, a total of 33 of the 91 subjects surveyed had one or more respiratory tract virus (36.3%, 95% CI 26.6-47.0%, Fig. 1 ). Furthermore, 10 of those cases were triple infections and 5 were quadruple infections (illustrated by color of bars in Fig. 1 ). Figure 2 indicates how frequently each pair of viruses were found in the same participant; co-infections were most common among enterovirus and parainfluenza virus 4 (Fig. 2) . We also compared and contrasted the clinical and community results. Parainfluenza virus 4, respiratory syncytial virus B and enterovirus were the most common viruses found in the clinical sample. These three infections resulted in 41 viruses detected in 15 subjects clinically, and eight infections detected in five people in the community. Together they infected 94% (15/16, 95% CI 67.7-99.7%) of clinical subjects, and 7% (5/75, 95% CI 2.5-15.5%) in the community (significant difference, p < 0.001). The most common virus detected in community samples was Coronavirus OC43; this virus was detected in 13.3% (95% CI 6.9-23.6%) people in the community and not in any of the clinical samples. However a different strain, coronavirus OC 229 E/NL63 was detected in 12.5% of the clinical subjects (2/16, 95% CI 2.2-39.6%) and not detected in the community. Double, triple and quadruple infections were another common feature of note. We identified ten different respiratory tract viruses among the subjects as shown in Fig. 1 . Samples collected from the Children's specialist hospital showed 100% prevalence rate of infection with one or more viruses. This might not be surprising, as the basic difference between the community and clinic samples was an increased severity of illness in the clinical sample. This may also explain the high level of co-infection found among the clinical subjects. The most prevalent virus in the clinical sample (coronavirus OC43) was not detected in the community sample. Further, there was a significant difference between prevalence of the most common viruses in the clinical sample (parainfluenza virus 4, respiratory syncytial virus B and enterovirus) and their prevalence in the community. Finally, some of the viruses detected in this study have not been detected and implicated with ARIs in Nigeria. There is no report, to the best of our knowledge, implicating coronavirus in ARIs in Nigeria, and it was detected in 12 subjects in this study. Although cases of double and triple infections were observed in a study in Nigeria in 2011 [28] , as far as we are aware, reports of quadruple infections are rare and have not been reported in Nigeria previously. Due to the unique nature of the data generated in this study and novelty of work in the setting, it is not possible to exactly compare results to other studies. For example, though we found a similar study regarding ARIs in clinical subjects in Burkina Faso [27] , due to the small sample size from this study it would not be feasible to infer or compare prevalence rates. Studies of ARI etiology have mostly been generally focused in areas of the world that are more developed [29] , and it is important to note that the availability of molecular diagnostic methods as employed in this study substantially improve the ability to detect viruses which hitherto have not been detected in Nigeria. Further, findings from this work also add to the growing body of research that shows value of community-data in infectious disease surveillance [8] . As most of the work to-date has been in higher resource areas of the world this study adds perspective from an area where healthcare resources are lower. In conclusion, results of this study provide evidence for active community surveillance to enhance public health surveillance and scientific understanding of ARIs. This is not only because a minority of children with severe infection are admitted to the hospital in areas such this in Nigeria, but also findings from this pilot study which indicate that viral circulation in the community may not get detected clinically [29] . This pilot study indicates that in areas of Nigeria, etiology of ARIs ascertained from clinical samples may not represent all of the ARIs circulating in the community. The main limitation of the study is the sample size. In particular, the sample is not equally representative across all ages. However, the sample size was big enough to ascertain significant differences in community and clinic sourced viruses, and provides a qualitative understanding of viral etiology in samples from the community and clinic. Moreover, the sample was largely concentrated on subjects under 6 years, who are amongst the groups at highest risk of ARIs. Despite the small sample size, samples here indicate that circulation patterns in the community may differ from those in the clinic. In addition, this study resulted in unique findings Given that resources are limited for research and practice, we hope these pilot results may motivate further systematic investigations into how community-generated data can best be used in ARI surveillance. Results of this study can inform a larger study, representative across demographic and locations to systematically assess the etiology of infection and differences in clinical and community cohorts. A larger study will also enable accounting for potential confounders such as environmental risk factors. Finally, while it may be intuitive, findings from this pilot study shed light on the scope of differences in ARI patterns including different types and strains of circulating viruses. Also, because PCR was used for viral detection, the study was limited to detection of viruses in the primer sets. Given that these are the most up-to-date and common viruses, this approach was deemed sufficient for this initial investigation. The study was conceived by RC and OK. RC and OK, MO and TD were involved in the design of the study, which was conducted by MO and TD. RC and OK analyzed the data. RC and OK wrote and revised the manuscript. All authors read and approved the final manuscript.

What was the prevalence of Coronavirus OC 229 E/NL63 in clinical subjects in Ilorin, Nigeria?

Frontiers in antiviral therapy and immunotherapy https://doi.org/10.1002/cti2.1115 SHA: facbfdfa7189ca9ff83dc30e5d241ab22e962dbf Authors: Heaton, Steven M Date: 2020 DOI: 10.1002/cti2.1115 License: cc-by Abstract: nan Text: Globally, recent decades have witnessed a growing disjunction, a 'Valley of Death' 1,2 no less, between broadening strides in fundamental biomedical research and their incommensurate reach into the clinic. Plumbing work on research funding and development pipelines through recent changes in the structure of government funding, 2 new public and private joint ventures and specialist undergraduate and postgraduate courses now aim to incorporate pathways to translation at the earliest stages. Reflecting this shift, the number of biomedical research publications targeting 'translational' concepts has increased exponentially, up 1800% between 2003 and 2014 3 and continuing to rise rapidly up to the present day. Fuelled by the availability of new research technologies, as well as changing disease, cost and other pressing issues of our time, further growth in this exciting space will undoubtedly continue. Despite recent advances in the therapeutic control of immune function and viral infection, current therapies are often challenging to develop, expensive to deploy and readily select for resistance-conferring mutants. Shaped by the hostvirus immunological 'arms race' and tempered in the forge of deep time, the biodiversity of our world is increasingly being harnessed for new biotechnologies and therapeutics. Simultaneously, a shift towards host-oriented antiviral therapies is currently underway. In this Clinical & Translational Immunology Special Feature, I illustrate a strategic vision integrating these themes to create new, effective, economical and robust antiviral therapies and immunotherapies, with both the realities and the opportunities afforded to researchers working in our changing world squarely in mind. Opening this CTI Special Feature, I outline ways these issues may be solved by creatively leveraging the so-called 'strengths' of viruses. Viral RNA polymerisation and reverse transcription enable resistance to treatment by conferring extraordinary genetic diversity. However, these exact processes ultimately restrict viral infectivity by strongly limiting virus genome sizes and their incorporation of new information. I coin this evolutionary dilemma the 'information economy paradox'. Many viruses attempt to resolve this by manipulating multifunctional or multitasking host cell proteins (MMHPs), thereby maximising host subversion and viral infectivity at minimal informational cost. 4 I argue this exposes an 'Achilles Heel' that may be safely targeted via host-oriented therapies to impose devastating informational and fitness barriers on escape mutant selection. Furthermore, since MMHPs are often conserved targets within and between virus families, MMHP-targeting therapies may exhibit both robust and broadspectrum antiviral efficacy. Achieving this through drug repurposing will break the vicious cycle of escalating therapeutic development costs and trivial escape mutant selection, both quickly and in multiple places. I also discuss alternative posttranslational and RNA-based antiviral approaches, designer vaccines, immunotherapy and the emerging field of neo-virology. 4 I anticipate international efforts in these areas over the coming decade will enable the tapping of useful new biological functions and processes, methods for controlling infection, and the deployment of symbiotic or subclinical viruses in new therapies and biotechnologies that are so crucially needed. Upon infection, pathogens stimulate expression of numerous host inflammatory factors that support recruitment and activation of immune cells. On the flip side, this same process also causes immunopathology when prolonged or deregulated. 5 In their contribution to this Special Feature, Yoshinaga and Takeuchi review endogenous RNA-binding proteins (RBPs) that post-transcriptionally control expression of crucial inflammatory factors in various tissues and their potential therapeutic applications. 6 These RBPs include tristetraprolin and AUF1, which promote degradation of AU-rich element (ARE)-containing mRNA; members of the Roquin and Regnase families, which respectively promote or effect degradation of mRNAs harbouring stem-loop structures; and the increasingly apparent role of the RNA methylation machinery in controlling inflammatory mRNA stability. These activities take place in various subcellular compartments and are differentially regulated during infection. In this way, mRNA-destabilising RBPs constitute a 'brake' on the immune system, which may ultimately be toggled therapeutically. I anticipate continued efforts in this area will lead to new methods of regaining control over inflammation in autoimmunity, selectively enhancing immunity in immunotherapy, and modulating RNA synthesis and virus replication during infection. Another mRNA under post-transcriptional regulation by Regnase-1 and Roquin is Furin, which encodes a conserved proprotein convertase crucial in human health and disease. Furin, along with other PCSK family members, is widely implicated in immune regulation, cancer and the entry, maturation or release of a broad array of evolutionarily diverse viruses including human papillomavirus (HPV), influenza (IAV), Ebola (EboV), dengue (DenV) and human immunodeficiency virus (HIV). Here, Braun and Sauter review the roles of furin in these processes, as well as the history and future of furin-targeting therapeutics. 7 They also discuss their recent work revealing how two IFN-cinducible factors exhibit broad-spectrum inhibition of IAV, measles (MV), zika (ZikV) and HIV by suppressing furin activity. 8 Over the coming decade, I expect to see an ever-finer spatiotemporal resolution of host-oriented therapies to achieve safe, effective and broad-spectrum yet costeffective therapies for clinical use. The increasing abundance of affordable, sensitive, high-throughput genome sequencing technologies has led to a recent boom in metagenomics and the cataloguing of the microbiome of our world. The MinION nanopore sequencer is one of the latest innovations in this space, enabling direct sequencing in a miniature form factor with only minimal sample preparation and a consumer-grade laptop computer. Nakagawa and colleagues here report on their latest experiments using this system, further improving its performance for use in resource-poor contexts for meningitis diagnoses. 9 While direct sequencing of viral genomic RNA is challenging, this system was recently used to directly sequence an RNA virus genome (IAV) for the first time. 10 I anticipate further improvements in the performance of such devices over the coming decade will transform virus surveillance efforts, the importance of which was underscored by the recent EboV and novel coronavirus (nCoV / COVID-19) outbreaks, enabling rapid deployment of antiviral treatments that take resistance-conferring mutations into account. Decades of basic immunology research have provided a near-complete picture of the main armaments in the human antiviral arsenal. Nevertheless, this focus on mammalian defences and pathologies has sidelined examination of the types and roles of viruses and antiviral defences that exist throughout our biosphere. One case in point is the CRISPR/Cas antiviral immune system of prokaryotes, which is now repurposed as a revolutionary gene-editing biotechnology in plants and animals. 11 Another is the ancient lineage of nucleocytosolic large DNA viruses (NCLDVs), which are emerging human pathogens that possess enormous genomes of up to several megabases in size encoding hundreds of proteins with unique and unknown functions. 12 Moreover, hundreds of human-and avian-infective viruses such as IAV strain H5N1 are known, but recent efforts indicate the true number may be in the millions and many harbour zoonotic potential. 13 It is increasingly clear that host-virus interactions have generated truly vast yet poorly understood and untapped biodiversity. Closing this Special Feature, Watanabe and Kawaoka elaborate on neo-virology, an emerging field engaged in cataloguing and characterising this biodiversity through a global consortium. 14 I predict these efforts will unlock a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution. When biomedical innovations fall into the 'Valley of Death', patients who are therefore not reached all too often fall with them. Being entrusted with the resources and expectation to conceive, deliver and communicate dividends to society is both cherished and eagerly pursued at every stage of our careers. Nevertheless, the road to research translation is winding and is built on a foundation of basic research. Supporting industry-academia collaboration and nurturing talent and skills in the Indo-Pacific region are two of the four pillars of the National Innovation and Science Agenda. 2 These frame Australia's Medical Research and Innovation Priorities, which include antimicrobial resistance, global health and health security, drug repurposing and translational research infrastructure, 15 capturing many of the key elements of this CTI Special Feature. Establishing durable international relationships that integrate diverse expertise is essential to delivering these outcomes. To this end, NHMRC has recently taken steps under the International Engagement Strategy 16 to increase cooperation with its counterparts overseas. These include the Japan Agency for Medical Research and Development (AMED), tasked with translating the biomedical research output of that country. Given the reciprocal efforts at accelerating bilateral engagement currently underway, 17 the prospects for new areas of international cooperation and mobility have never been more exciting nor urgent. With the above in mind, all contributions to this CTI Special Feature I have selected from research presented by fellow invitees to the 2018 Awaji International Forum on Infection and Immunity (AIFII) and 2017 Consortium of Biological Sciences (ConBio) conferences in Japan. Both Australia and Japan have strong traditions in immunology and related disciplines, and I predict that the quantity, quality and importance of our bilateral cooperation will accelerate rapidly over the short to medium term. By expanding and cooperatively leveraging our respective research strengths, our efforts may yet solve the many pressing disease, cost and other sustainability issues of our time.

What is the Japan AMED tasked with?

Optimization Method for Forecasting Confirmed Cases of COVID-19 in China https://doi.org/10.3390/jcm9030674 SHA: 1d7f8850c5244fdc9b387038e7eeae9bcbbde6d2 Authors: Al-Qaness, Mohammed A. A.; Ewees, Ahmed A.; Fan, Hong; Abd El Aziz, Mohamed Date: 2020 DOI: 10.3390/jcm9030674 License: cc-by Abstract: In December 2019, a novel coronavirus, called COVID-19, was discovered in Wuhan, China, and has spread to different cities in China as well as to 24 other countries. The number of confirmed cases is increasing daily and reached 34,598 on 8 February 2020. In the current study, we present a new forecasting model to estimate and forecast the number of confirmed cases of COVID-19 in the upcoming ten days based on the previously confirmed cases recorded in China. The proposed model is an improved adaptive neuro-fuzzy inference system (ANFIS) using an enhanced flower pollination algorithm (FPA) by using the salp swarm algorithm (SSA). In general, SSA is employed to improve FPA to avoid its drawbacks (i.e., getting trapped at the local optima). The main idea of the proposed model, called FPASSA-ANFIS, is to improve the performance of ANFIS by determining the parameters of ANFIS using FPASSA. The FPASSA-ANFIS model is evaluated using the World Health Organization (WHO) official data of the outbreak of the COVID-19 to forecast the confirmed cases of the upcoming ten days. More so, the FPASSA-ANFIS model is compared to several existing models, and it showed better performance in terms of Mean Absolute Percentage Error (MAPE), Root Mean Squared Relative Error (RMSRE), Root Mean Squared Relative Error (RMSRE), coefficient of determination ( R 2 ), and computing time. Furthermore, we tested the proposed model using two different datasets of weekly influenza confirmed cases in two countries, namely the USA and China. The outcomes also showed good performances. Text: A large family of viruses, called coronaviruses, are severe pathogens for human beings, which infect respiratory, hepatic, gastrointestinal, and neurologic diseases. They are distributed among humans, birds, livestock, mice, bats, and other wild animals [1] [2] [3] . The outbreaks of two previous coronaviruses, SARS-CoV and MERS-CoV in 2003 and 2012, respectively, have approved the transmission from animal to animal, and human to human [4] . In December 2019, the World Health Organization (WHO) received notifications from China for many cases of respiratory illness that were linked to some people who had visited a seafood market in Wuhan [5] . Currently, Wuhan city suffers from the spreading of a novel coronavirus, called COVID-19 (previously, it was called 2019-nCoV). In [6] , the authors concluded that COVID-19 likely originated in bats, because it is more similar to two bat-derived coronavirus strains. However, the source of the COVID-19 is not confirmed yet, and it communities, Hong Kong and Toronto, were 1.2 and 1.32, respectively. Ong et al. [20] proposed a monitoring and forecasting model for influenza A (H1N1-2009). Furthermore, Nah et al. [21] proposed a probability-based model to predict the spread of the MERS. The Adaptive Neuro-Fuzzy Inference System (ANFIS) [22] is widely applied in time series prediction and forecasting problems, and it showed good performance in many existing applications. It offers flexibility in determining nonlinearity in the time series data, as well as combining the properties of both artificial neural networks (ANN) and fuzzy logic systems. It has been applied in various forecasting applications, for example, in [23] , a stock price forecasting model was proposed using ANFIS and empirical mode decomposition. Chen et al. [24] proposed a TAIEX time series forecasting model based on a hybrid of ANFIS and ordered weighted averaging (OWA). In [25] , another time series forecasting method was presented for electricity prices based on ANFIS. Svalina et al. [26] proposed an ANFIS based forecasting model for close price indices for a stock market for five days. Ekici and Aksoy [27] presented an ANFIS based building energy consumption forecasting model. More so, ANFIS is also applied to forecast electricity loads [28] . Kumar et al. [29] proposed an ANFIS based model to forecast return products. Ho and Tsai [30] applied ANFIS to forecast product development performance. However, estimating ANFIS parameters is a challenge that needs to be improved. Therefore, in previous studies, some individual swarm intelligence (SI) methods have been applied to the ANFIS parameters to enhance time series forecasting because these parameters have a significant effect on the performance of ANFIS. The SI methods include the particle swarm optimization (PSO) [31, 32] , social-spider optimization [33] , sine-cosine algorithm (SCA) [34] , and multi-verse optimizer (MVO) [35] . For example, in [34] SCA algorithm was applied to improve the ANFIS model to forecast oil consumption in three countries, namely, Canada, Germany, and Japan. In the same context, in [35] , The MVO algorithm was used to enhance the ANFIS model to forecast oil consumption in two countries. In addition, in [36] the PSO was used with ANFIS to predict biochar yield. However, individual SI algorithms may stock at local optima. Therefore, one solution is to apply hybrid SI algorithms to avoid this problem. In [37] , a hybrid of two SI algorithms, namely GA and SSA, was presented to improve the ANFIS model. The proposed new model called GA-SSA-ANFIS was applied to forecast crude oil prices for long-term time series data. However, the previously mentioned methods suffer from some limitations that can affect the performance of the forecasting output such as slow convergence and the ability to balance between exploration and exploitation phases can influence the quality of the final output. This motivated us to propose an alternative forecasting method dependent on the hybridization concept. This concept avoids the limitations of traditional SI techniques by combining the strengths of different techniques, and this produces new SI techniques that are better than traditional ones. In the current study, we propose an improved ANFIS model based on a modified flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). The FPA is an optimization algorithm proposed by Yang [38] , which was inspired by the flow pollination process of the flowering plants. The FPA was employed in various optimization applications, for example to estimate solar PV parameter [39, 40] , solving sudoku puzzles [41] , feature selection [42] , antenna design [43] , and other applications [44] [45] [46] [47] . Moreover, SSA is also an optimization algorithm proposed by Mirjalili et al. [48] inspired by the behavior of salp chains. In recent years, the SSA was utilized to solve different optimization problems, such as feature selection [49, 50] , data classification [51] , image segmentation [52] , and others [53, 54] . The proposed method called FPASSA is a hybrid of FPA and SSA, in which the SSA is applied as a local search method for FPA. The proposed FPASSA starts by receiving the historical COVID-19 dataset. Then a set of solutions is generated where each of them represents the value for the parameters of the ANFIS model. Then the quality of each solution is calculated using the fitness value, and the solution that has the best fitness value is chosen to represent the best solution. Then the probability of each solution is computed. Then the current solution will be updated, either using global or local strategy in FPA. However, in the case of local strategy, the operators of SSA or FPA will be used according to the probability of the fitness value for each solution. The process of updating the solutions is repeated until reaching the stop condition, and the best parameter configurations are used to forecast the number of confirmed cases of COVID-19. The main contribution points of the current study are as follows: 1. We propose an efficient forecasting model to forecast the confirmed cases of the COVID-19 in China for the upcoming ten days based on previously confirmed cases. An improved ANFIS model is proposed using a modified FPA algorithm, using SSA. We compare the proposed model with the original ANFIS and existing modified ANFIS models, such as PSO, GA, ABC, and FPA. The rest of this study is organized as follows. The preliminaries of ANFIS, FPA, and SSA are described in Section 2. Section 3 presents the proposed FPASSA, and Section 4 presents the experimental setup and results. We conclude this study in Section 5. The principles of the ANFIS are given in this section. The ANFIS model links the fuzzy logic and neural networks [22] . It generates a mapping between the input and output by applying IF-THEN rules (it is also called Takagi-Sugeno inference model). Figure 1 illustrates the ANFIS model where, y and x define the inputs to Layer 1 whereas, O 1i is its output of node i that is computed as follows: where µ denotes the generalized Gaussian membership functions. A i and B i define the membership values of µ. α i and ρ i denote the premise parameters set. The output of Layer 2 (it is also known as the firing strength of a rule) is calculated as follows: Meanwhile, the output of Layer 3 (it is also known as the normalized firing strength) is calculated as follows: The output of Layer 4 (it is also known as an adaptive node) is calculated as follows: where r i , q i , and p i define the consequent parameters of the node i. Layer 5 contains only one node; its output is computed as: Flower Pollination Algorithm is an optimization method proposed by Yang [38] . It simulates the transfer of flowers' pollen by pollinators in nature. This algorithm utilizes the two types of pollination (i.e., self-pollination and cross-pollination). In self-pollination, the pollination occurs with no pollinators, whereas, in cross-pollination, the pollens are moved between different plants. In more detail, the self-pollination can be represented as a local pollination while the cross-pollination can be called global pollination. The global pollination or cross-pollination can be mathematically formed as follows: where x t i defines the pollen i at iteration t. L denotes the pollination's strength or the step size. F * is the target position or best solution. In some cases, insects can fly with different distance steps for a long space; therefore, Levy fly distribution is applied to simulate this movement. where λ = 1.5. Γ(λ) denotes the gamma function. This distribution is available for large steps s > 0. The self-pollination or local pollination can be mathematically formed as follows: where x t i and x k i represent pollens from different flower in the same plant. in the range [0,1] The process of pollination can be done using cross-pollination or self-pollination. Therefore, the random variable p, in the range [0, 1], is used to determine this process. SSA is an optimization technique introduced by [48] . It simulates the Salps' behavior in nature. This behavior is called salp chain. The mathematical model of SSA begins by splinting its population into a leader group and followers group. The leader is the front salp, whereas, the followers are the other salps. The search space is determined in n-dimensions with n variables. Equation (10) works to update the salps' positions. where x 1 j denotes the leader's position in j-th dimension. F j is the target position. ub j and lb j represent the max and min bounds, respectively. c 2 and c 3 denote random numbers in [0, 1]. c 1 is an important parameter; it balances between the exploration and exploitation phases. It is computed as follows: where the current loop number is t and the max loop' number is t max . Then, the followers' position is updated as follows: where x i j defines the i-th position of the follower in j-th dimension. i > 1. This section explains the proposed FPASSA-ANFIS method. It is a time series method for forecasting the confirmed cases of the COVID-19, as given in Figure 2 . The FPASSA-ANFIS utilizes the improved FPA to train the ANFIS model by optimizing its parameters. The FPASSA-ANFIS contains five layers as the classic ANFIS model. Layer 1 contains the input variables (the historical COVID-19 confirmed cases). Whereas Layer 5 produces the forecasted values. In the learning phase, the FPASSA is used to select the best weights between Layer 4 and Layer 5. The FPASSA-ANFIS starts by formatting the input data in a time series form. In our case, the autocorrelation function (ACF) was considered. ACF is one of the methods applied to find patterns in the data; it presents information about the correlation between points separated by various time lags. Therefore, in this paper, the variables with ACF greater than 0.2 are considered i.e., 5-lags. Besides, the training data contains 75% of the dataset, whereas the testing data contains 25% of them. The number of clusters is defined by the fuzzy c-mean (FCM) method to construct the ANFIS model. The parameters of the ANFIS model are prepared by the FPASSA algorithm. In the training phase, the calculation error (as in Equation (13)) between the real data and the predicted data is used to evaluate the parameters' quality. where T is the real data, and P is the predicted data. N s is the sample length. The smaller values of the objective function indicate good ANFIS's parameter. On the other hand, the updating phase of the followers' positions in the SSA algorithm is applied to improve the global pollination phase in the FPA algorithm. In this improvement, there is a random variable (r) used to switch between both phases. If r > 0.5, then the operators of the SSA is used; otherwise, the operators of the FPA are used. In general, The FPASSA starts by constructing the population (X); afterward, the objective function is calculated for each solution. The solution with the lowest error value is saved to the next iteration. This sequence is repeated until meeting the stop condition, which in this paper, is the maximum number of iterations. Then the best solution is passed to train the parameters of the ANFIS model. After finishing the training phase, the testing phase is started with the best solution to compute the final output. The performance of the proposed method is evaluated by comparing the real data with the predicted data using the performance measures. Finally, the FPASSA produces a foretasted value for confirmed cases of COVID-19 in China in the next day. The steps of the proposed FPASSA are presented in Algorithm 1. Input: Historical COVID-19 dataset, size of population N, total number of iterations t max . Divide the data into training and testing sets. Using Fuzzy c-mean method to determine the number of membership functions. Constructing the ANFIS network. Set the initial value for N solutions (X). Return the best solution that represents the best configuration for ANFIS. Apply the testing set to the best ANFIS model. Forecasting the COVID-19 for the next ten days. This section presents the description of the used dataset, the performance measures, the parameter setting for all methods, the experiment results, and discussions. The main dataset of this study is COVID-19 dataset. It was collected from the WHO website (https: //www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). It contains the daily confirmed cases in China from 21 January 2020 to 18 February 2020, as shown in Table 1 . We used 75% from the dataset to train the model while the rest is used to test it. Moreover, we evaluated the performance of the proposed method using two datasets of weekly influenza confirmed cases. The first one is called DS1; it was collected from the Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/flu/weekly/). It starts from week number 40 in 2015 and continues until week number 6 in 2020. Whereas, the second one is called DS2. It was collected from the WHO website (https://www.who.int/influenza). It contains the data of weekly influenza confirmed cases in China from week number 1 in 2016 to week number 8 in 2020. The quality of the proposed method is evaluated using a set of performance metrics as follows: • Root Mean Square Error (RMSE): where Yp and Y are the predicted and original values, respectively. • Mean Absolute Error (MAE): • Mean Absolute Percentage Error (MAPE): • Root Mean Squared Relative Error (RMSRE): N s represents the sample size of the data. • Coefficient of Determination (R 2 ): where Y represents the average of Y. The lowest value of RMSE, MAE, MAPE, and RMSRE refers to the best method. The higher value of R 2 indicates better correlation for the method. This paper aims to assess the ability of the FPASSA to forecast the COVID-19 by comparing its performance with other methods, namely the ANFIS and the trained ANFIS models using PSO, GA, ABC, FPA, and FPASSA. The parameters' setting for these models is listed in Table 2 . The common parameters, such as population size, are set to 25 and 100 iterations are applied. Besides, each algorithm is performed for 30 independent runs to fair comparisons. The selected parameters are chosen because they produced good behavior in previous experiments, such as [34, 35, 55, 56] . Table 2 . Parameters' setting. Parameters Setting Max. epochs = 100, Error goal = 0, Initial step = 0.01, Decrease rate = 0.9, Increase rate = 1. In this section, the performance of the proposed FPASSA to predict the DS1 and DS2 is discussed. It can be concluded from Table 3 that the performance of FPASSA outperformed the compared methods in all measures, whereas the FPA is ranked second. The results of DS2 indicate that the FPASSA is ranked first in terms of RMSE, MAPE, R 2 , and the CPU time. Whereas, the PSO is ranked second, followed by the FPA, GA, then ABC. These results denote that the proposed method can optimize the parameters of the ANFIS model effectively and produce good results in terms of the performance measures. Comparison results between the proposed FPASSA and other models to forecast COVID-19 are given in Table 4 . It can be concluded that the FPASSA outperforms other models. For example, by analyzing the results of RMSE, MAE, MAPE, RMSRE, and CPU time(s) it can be observed that the FPASSA achieves the smallest value among the comparison algorithms, and this indicates the high quality of the FPASSA. Meanwhile, the FPA allocates the second rank, which provides better results than the rest of the methods. Moreover, the value of R 2 refers to the high correlation between the prediction obtained by the proposed FPASSA method and the original COVID-19, which has nearly 0.97. This can also be noticed from Figure 3 , which depicts the training of the algorithms using the historical data of the COVID-19 as well as their forecasting values for ten days. Table 5 depicts the forecasting value for the confirmed cases of the COVID-19 in China from 19/2/2020 to 28/2/2020. From these results, it can be noticed that the outbreak will reach its highest level on the day 28/2/2020. The average percentage of the increase over the forecasted period is 10%, the highest percentage is 12% on 28/2/2020, and the lowest percentage is 8.7% on 19/2/2020. From the previous results, it can be concluded that the proposed FPASSA-ANFIS has a high ability to forecast the COVID-19 dataset. These results avoid the limitations of traditional ANFIS because of the combination with the modified FPA method. Moreover, the operators of SSA are combined with the local strategy of FPA to enhance their exploitation ability. However, the time computational of the proposed FPASSA method still requires more improvements. This paper proposed a modified version for the flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). This modified version, called FPASSA, is applied to improve the performance of the ANFIS through determining the optimal value for its parameters. The developed FPASSA-ANFIS model is applied as a forecasting technique for a novel coronavirus, called COVID-19, that was discovered in Wuhan, China at the end of last year and January of the current year. The proposed FPASSA-ANFIS model has a high ability to predict the number of confirmed cases within ten days. Besides, FPASSA-ANFIS outperforms other forecasting models in terms of RMSE, MAE, MAPE, RMSRE, and R 2 . Furthermore, two datasets of weekly influenza confirmed cases in the USA and China were used to evaluate the proposed method, and the evaluation outcomes showed its good performance. According to the promising results obtained by the proposed FPASSA-ANFIS, it can be applied in different forecasting applications.

For what SCA algorithm was applied to improve the ANFIS model ?

First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164/ SHA: ce358c18aac69fc83c7b2e9a7dca4a43b0f60e2e Authors: Spiteri, Gianfranco; Fielding, James; Diercke, Michaela; Campese, Christine; Enouf, Vincent; Gaymard, Alexandre; Bella, Antonino; Sognamiglio, Paola; Sierra Moros, Maria José; Riutort, Antonio Nicolau; Demina, Yulia V.; Mahieu, Romain; Broas, Markku; Bengnér, Malin; Buda, Silke; Schilling, Julia; Filleul, Laurent; Lepoutre, Agnès; Saura, Christine; Mailles, Alexandra; Levy-Bruhl, Daniel; Coignard, Bruno; Bernard-Stoecklin, Sibylle; Behillil, Sylvie; van der Werf, Sylvie; Valette, Martine; Lina, Bruno; Riccardo, Flavia; Nicastri, Emanuele; Casas, Inmaculada; Larrauri, Amparo; Salom Castell, Magdalena; Pozo, Francisco; Maksyutov, Rinat A.; Martin, Charlotte; Van Ranst, Marc; Bossuyt, Nathalie; Siira, Lotta; Sane, Jussi; Tegmark-Wisell, Karin; Palmérus, Maria; Broberg, Eeva K.; Beauté, Julien; Jorgensen, Pernille; Bundle, Nick; Pereyaslov, Dmitriy; Adlhoch, Cornelia; Pukkila, Jukka; Pebody, Richard; Olsen, Sonja; Ciancio, Bruno Christian Date: 2020-03-05 DOI: 10.2807/1560-7917.es.2020.25.9.2000178 License: cc-by Abstract: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters’ index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. Text: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. A cluster of pneumonia of unknown origin was identified in Wuhan, China, in December 2019 [1] . On 12 January 2020, Chinese authorities shared the sequence of a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from some clustered cases [2] . Since then, the disease caused by SARS-CoV-2 has been named coronavirus disease 2019 (COVID -19) . As at 21 February 2020, the virus had spread rapidly mostly within China but also to 28 other countries, including in the World Health Organization (WHO) European Region [3] [4] [5] . Here we describe the epidemiology of the first cases of COVID-19 in this region, excluding cases reported in the United Kingdom (UK), as at 21 February 2020. The study includes a comparison between cases detected among travellers from China and cases whose infection was acquired due to subsequent local transmission. On 27 January 2020, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe asked countries to complete a WHO standard COVID-19 case report form for all confirmed and probable cases according to WHO criteria [6] [7] [8] . The overall aim of surveillance at this time was to support the global strategy of containment of COVID-19 with rapid identification and follow-up of cases linked to affected countries in order to minimise onward transmission. The surveillance objectives were to: describe the key epidemiological and clinical characteristics of COVID-19 cases detected in Europe; inform country preparedness; and improve further case detection and management. Data collected included demographics, history of recent travel to affected areas, close contact with a probable or confirmed COVID-19 case, underlying conditions, signs and symptoms of disease at onset, type of specimens from which the virus was detected, and clinical outcome. The WHO case definition was adopted for surveillance: a confirmed case was a person with laboratory confirmation of SARS-CoV-2 infection (ECDC recommended two separate SARS-CoV-2 RT-PCR tests), irrespective of clinical signs and symptoms, whereas a probable case was a suspect case for whom testing for SARS-CoV-2 was inconclusive or positive using a pan-coronavirus assay [8] . By 31 January 2020, 47 laboratories in 31 countries, including 38 laboratories in 24 European Union and European Economic Area (EU/EEA) countries, had diagnostic capability for SARS-CoV-2 available (close to 60% of countries in the WHO European Region), with cross-border shipment arrangements in place for many of those lacking domestic testing capacity. The remaining six EU/EEA countries were expected to have diagnostic testing available by mid-February [9] . As at 09:00 on 21 February 2020, 47 confirmed cases of COVID-19 were reported in the WHO European Region and one of these cases had died [4] . Data on 38 of these cases (i.e. all except the nine reported in the UK) are included in this analysis. The first three cases detected were reported in France on 24 January 2020 and had onset of symptoms on 17, 19 and 23 January respectively [10] . The first death was reported on 15 February in France. As at 21 February, nine countries had reported cases ( Figure) : Belgium (1), Finland (1), France (12), Germany (16), Italy (3), Russia (2), Spain (2), Sweden (1) and the UK (9 -not included further). The place of infection (assessed at national level based on an incubation period presumed to be up to 14 days [11] , travel history and contact with probable or confirmed cases as per the case definition) was reported for 35 cases (missing for three cases), of whom 14 were infected in China (Hubei province: 10 cases; Shandong province: one case; province not reported for three cases). The remaining 21 cases were infected in Europe. Of these, 14 were linked to a cluster in Bavaria, Germany, and seven to a cluster in Haute-Savoie, France [12, 13] . Cases from the Bavarian cluster were reported from Germany and Spain, whereas cases from the Haute-Savoie cluster were reported from France All but two cases were hospitalised (35 of 37 where information on hospitalisation was reported), although it is likely that most were hospitalised to isolate the person rather than because of severe disease. The time from onset of symptoms to hospitalisation (and isolation) ranged between 0 and 10 days with a mean of 3.7 days (reported for 29 cases). The mean number of days to hospitalisation was 2.5 days for cases imported from China, but 4.6 days for those infected in Europe. This was mostly a result of delays in identifying the index cases of the two clusters in France and Germany. In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six took only a mean of 2 days to be hospitalised. Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc. were reported for any of the 38 cases. All hospitalised cases had a benign clinical evolution except four, two reported in Italy and two reported in France, all of whom developed viral pneumonia. All three cases who were aged 65 years or over were admitted to intensive care and required respiratory support and one French case died. The case who died was hospitalised for 21 days and required intensive care and mechanical ventilation for 19 days. The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] . As at 5 March 2020, there are 4,250 cases including 113 deaths reported among 38 countries in the WHO European region [19] . In our analysis of early cases, we observed transmission in two broad contexts: sporadic cases among travellers from China (14 cases) and cases who acquired infection due to subsequent local transmission in Europe (21 cases). Our analysis shows that the time from symptom onset to hospitalisation/case isolation was about 3 days longer for locally acquired cases than for imported cases. People returning from affected areas are likely to have a low threshold to seek care and be tested when symptomatic, however delays in identifying the index cases of the two clusters in France and Germany meant that locally acquired cases took longer to be detected and isolated. Once the exposure is determined and contacts identified and quarantined (171 contacts in France and 200 in Germany for the clusters in Haute-Savoie and Bavaria, respectively), further cases are likely to be rapidly detected and isolated when they develop symptoms [15, 20] . In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six were hospitalised after a mean of 2 days. Locally acquired cases require significant resources for contact tracing and quarantine, and countries should be prepared to allocate considerable public health resources during the containment phase, should local clusters emerge in their population. In addition, prompt sharing of information on cases and contacts through international notification systems such as the International Health Regulations (IHR) mechanism and the European Commission's European Early Warning and Response System is essential to contain international spread of infection. All of the imported cases had a history of travel to China. This was consistent with the epidemiological situation in Asia, and supported the recommendation for testing of suspected cases with travel history to China and potentially other areas of presumed ongoing community transmission. The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic. Hospital-based surveillance could help estimate the incidence of severe cases and identify risk factors for severity and death. Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2. This approach will be used globally to help identify evidence of widespread community transmission and, should the virus spread and containment no longer be deemed feasible, to monitor intensity of disease transmission, trends and its geographical spread. Additional research is needed to complement surveillance data to build knowledge on the infectious period, modes of transmission, basic and effective reproduction numbers, and effectiveness of prevention and case management options also in settings outside of China. Such special studies are being conducted globally, including a cohort study on citizens repatriated from China to Europe, with the aim to extrapolate disease incidence and risk factors for infection in areas with community transmission. Countries together with ECDC and WHO, should use all opportunities to address these questions in a coordinated fashion at the European and global level. provided input to the outline, multiple versions of the manuscript and gave approval to the final draft.

What is the adopted WHO case definition?

Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521148/ SHA: ee14de143337eec0e9708f8139bfac2b7b8fdd27 Authors: Wang, Ning; Luo, Chuming; Liu, Haizhou; Yang, Xinglou; Hu, Ben; Zhang, Wei; Li, Bei; Zhu, Yan; Zhu, Guangjian; Shen, Xurui; Peng, Cheng; Shi, Zhengli Date: 2019-04-24 DOI: 10.3390/v11040379 License: cc-by Abstract: Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder (https://www.ncbi.nlm.nih.gov/ orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform (http://services.cbu.uib.no/tools/kaks) with default parameters [27] . The protein homology detection was analyzed using HHpred (https://toolkit.tuebingen.mpg.de/#/tools/hhpred) with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, www.atcc.org). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/4/379/s1, Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3

What plays a role in regulating the immune response to a viral infection?

Etiology of Influenza-Like Illnesses from Sentinel Network Practitioners in Réunion Island, 2011-2012 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031398/ SHA: f5ff89ebfdd0375d034c112c6c1c7e163fa69a0c Authors: Brottet, Elise; Jaffar-Bandjee, Marie-Christine; Li-Pat-Yuen, Ghislaine; Filleul, Laurent Date: 2016-09-21 DOI: 10.1371/journal.pone.0163377 License: cc-by Abstract: In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 Statistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time.

What could not be tested for?

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067204/ SHA: c097a8a9a543d69c34f10e5c3fd78019e560026a Authors: Chan, Jasper Fuk-Woo; Kok, Kin-Hang; Zhu, Zheng; Chu, Hin; To, Kelvin Kai-Wang; Yuan, Shuofeng; Yuen, Kwok-Yung Date: 2020-01-28 DOI: 10.1080/22221751.2020.1719902 License: cc-by Abstract: A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Deltacoronavirus (δCoV), and Gammacoronavirus (γCoV) [1] . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV (SARS-CoV) which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV (MERS-CoV) which has caused a persistent epidemic in the Arabian Peninsula since 2012 [2, 3] . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host [the Himalayan palm civet (Paguma larvata) for SARS-CoV and the dromedary camel (Camelus dromedarius) for MERS-CoV] before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV (HCoV-229E and HKU-NL63) and 4 βCoV (HCoV-OC43 [ HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly [4] . In contrast, SARS-CoV (lineage B βCoV) and MERS-CoV (lineage C βCoV) may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively [5, 6] . On 31 December 2019, the World Health Organization (WHO) was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China [7] . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan [8, 9] . The earliest date of symptom onset was 1 December 2019 [10] . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 (32%) required intensive care and 6 (15%) died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans [10] . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan [11] . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank (accession no. MN975262) ( Table 1 ). The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees [12] . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [13] . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site [14] . All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X [15] . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction (PSIPRED) [16] . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). Secondary structure prediction in the 5 ′ -untranslated region (UTR) and 3 ′ -UTR was performed using the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/ RNAWebSuite/RNAfold.cgi) with minimum free energy (MFE) and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other (Table 2 ). There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV (Table 3) . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain (NTD) and receptor-binding domain (RBD), while the S2 subunit contains conserved fusion peptide (FP), heptad repeat (HR) 1 and 2, transmembrane domain (TM), and cytoplasmic domain (CP). We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs (SL-CoV ZXC21 and ZC45) and human SARS-CoV (Figure 2 ). Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials [18] . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV (Figure 3(A) ), the core domain of RBD (excluding the external subdomain) are highly conserved (Figure 3(B) ). Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain (RBD) when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b ( Figure 4 ). It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types [19] , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points [20] . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling [21] . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV [22] . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 [23] . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients [24] . In addition, we have previously identified two bat SARS-related-CoV (Bat-CoV YNLF_31C and YNLF_34C) and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV [25] . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 (human SARS-CoV GZ02), the mid-and late-phase SARS-CoV that includes the split orf8b (human SARS-CoV Tor2), civet SARS-CoV (paguma SARS-CoV), two bat SARS-related-CoV containing full-length orf8 (bat-CoV YNLF_31C and YNLF_34C), 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45), and bat SARS-related-CoV HKU3-1 ( Figure 5(A) ). As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5(B) ) which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes [26] , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet(s) containing six strands ( Figure 5(C) ). The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 (MG772934.1), and 82% with human SARS-CoV BJ01 2003 (AY278488) and human SARS-CoV Tor2 (AY274119). The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes (S, E, M, and N) were shown (Figure 6(A-E) ). For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 (A-C), the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif [27] . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b (marked in red), which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR (Figure 7(D-F) ). The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs [27] . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV.

What do the S1 and S2 subunits of spike glycoprotein contain?

Exploring the Innate Immunological Response of an Alternative Nonhuman Primate Model of Infectious Disease; the Common Marmoset https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129158/ SHA: f4c43e4ae49ca69dbac32620bd0a73ecbb683b91 Authors: Nelson, M.; Loveday, M. Date: 2014-07-22 DOI: 10.1155/2014/913632 License: cc-by Abstract: The common marmoset (Callithrix jacchus) is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset (Callithrix jacchus), a New World monkey (NWM) species is a small, arboreal nonhuman primate (NHP), native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans [1] . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys (OWM) (e.g., rhesus and cynomolgus macaques). Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates [2] . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus [3] , Hepatitis C virus [4] , Dengue virus [5] , Herpesvirus [6] , Junin virus [7] Rift Valley Fever [8] , and SARS [9] . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus [10] , Bacillus anthracis [11] , Francisella tularensis [12, 13] , Burkholderia pseudomallei [14] , Marburg haemorrhagic fever virus [15, 16] , Ebola haemorrhagic fever virus [16] , and Variola virus [17] . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever [18] and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively [19, 20] . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man [21] . Immunologically, there is a high degree of homology between humans and marmosets [22] . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules (e.g., CD80, CD86 etc.) and those of marmosets [23] . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology [24, 25] . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood [26] [27] [28] . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models [5, 6, 8, 17] or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model [17] ; neutrophils and macrophages in the Herpes model [6] ; or CD3+ and CD20+ cells in the Lassa model [3] . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets (C. jacchus) were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions [12] . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 (SP34-2), CD8 (LT8), CD11c (SHCL3), CD14 (M5E2), CD16 (3G8), CD20 (Bly1), CD45RA (5H9), CD54 (HCD54), CD56 (B159), CD69 (FN50), CD163 (GHI/61), and MCHII (L243) (BD Bioscience, Insight Bioscience, AbD serotec). Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry (FACScanto II BD) within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially (BD cytokine flex beads and the Luminex system). These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 [29] . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD [13] . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response (e.g., neutrophils, macrophages, and NK cells) and the adaptive response (T and B cells) with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size (forward) and granularity (side) scatter (Figure 1(a) ). Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen (CD45) normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments (data not shown). Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin (Figure 1(b) ). Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets (Figure 2 (a), Table 1 ); approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 (a) with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets (Figures 2(b) and 2(c) ). Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue (8% both). Healthy mouse spleens typically have approximately 1-2% granulocytes [30] . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells [34] . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens [30] . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples [27] and information available for human blood [32, 33] (Table 1 ). However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively [35] compared to the 30% observed in this study and the work previously reported by Brok et al. [27] . Brok's study involved a small number of animals (eight) and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils [31] , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% (Table 1 ) [27, 31] . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection [36] . A cross-species comparison suggests that monocytes comprise 3% of leukocytes ( Table 1) . Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus [13] [14] [15] . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset (present data) Marmoset [27] Mouse 4 [30, 31] Human Asian [32] Human Caucasian [33] Number the lungs, spleen, and blood as the disease progressed (TNF and IFN were not measured in this study). Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus [15] . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time (TNF was not measured). In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells [37] [38] [39] [40] . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells [36, 41] . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs (e.g., NIH NHP reagent resource, http://www.nhpreagents.org/NHP/default.aspx), has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 (ICAM-1) associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II (HLA-DR). CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets [37, 42] . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation ( = 22) or by a systemic route ( = 12) and humanely killed once they had reached a humane endpoint (between day 4 and day 5 after challenge). Figure 3 illustrates the cellular activity in representative tissues following inhalational (Figures 3(b) and 3(e)) or systemic challenge (Figures 3(c) and 3(f)) and in naïve samples (Figures 3(a) and 3(d) ). Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the lung and spleen samples. All of the cytokines (with the exception of Rantes) were expressed at high levels (ng/mg) in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens.

What is the conclusion of the study?

Accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064339/ SHA: f2cc0d63ff2c4aaa127c4caae21d8f3a0067e3d5 Authors: Brook, Cara E; Boots, Mike; Chandran, Kartik; Dobson, Andrew P; Drosten, Christian; Graham, Andrea L; Grenfell, Bryan T; Müller, Marcel A; Ng, Melinda; Wang, Lin-Fa; van Leeuwen, Anieke Date: 2020-02-03 DOI: 10.7554/elife.48401 License: cc-by Abstract: Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations: We defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of " > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and "; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of ": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2): Pathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model: At equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of " that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of ", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + ") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and " were fixed at 0 while b was estimated; under induced immune assumptions, " was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, ", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition ("). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, ", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with ") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of ", any parameter tests of " values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from " prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation: where P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium: Because and " both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and ", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and " derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for ", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, ". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 To derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and " by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and " across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and " (" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (" = r = 0) version of the theoretical model and, subsequently, an induced immunity (" = 0; r >0) and constitutive immunity (" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and ") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and " by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare

In non-flying mammals, what what would be elicited by IFN expression upon viral infection?

Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/ SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A. Date: 2013-08-05 DOI: 10.1371/journal.pone.0069982 License: cc-by Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a). Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms. The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] . Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] . Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents. The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform. The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools. N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads. Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1- The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation, Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain. For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day. After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times. Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] . Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain. Type of inhibition Ki (mM) IC 50 (mM) Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases. These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d). The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases. The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors. In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy. Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally. File S1 Types of inhibitions with Ki (Compounds 3a-3d). (DOC)

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.

What represses murine hepatitis virus strain 3 (MHV-3) infection?

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.

What is a function associated with IFITM5?

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

How was the ELISA-array assay validated?

Estimating the number of infections and the impact of non- pharmaceutical interventions on COVID-19 in 11 European countries 30 March 2020 Imperial College COVID-19 Response Team Seth Flaxmani Swapnil Mishra*, Axel Gandy*, H JulietteT Unwin, Helen Coupland, Thomas A Mellan, Harrison Zhu, Tresnia Berah, Jeffrey W Eaton, Pablo N P Guzman, Nora Schmit, Lucia Cilloni, Kylie E C Ainslie, Marc Baguelin, Isobel Blake, Adhiratha Boonyasiri, Olivia Boyd, Lorenzo Cattarino, Constanze Ciavarella, Laura Cooper, Zulma Cucunuba’, Gina Cuomo—Dannenburg, Amy Dighe, Bimandra Djaafara, Ilaria Dorigatti, Sabine van Elsland, Rich FitzJohn, Han Fu, Katy Gaythorpe, Lily Geidelberg, Nicholas Grassly, Wi|| Green, Timothy Hallett, Arran Hamlet, Wes Hinsley, Ben Jeffrey, David Jorgensen, Edward Knock, Daniel Laydon, Gemma Nedjati—Gilani, Pierre Nouvellet, Kris Parag, Igor Siveroni, Hayley Thompson, Robert Verity, Erik Volz, Caroline Walters, Haowei Wang, Yuanrong Wang, Oliver Watson, Peter Winskill, Xiaoyue Xi, Charles Whittaker, Patrick GT Walker, Azra Ghani, Christl A. Donnelly, Steven Riley, Lucy C Okell, Michaela A C Vollmer, NeilM.Ferguson1and Samir Bhatt*1 Department of Infectious Disease Epidemiology, Imperial College London Department of Mathematics, Imperial College London WHO Collaborating Centre for Infectious Disease Modelling MRC Centre for Global Infectious Disease Analysis Abdul LatifJameeI Institute for Disease and Emergency Analytics, Imperial College London Department of Statistics, University of Oxford *Contributed equally 1Correspondence: nei|.ferguson@imperial.ac.uk, s.bhatt@imperial.ac.uk Summary Following the emergence of a novel coronavirus (SARS-CoV-Z) and its spread outside of China, Europe is now experiencing large epidemics. In response, many European countries have implemented unprecedented non-pharmaceutical interventions including case isolation, the closure of schools and universities, banning of mass gatherings and/or public events, and most recently, widescale social distancing including local and national Iockdowns. In this report, we use a semi-mechanistic Bayesian hierarchical model to attempt to infer the impact of these interventions across 11 European countries. Our methods assume that changes in the reproductive number— a measure of transmission - are an immediate response to these interventions being implemented rather than broader gradual changes in behaviour. Our model estimates these changes by calculating backwards from the deaths observed over time to estimate transmission that occurred several weeks prior, allowing for the time lag between infection and death. One of the key assumptions of the model is that each intervention has the same effect on the reproduction number across countries and over time. This allows us to leverage a greater amount of data across Europe to estimate these effects. It also means that our results are driven strongly by the data from countries with more advanced epidemics, and earlier interventions, such as Italy and Spain. We find that the slowing growth in daily reported deaths in Italy is consistent with a significant impact of interventions implemented several weeks earlier. In Italy, we estimate that the effective reproduction number, Rt, dropped to close to 1 around the time of Iockdown (11th March), although with a high level of uncertainty. Overall, we estimate that countries have managed to reduce their reproduction number. Our estimates have wide credible intervals and contain 1 for countries that have implemented a|| interventions considered in our analysis. This means that the reproduction number may be above or below this value. With current interventions remaining in place to at least the end of March, we estimate that interventions across all 11 countries will have averted 59,000 deaths up to 31 March [95% credible interval 21,000-120,000]. Many more deaths will be averted through ensuring that interventions remain in place until transmission drops to low levels. We estimate that, across all 11 countries between 7 and 43 million individuals have been infected with SARS-CoV-Z up to 28th March, representing between 1.88% and 11.43% ofthe population. The proportion of the population infected to date — the attack rate - is estimated to be highest in Spain followed by Italy and lowest in Germany and Norway, reflecting the relative stages of the epidemics. Given the lag of 2-3 weeks between when transmission changes occur and when their impact can be observed in trends in mortality, for most of the countries considered here it remains too early to be certain that recent interventions have been effective. If interventions in countries at earlier stages of their epidemic, such as Germany or the UK, are more or less effective than they were in the countries with advanced epidemics, on which our estimates are largely based, or if interventions have improved or worsened over time, then our estimates of the reproduction number and deaths averted would change accordingly. It is therefore critical that the current interventions remain in place and trends in cases and deaths are closely monitored in the coming days and weeks to provide reassurance that transmission of SARS-Cov-Z is slowing. SUGGESTED CITATION Seth Flaxman, Swapnil Mishra, Axel Gandy et 0/. Estimating the number of infections and the impact of non— pharmaceutical interventions on COVID—19 in 11 European countries. Imperial College London (2020), doi: https://doi.org/10.25561/77731 1 Introduction Following the emergence of a novel coronavirus (SARS-CoV-Z) in Wuhan, China in December 2019 and its global spread, large epidemics of the disease, caused by the virus designated COVID-19, have emerged in Europe. In response to the rising numbers of cases and deaths, and to maintain the capacity of health systems to treat as many severe cases as possible, European countries, like those in other continents, have implemented or are in the process of implementing measures to control their epidemics. These large-scale non-pharmaceutical interventions vary between countries but include social distancing (such as banning large gatherings and advising individuals not to socialize outside their households), border closures, school closures, measures to isolate symptomatic individuals and their contacts, and large-scale lockdowns of populations with all but essential internal travel banned. Understanding firstly, whether these interventions are having the desired impact of controlling the epidemic and secondly, which interventions are necessary to maintain control, is critical given their large economic and social costs. The key aim ofthese interventions is to reduce the effective reproduction number, Rt, ofthe infection, a fundamental epidemiological quantity representing the average number of infections, at time t, per infected case over the course of their infection. Ith is maintained at less than 1, the incidence of new infections decreases, ultimately resulting in control of the epidemic. If Rt is greater than 1, then infections will increase (dependent on how much greater than 1 the reproduction number is) until the epidemic peaks and eventually declines due to acquisition of herd immunity. In China, strict movement restrictions and other measures including case isolation and quarantine began to be introduced from 23rd January, which achieved a downward trend in the number of confirmed new cases during February, resulting in zero new confirmed indigenous cases in Wuhan by March 19th. Studies have estimated how Rt changed during this time in different areas ofChina from around 2-4 during the uncontrolled epidemic down to below 1, with an estimated 7-9 fold decrease in the number of daily contacts per person.1'2 Control measures such as social distancing, intensive testing, and contact tracing in other countries such as Singapore and South Korea have successfully reduced case incidence in recent weeks, although there is a riskthe virus will spread again once control measures are relaxed.3'4 The epidemic began slightly laterin Europe, from January or later in different regions.5 Countries have implemented different combinations of control measures and the level of adherence to government recommendations on social distancing is likely to vary between countries, in part due to different levels of enforcement. Estimating reproduction numbers for SARS-CoV-Z presents challenges due to the high proportion of infections not detected by health systems”7 and regular changes in testing policies, resulting in different proportions of infections being detected over time and between countries. Most countries so far only have the capacity to test a small proportion of suspected cases and tests are reserved for severely ill patients or for high-risk groups (e.g. contacts of cases). Looking at case data, therefore, gives a systematically biased view of trends. An alternative way to estimate the course of the epidemic is to back-calculate infections from observed deaths. Reported deaths are likely to be more reliable, although the early focus of most surveillance systems on cases with reported travel histories to China may mean that some early deaths will have been missed. Whilst the recent trends in deaths will therefore be informative, there is a time lag in observing the effect of interventions on deaths since there is a 2-3-week period between infection, onset of symptoms and outcome. In this report, we fit a novel Bayesian mechanistic model of the infection cycle to observed deaths in 11 European countries, inferring plausible upper and lower bounds (Bayesian credible intervals) of the total populations infected (attack rates), case detection probabilities, and the reproduction number over time (Rt). We fit the model jointly to COVID-19 data from all these countries to assess whether there is evidence that interventions have so far been successful at reducing Rt below 1, with the strong assumption that particular interventions are achieving a similar impact in different countries and that the efficacy of those interventions remains constant over time. The model is informed more strongly by countries with larger numbers of deaths and which implemented interventions earlier, therefore estimates of recent Rt in countries with more recent interventions are contingent on similar intervention impacts. Data in the coming weeks will enable estimation of country-specific Rt with greater precision. Model and data details are presented in the appendix, validation and sensitivity are also presented in the appendix, and general limitations presented below in the conclusions. 2 Results The timing of interventions should be taken in the context of when an individual country’s epidemic started to grow along with the speed with which control measures were implemented. Italy was the first to begin intervention measures, and other countries followed soon afterwards (Figure 1). Most interventions began around 12th-14th March. We analyzed data on deaths up to 28th March, giving a 2-3-week window over which to estimate the effect of interventions. Currently, most countries in our study have implemented all major non-pharmaceutical interventions. For each country, we model the number of infections, the number of deaths, and Rt, the effective reproduction number over time, with Rt changing only when an intervention is introduced (Figure 2- 12). Rt is the average number of secondary infections per infected individual, assuming that the interventions that are in place at time t stay in place throughout their entire infectious period. Every country has its own individual starting reproduction number Rt before interventions take place. Specific interventions are assumed to have the same relative impact on Rt in each country when they were introduced there and are informed by mortality data across all countries. Figure l: Intervention timings for the 11 European countries included in the analysis. For further details see Appendix 8.6. 2.1 Estimated true numbers of infections and current attack rates In all countries, we estimate there are orders of magnitude fewer infections detected (Figure 2) than true infections, mostly likely due to mild and asymptomatic infections as well as limited testing capacity. In Italy, our results suggest that, cumulatively, 5.9 [1.9-15.2] million people have been infected as of March 28th, giving an attack rate of 9.8% [3.2%-25%] of the population (Table 1). Spain has recently seen a large increase in the number of deaths, and given its smaller population, our model estimates that a higher proportion of the population, 15.0% (7.0 [18-19] million people) have been infected to date. Germany is estimated to have one of the lowest attack rates at 0.7% with 600,000 [240,000-1,500,000] people infected. Imperial College COVID-19 Response Team Table l: Posterior model estimates of percentage of total population infected as of 28th March 2020. Country % of total population infected (mean [95% credible intervall) Austria 1.1% [0.36%-3.1%] Belgium 3.7% [1.3%-9.7%] Denmark 1.1% [0.40%-3.1%] France 3.0% [1.1%-7.4%] Germany 0.72% [0.28%-1.8%] Italy 9.8% [3.2%-26%] Norway 0.41% [0.09%-1.2%] Spain 15% [3.7%-41%] Sweden 3.1% [0.85%-8.4%] Switzerland 3.2% [1.3%-7.6%] United Kingdom 2.7% [1.2%-5.4%] 2.2 Reproduction numbers and impact of interventions Averaged across all countries, we estimate initial reproduction numbers of around 3.87 [3.01-4.66], which is in line with other estimates.1'8 These estimates are informed by our choice of serial interval distribution and the initial growth rate of observed deaths. A shorter assumed serial interval results in lower starting reproduction numbers (Appendix 8.4.2, Appendix 8.4.6). The initial reproduction numbers are also uncertain due to (a) importation being the dominant source of new infections early in the epidemic, rather than local transmission (b) possible under-ascertainment in deaths particularly before testing became widespread. We estimate large changes in Rt in response to the combined non-pharmaceutical interventions. Our results, which are driven largely by countries with advanced epidemics and larger numbers of deaths (e.g. Italy, Spain), suggest that these interventions have together had a substantial impact on transmission, as measured by changes in the estimated reproduction number Rt. Across all countries we find current estimates of Rt to range from a posterior mean of 0.97 [0.14-2.14] for Norway to a posterior mean of2.64 [1.40-4.18] for Sweden, with an average of 1.43 across the 11 country posterior means, a 64% reduction compared to the pre-intervention values. We note that these estimates are contingent on intervention impact being the same in different countries and at different times. In all countries but Sweden, under the same assumptions, we estimate that the current reproduction number includes 1 in the uncertainty range. The estimated reproduction number for Sweden is higher, not because the mortality trends are significantly different from any other country, but as an artefact of our model, which assumes a smaller reduction in Rt because no full lockdown has been ordered so far. Overall, we cannot yet conclude whether current interventions are sufficient to drive Rt below 1 (posterior probability of being less than 1.0 is 44% on average across the countries). We are also unable to conclude whether interventions may be different between countries or over time. There remains a high level of uncertainty in these estimates. It is too early to detect substantial intervention impact in many countries at earlier stages of their epidemic (e.g. Germany, UK, Norway). Many interventions have occurred only recently, and their effects have not yet been fully observed due to the time lag between infection and death. This uncertainty will reduce as more data become available. For all countries, our model fits observed deaths data well (Bayesian goodness of fit tests). We also found that our model can reliably forecast daily deaths 3 days into the future, by withholding the latest 3 days of data and comparing model predictions to observed deaths (Appendix 8.3). The close spacing of interventions in time made it statistically impossible to determine which had the greatest effect (Figure 1, Figure 4). However, when doing a sensitivity analysis (Appendix 8.4.3) with uninformative prior distributions (where interventions can increase deaths) we find similar impact of Imperial College COVID-19 Response Team interventions, which shows that our choice of prior distribution is not driving the effects we see in the main analysis. Figure 2: Country-level estimates of infections, deaths and Rt. Left: daily number of infections, brown bars are reported infections, blue bands are predicted infections, dark blue 50% credible interval (CI), light blue 95% CI. The number of daily infections estimated by our model drops immediately after an intervention, as we assume that all infected people become immediately less infectious through the intervention. Afterwards, if the Rt is above 1, the number of infections will starts growing again. Middle: daily number of deaths, brown bars are reported deaths, blue bands are predicted deaths, CI as in left plot. Right: time-varying reproduction number Rt, dark green 50% CI, light green 95% CI. Icons are interventions shown at the time they occurred. Imperial College COVID-19 Response Team Table 2: Totalforecasted deaths since the beginning of the epidemic up to 31 March in our model and in a counterfactual model (assuming no intervention had taken place). Estimated averted deaths over this time period as a result of the interventions. Numbers in brackets are 95% credible intervals. 2.3 Estimated impact of interventions on deaths Table 2 shows total forecasted deaths since the beginning of the epidemic up to and including 31 March under ourfitted model and under the counterfactual model, which predicts what would have happened if no interventions were implemented (and R, = R0 i.e. the initial reproduction number estimated before interventions). Again, the assumption in these predictions is that intervention impact is the same across countries and time. The model without interventions was unable to capture recent trends in deaths in several countries, where the rate of increase had clearly slowed (Figure 3). Trends were confirmed statistically by Bayesian leave-one-out cross-validation and the widely applicable information criterion assessments —WA|C). By comparing the deaths predicted under the model with no interventions to the deaths predicted in our intervention model, we calculated the total deaths averted up to the end of March. We find that, across 11 countries, since the beginning of the epidemic, 59,000 [21,000-120,000] deaths have been averted due to interventions. In Italy and Spain, where the epidemic is advanced, 38,000 [13,000- 84,000] and 16,000 [5,400-35,000] deaths have been averted, respectively. Even in the UK, which is much earlier in its epidemic, we predict 370 [73-1,000] deaths have been averted. These numbers give only the deaths averted that would have occurred up to 31 March. lfwe were to include the deaths of currently infected individuals in both models, which might happen after 31 March, then the deaths averted would be substantially higher. Figure 3: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for (a) Italy and (b) Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. Other countries are shown in Appendix 8.6. 03/0 25% 50% 753% 100% (no effect on transmissibility) (ends transmissibility Relative % reduction in R. Figure 4: Our model includes five covariates for governmental interventions, adjusting for whether the intervention was the first one undertaken by the government in response to COVID-19 (red) or was subsequent to other interventions (green). Mean relative percentage reduction in Rt is shown with 95% posterior credible intervals. If 100% reduction is achieved, Rt = 0 and there is no more transmission of COVID-19. No effects are significantly different from any others, probably due to the fact that many interventions occurred on the same day or within days of each other as shown in Figure l. 3 Discussion During this early phase of control measures against the novel coronavirus in Europe, we analyze trends in numbers of deaths to assess the extent to which transmission is being reduced. Representing the COVlD-19 infection process using a semi-mechanistic, joint, Bayesian hierarchical model, we can reproduce trends observed in the data on deaths and can forecast accurately over short time horizons. We estimate that there have been many more infections than are currently reported. The high level of under-ascertainment of infections that we estimate here is likely due to the focus on testing in hospital settings rather than in the community. Despite this, only a small minority of individuals in each country have been infected, with an attack rate on average of 4.9% [l.9%-ll%] with considerable variation between countries (Table 1). Our estimates imply that the populations in Europe are not close to herd immunity ("50-75% if R0 is 2-4). Further, with Rt values dropping substantially, the rate of acquisition of herd immunity will slow down rapidly. This implies that the virus will be able to spread rapidly should interventions be lifted. Such estimates of the attack rate to date urgently need to be validated by newly developed antibody tests in representative population surveys, once these become available. We estimate that major non-pharmaceutical interventions have had a substantial impact on the time- varying reproduction numbers in countries where there has been time to observe intervention effects on trends in deaths (Italy, Spain). lfadherence in those countries has changed since that initial period, then our forecast of future deaths will be affected accordingly: increasing adherence over time will have resulted in fewer deaths and decreasing adherence in more deaths. Similarly, our estimates of the impact ofinterventions in other countries should be viewed with caution if the same interventions have achieved different levels of adherence than was initially the case in Italy and Spain. Due to the implementation of interventions in rapid succession in many countries, there are not enough data to estimate the individual effect size of each intervention, and we discourage attributing associations to individual intervention. In some cases, such as Norway, where all interventions were implemented at once, these individual effects are by definition unidentifiable. Despite this, while individual impacts cannot be determined, their estimated joint impact is strongly empirically justified (see Appendix 8.4 for sensitivity analysis). While the growth in daily deaths has decreased, due to the lag between infections and deaths, continued rises in daily deaths are to be expected for some time. To understand the impact of interventions, we fit a counterfactual model without the interventions and compare this to the actual model. Consider Italy and the UK - two countries at very different stages in their epidemics. For the UK, where interventions are very recent, much of the intervention strength is borrowed from countries with older epidemics. The results suggest that interventions will have a large impact on infections and deaths despite counts of both rising. For Italy, where far more time has passed since the interventions have been implemented, it is clear that the model without interventions does not fit well to the data, and cannot explain the sub-linear (on the logarithmic scale) reduction in deaths (see Figure 10). The counterfactual model for Italy suggests that despite mounting pressure on health systems, interventions have averted a health care catastrophe where the number of new deaths would have been 3.7 times higher (38,000 deaths averted) than currently observed. Even in the UK, much earlier in its epidemic, the recent interventions are forecasted to avert 370 total deaths up to 31 of March. 4 Conclusion and Limitations Modern understanding of infectious disease with a global publicized response has meant that nationwide interventions could be implemented with widespread adherence and support. Given observed infection fatality ratios and the epidemiology of COVlD-19, major non-pharmaceutical interventions have had a substantial impact in reducing transmission in countries with more advanced epidemics. It is too early to be sure whether similar reductions will be seen in countries at earlier stages of their epidemic. While we cannot determine which set of interventions have been most successful, taken together, we can already see changes in the trends of new deaths. When forecasting 3 days and looking over the whole epidemic the number of deaths averted is substantial. We note that substantial innovation is taking place, and new more effective interventions or refinements of current interventions, alongside behavioral changes will further contribute to reductions in infections. We cannot say for certain that the current measures have controlled the epidemic in Europe; however, if current trends continue, there is reason for optimism. Our approach is semi-mechanistic. We propose a plausible structure for the infection process and then estimate parameters empirically. However, many parameters had to be given strong prior distributions or had to be fixed. For these assumptions, we have provided relevant citations to previous studies. As more data become available and better estimates arise, we will update these in weekly reports. Our choice of serial interval distribution strongly influences the prior distribution for starting R0. Our infection fatality ratio, and infection-to-onset-to-death distributions strongly influence the rate of death and hence the estimated number of true underlying cases. We also assume that the effect of interventions is the same in all countries, which may not be fully realistic. This assumption implies that countries with early interventions and more deaths since these interventions (e.g. Italy, Spain) strongly influence estimates of intervention impact in countries at earlier stages of their epidemic with fewer deaths (e.g. Germany, UK). We have tried to create consistent definitions of all interventions and document details of this in Appendix 8.6. However, invariably there will be differences from country to country in the strength of their intervention — for example, most countries have banned gatherings of more than 2 people when implementing a lockdown, whereas in Sweden the government only banned gatherings of more than 10 people. These differences can skew impacts in countries with very little data. We believe that our uncertainty to some degree can cover these differences, and as more data become available, coefficients should become more reliable. However, despite these strong assumptions, there is sufficient signal in the data to estimate changes in R, (see the sensitivity analysis reported in Appendix 8.4.3) and this signal will stand to increase with time. In our Bayesian hierarchical framework, we robustly quantify the uncertainty in our parameter estimates and posterior predictions. This can be seen in the very wide credible intervals in more recent days, where little or no death data are available to inform the estimates. Furthermore, we predict intervention impact at country-level, but different trends may be in place in different parts of each country. For example, the epidemic in northern Italy was subject to controls earlier than the rest of the country. 5 Data Our model utilizes daily real-time death data from the ECDC (European Centre of Disease Control), where we catalogue case data for 11 European countries currently experiencing the epidemic: Austria, Belgium, Denmark, France, Germany, Italy, Norway, Spain, Sweden, Switzerland and the United Kingdom. The ECDC provides information on confirmed cases and deaths attributable to COVID-19. However, the case data are highly unrepresentative of the incidence of infections due to underreporting as well as systematic and country-specific changes in testing. We, therefore, use only deaths attributable to COVID-19 in our model; we do not use the ECDC case estimates at all. While the observed deaths still have some degree of unreliability, again due to changes in reporting and testing, we believe the data are ofsufficient fidelity to model. For population counts, we use UNPOP age-stratified counts.10 We also catalogue data on the nature and type of major non-pharmaceutical interventions. We looked at the government webpages from each country as well as their official public health division/information webpages to identify the latest advice/laws being issued by the government and public health authorities. We collected the following: School closure ordered: This intervention refers to nationwide extraordinary school closures which in most cases refer to both primary and secondary schools closing (for most countries this also includes the closure of otherforms of higher education or the advice to teach remotely). In the case of Denmark and Sweden, we allowed partial school closures of only secondary schools. The date of the school closure is taken to be the effective date when the schools started to be closed (ifthis was on a Monday, the date used was the one of the previous Saturdays as pupils and students effectively stayed at home from that date onwards). Case-based measures: This intervention comprises strong recommendations or laws to the general public and primary care about self—isolation when showing COVID-19-like symptoms. These also include nationwide testing programs where individuals can be tested and subsequently self—isolated. Our definition is restricted to nationwide government advice to all individuals (e.g. UK) or to all primary care and excludes regional only advice. These do not include containment phase interventions such as isolation if travelling back from an epidemic country such as China. Public events banned: This refers to banning all public events of more than 100 participants such as sports events. Social distancing encouraged: As one of the first interventions against the spread of the COVID-19 pandemic, many governments have published advice on social distancing including the recommendation to work from home wherever possible, reducing use ofpublictransport and all other non-essential contact. The dates used are those when social distancing has officially been recommended by the government; the advice may include maintaining a recommended physical distance from others. Lockdown decreed: There are several different scenarios that the media refers to as lockdown. As an overall definition, we consider regulations/legislations regarding strict face-to-face social interaction: including the banning of any non-essential public gatherings, closure of educational and public/cultural institutions, ordering people to stay home apart from exercise and essential tasks. We include special cases where these are not explicitly mentioned on government websites but are enforced by the police (e.g. France). The dates used are the effective dates when these legislations have been implemented. We note that lockdown encompasses other interventions previously implemented. First intervention: As Figure 1 shows, European governments have escalated interventions rapidly, and in some examples (Norway/Denmark) have implemented these interventions all on a single day. Therefore, given the temporal autocorrelation inherent in government intervention, we include a binary covariate for the first intervention, which can be interpreted as a government decision to take major action to control COVID-19. A full list of the timing of these interventions and the sources we have used can be found in Appendix 8.6. 6 Methods Summary A Visual summary of our model is presented in Figure 5 (details in Appendix 8.1 and 8.2). Replication code is available at https://github.com/|mperia|CollegeLondon/covid19model/releases/tag/vl.0 We fit our model to observed deaths according to ECDC data from 11 European countries. The modelled deaths are informed by an infection-to-onset distribution (time from infection to the onset of symptoms), an onset-to-death distribution (time from the onset of symptoms to death), and the population-averaged infection fatality ratio (adjusted for the age structure and contact patterns of each country, see Appendix). Given these distributions and ratios, modelled deaths are a function of the number of infections. The modelled number of infections is informed by the serial interval distribution (the average time from infection of one person to the time at which they infect another) and the time-varying reproduction number. Finally, the time-varying reproduction number is a function of the initial reproduction number before interventions and the effect sizes from interventions. Figure 5: Summary of model components. Following the hierarchy from bottom to top gives us a full framework to see how interventions affect infections, which can result in deaths. We use Bayesian inference to ensure our modelled deaths can reproduce the observed deaths as closely as possible. From bottom to top in Figure 5, there is an implicit lag in time that means the effect of very recent interventions manifest weakly in current deaths (and get stronger as time progresses). To maximise the ability to observe intervention impact on deaths, we fit our model jointly for all 11 European countries, which results in a large data set. Our model jointly estimates the effect sizes of interventions. We have evaluated the effect ofour Bayesian prior distribution choices and evaluate our Bayesian posterior calibration to ensure our results are statistically robust (Appendix 8.4). 7 Acknowledgements Initial research on covariates in Appendix 8.6 was crowdsourced; we thank a number of people across the world for help with this. This work was supported by Centre funding from the UK Medical Research Council under a concordat with the UK Department for International Development, the NIHR Health Protection Research Unit in Modelling Methodology and CommunityJameel. 8 Appendix: Model Specifics, Validation and Sensitivity Analysis 8.1 Death model We observe daily deaths Dam for days t E 1, ...,n and countries m E 1, ...,p. These daily deaths are modelled using a positive real-Valued function dam = E(Dam) that represents the expected number of deaths attributed to COVID-19. Dam is assumed to follow a negative binomial distribution with The expected number of deaths (1 in a given country on a given day is a function of the number of infections C occurring in previous days. At the beginning of the epidemic, the observed deaths in a country can be dominated by deaths that result from infection that are not locally acquired. To avoid biasing our model by this, we only include observed deaths from the day after a country has cumulatively observed 10 deaths in our model. To mechanistically link ourfunction for deaths to infected cases, we use a previously estimated COVID- 19 infection-fatality-ratio ifr (probability of death given infection)9 together with a distribution oftimes from infection to death TE. The ifr is derived from estimates presented in Verity et al11 which assumed homogeneous attack rates across age-groups. To better match estimates of attack rates by age generated using more detailed information on country and age-specific mixing patterns, we scale these estimates (the unadjusted ifr, referred to here as ifr’) in the following way as in previous work.4 Let Ca be the number of infections generated in age-group a, Na the underlying size of the population in that age group and AR“ 2 Ca/Na the age-group-specific attack rate. The adjusted ifr is then given by: ifra = fififié, where AR50_59 is the predicted attack-rate in the 50-59 year age-group after incorporating country-specific patterns of contact and mixing. This age-group was chosen as the reference as it had the lowest predicted level of underreporting in previous analyses of data from the Chinese epidemic“. We obtained country-specific estimates of attack rate by age, AR“, for the 11 European countries in our analysis from a previous study which incorporates information on contact between individuals of different ages in countries across Europe.12 We then obtained overall ifr estimates for each country adjusting for both demography and age-specific attack rates. Using estimated epidemiological information from previous studies,“'11 we assume TE to be the sum of two independent random times: the incubation period (infection to onset of symptoms or infection- to-onset) distribution and the time between onset of symptoms and death (onset-to-death). The infection-to-onset distribution is Gamma distributed with mean 5.1 days and coefficient of variation 0.86. The onset-to-death distribution is also Gamma distributed with a mean of 18.8 days and a coefficient of va riation 0.45. ifrm is population averaged over the age structure of a given country. The infection-to-death distribution is therefore given by: um ~ ifrm ~ (Gamma(5.1,0.86) + Gamma(18.8,0.45)) Figure 6 shows the infection-to-death distribution and the resulting survival function that integrates to the infection fatality ratio. Figure 6: Left, infection-to-death distribution (mean 23.9 days). Right, survival probability of infected individuals per day given the infection fatality ratio (1%) and the infection-to-death distribution on the left. Using the probability of death distribution, the expected number of deaths dam, on a given day t, for country, m, is given by the following discrete sum: The number of deaths today is the sum of the past infections weighted by their probability of death, where the probability of death depends on the number of days since infection. 8.2 Infection model The true number of infected individuals, C, is modelled using a discrete renewal process. This approach has been used in numerous previous studies13'16 and has a strong theoretical basis in stochastic individual-based counting processes such as Hawkes process and the Bellman-Harris process.”18 The renewal model is related to the Susceptible-Infected-Recovered model, except the renewal is not expressed in differential form. To model the number ofinfections over time we need to specify a serial interval distribution g with density g(T), (the time between when a person gets infected and when they subsequently infect another other people), which we choose to be Gamma distributed: g ~ Gamma (6.50.62). The serial interval distribution is shown below in Figure 7 and is assumed to be the same for all countries. Figure 7: Serial interval distribution g with a mean of 6.5 days. Given the serial interval distribution, the number of infections Eamon a given day t, and country, m, is given by the following discrete convolution function: _ t—1 Cam — Ram ZT=0 Cr,mgt—‘r r where, similarto the probability ofdeath function, the daily serial interval is discretized by fs+0.5 1.5 gs = T=s—0.Sg(T)dT fors = 2,3, and 91 = fT=Og(T)dT. Infections today depend on the number of infections in the previous days, weighted by the discretized serial interval distribution. This weighting is then scaled by the country-specific time-Varying reproduction number, Ram, that models the average number of secondary infections at a given time. The functional form for the time-Varying reproduction number was chosen to be as simple as possible to minimize the impact of strong prior assumptions: we use a piecewise constant function that scales Ram from a baseline prior R0,m and is driven by known major non-pharmaceutical interventions occurring in different countries and times. We included 6 interventions, one of which is constructed from the other 5 interventions, which are timings of school and university closures (k=l), self—isolating if ill (k=2), banning of public events (k=3), any government intervention in place (k=4), implementing a partial or complete lockdown (k=5) and encouraging social distancing and isolation (k=6). We denote the indicator variable for intervention k E 1,2,3,4,5,6 by IkI’m, which is 1 if intervention k is in place in country m at time t and 0 otherwise. The covariate ”any government intervention” (k=4) indicates if any of the other 5 interventions are in effect,i.e.14’t’m equals 1 at time t if any of the interventions k E 1,2,3,4,5 are in effect in country m at time t and equals 0 otherwise. Covariate 4 has the interpretation of indicating the onset of major government intervention. The effect of each intervention is assumed to be multiplicative. Ram is therefore a function ofthe intervention indicators Ik’t’m in place at time t in country m: Ram : R0,m eXp(— 212:1 O(Rheum)- The exponential form was used to ensure positivity of the reproduction number, with R0,m constrained to be positive as it appears outside the exponential. The impact of each intervention on Ram is characterised by a set of parameters 0(1, ...,OL6, with independent prior distributions chosen to be ock ~ Gamma(. 5,1). The impacts ock are shared between all m countries and therefore they are informed by all available data. The prior distribution for R0 was chosen to be R0,m ~ Normal(2.4, IKI) with K ~ Normal(0,0.5), Once again, K is the same among all countries to share information. We assume that seeding of new infections begins 30 days before the day after a country has cumulatively observed 10 deaths. From this date, we seed our model with 6 sequential days of infections drawn from cl’m,...,66’m~EXponential(T), where T~Exponential(0.03). These seed infections are inferred in our Bayesian posterior distribution. We estimated parameters jointly for all 11 countries in a single hierarchical model. Fitting was done in the probabilistic programming language Stan,19 using an adaptive Hamiltonian Monte Carlo (HMC) sampler. We ran 8 chains for 4000 iterations with 2000 iterations of warmup and a thinning factor 4 to obtain 2000 posterior samples. Posterior convergence was assessed using the Rhat statistic and by diagnosing divergent transitions of the HMC sampler. Prior-posterior calibrations were also performed (see below). 8.3 Validation We validate accuracy of point estimates of our model using cross-Validation. In our cross-validation scheme, we leave out 3 days of known death data (non-cumulative) and fit our model. We forecast what the model predicts for these three days. We present the individual forecasts for each day, as well as the average forecast for those three days. The cross-validation results are shown in the Figure 8. Figure 8: Cross-Validation results for 3-day and 3-day aggregatedforecasts Figure 8 provides strong empirical justification for our model specification and mechanism. Our accurate forecast over a three-day time horizon suggests that our fitted estimates for Rt are appropriate and plausible. Along with from point estimates we all evaluate our posterior credible intervals using the Rhat statistic. The Rhat statistic measures whether our Markov Chain Monte Carlo (MCMC) chains have converged to the equilibrium distribution (the correct posterior distribution). Figure 9 shows the Rhat statistics for all of our parameters Figure 9: Rhat statistics - values close to 1 indicate MCMC convergence. Figure 9 indicates that our MCMC have converged. In fitting we also ensured that the MCMC sampler experienced no divergent transitions - suggesting non pathological posterior topologies. 8.4 SensitivityAnalysis 8.4.1 Forecasting on log-linear scale to assess signal in the data As we have highlighted throughout in this report, the lag between deaths and infections means that it ta kes time for information to propagate backwa rds from deaths to infections, and ultimately to Rt. A conclusion of this report is the prediction of a slowing of Rt in response to major interventions. To gain intuition that this is data driven and not simply a consequence of highly constrained model assumptions, we show death forecasts on a log-linear scale. On this scale a line which curves below a linear trend is indicative of slowing in the growth of the epidemic. Figure 10 to Figure 12 show these forecasts for Italy, Spain and the UK. They show this slowing down in the daily number of deaths. Our model suggests that Italy, a country that has the highest death toll of COVID-19, will see a slowing in the increase in daily deaths over the coming week compared to the early stages of the epidemic. We investigated the sensitivity of our estimates of starting and final Rt to our assumed serial interval distribution. For this we considered several scenarios, in which we changed the serial interval distribution mean, from a value of 6.5 days, to have values of 5, 6, 7 and 8 days. In Figure 13, we show our estimates of R0, the starting reproduction number before interventions, for each of these scenarios. The relative ordering of the Rt=0 in the countries is consistent in all settings. However, as expected, the scale of Rt=0 is considerably affected by this change — a longer serial interval results in a higher estimated Rt=0. This is because to reach the currently observed size of the epidemics, a longer assumed serial interval is compensated by a higher estimated R0. Additionally, in Figure 14, we show our estimates of Rt at the most recent model time point, again for each ofthese scenarios. The serial interval mean can influence Rt substantially, however, the posterior credible intervals of Rt are broadly overlapping. Figure 13: Initial reproduction number R0 for different serial interval (SI) distributions (means between 5 and 8 days). We use 6.5 days in our main analysis. Figure 14: Rt on 28 March 2020 estimated for all countries, with serial interval (SI) distribution means between 5 and 8 days. We use 6.5 days in our main analysis. 8.4.3 Uninformative prior sensitivity on or We ran our model using implausible uninformative prior distributions on the intervention effects, allowing the effect of an intervention to increase or decrease Rt. To avoid collinearity, we ran 6 separate models, with effects summarized below (compare with the main analysis in Figure 4). In this series of univariate analyses, we find (Figure 15) that all effects on their own serve to decrease Rt. This gives us confidence that our choice of prior distribution is not driving the effects we see in the main analysis. Lockdown has a very large effect, most likely due to the fact that it occurs after other interventions in our dataset. The relatively large effect sizes for the other interventions are most likely due to the coincidence of the interventions in time, such that one intervention is a proxy for a few others. Figure 15: Effects of different interventions when used as the only covariate in the model. 8.4.4 To assess prior assumptions on our piecewise constant functional form for Rt we test using a nonparametric function with a Gaussian process prior distribution. We fit a model with a Gaussian process prior distribution to data from Italy where there is the largest signal in death data. We find that the Gaussian process has a very similartrend to the piecewise constant model and reverts to the mean in regions of no data. The correspondence of a completely nonparametric function and our piecewise constant function suggests a suitable parametric specification of Rt. Nonparametric fitting of Rf using a Gaussian process: 8.4.5 Leave country out analysis Due to the different lengths of each European countries’ epidemic, some countries, such as Italy have much more data than others (such as the UK). To ensure that we are not leveraging too much information from any one country we perform a ”leave one country out” sensitivity analysis, where we rerun the model without a different country each time. Figure 16 and Figure 17 are examples for results for the UK, leaving out Italy and Spain. In general, for all countries, we observed no significant dependence on any one country. Figure 16: Model results for the UK, when not using data from Italy for fitting the model. See the Figure 17: Model results for the UK, when not using data from Spain for fitting the model. See caption of Figure 2 for an explanation of the plots. 8.4.6 Starting reproduction numbers vs theoretical predictions To validate our starting reproduction numbers, we compare our fitted values to those theoretically expected from a simpler model assuming exponential growth rate, and a serial interval distribution mean. We fit a linear model with a Poisson likelihood and log link function and extracting the daily growth rate r. For well-known theoretical results from the renewal equation, given a serial interval distribution g(r) with mean m and standard deviation 5, given a = mZ/S2 and b = m/SZ, and a subsequently R0 = (1 + %) .Figure 18 shows theoretically derived R0 along with our fitted estimates of Rt=0 from our Bayesian hierarchical model. As shown in Figure 18 there is large correspondence between our estimated starting reproduction number and the basic reproduction number implied by the growth rate r. R0 (red) vs R(FO) (black) Figure 18: Our estimated R0 (black) versus theoretically derived Ru(red) from a log-linear regression fit. 8.5 Counterfactual analysis — interventions vs no interventions Figure 19: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for all countries except Italy and Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. DOI: https://doi.org/10.25561/77731 Page 28 of 35 30 March 2020 Imperial College COVID-19 Response Team 8.6 Data sources and Timeline of Interventions Figure 1 and Table 3 display the interventions by the 11 countries in our study and the dates these interventions became effective. Table 3: Timeline of Interventions. Country Type Event Date effective School closure ordered Nationwide school closures.20 14/3/2020 Public events banned Banning of gatherings of more than 5 people.21 10/3/2020 Banning all access to public spaces and gatherings Lockdown of more than 5 people. Advice to maintain 1m ordered distance.22 16/3/2020 Social distancing encouraged Recommendation to maintain a distance of 1m.22 16/3/2020 Case-based Austria measures Implemented at lockdown.22 16/3/2020 School closure ordered Nationwide school closures.23 14/3/2020 Public events All recreational activities cancelled regardless of banned size.23 12/3/2020 Citizens are required to stay at home except for Lockdown work and essential journeys. Going outdoors only ordered with household members or 1 friend.24 18/3/2020 Public transport recommended only for essential Social distancing journeys, work from home encouraged, all public encouraged places e.g. restaurants closed.23 14/3/2020 Case-based Everyone should stay at home if experiencing a Belgium measures cough or fever.25 10/3/2020 School closure Secondary schools shut and universities (primary ordered schools also shut on 16th).26 13/3/2020 Public events Bans of events >100 people, closed cultural banned institutions, leisure facilities etc.27 12/3/2020 Lockdown Bans of gatherings of >10 people in public and all ordered public places were shut.27 18/3/2020 Limited use of public transport. All cultural Social distancing institutions shut and recommend keeping encouraged appropriate distance.28 13/3/2020 Case-based Everyone should stay at home if experiencing a Denmark measures cough or fever.29 12/3/2020 School closure ordered Nationwide school closures.30 14/3/2020 Public events banned Bans of events >100 people.31 13/3/2020 Lockdown Everybody has to stay at home. Need a self- ordered authorisation form to leave home.32 17/3/2020 Social distancing encouraged Advice at the time of lockdown.32 16/3/2020 Case-based France measures Advice at the time of lockdown.32 16/03/2020 School closure ordered Nationwide school closures.33 14/3/2020 Public events No gatherings of >1000 people. Otherwise banned regional restrictions only until lockdown.34 22/3/2020 Lockdown Gatherings of > 2 people banned, 1.5 m ordered distance.35 22/3/2020 Social distancing Avoid social interaction wherever possible encouraged recommended by Merkel.36 12/3/2020 Advice for everyone experiencing symptoms to Case-based contact a health care agency to get tested and Germany measures then self—isolate.37 6/3/2020 School closure ordered Nationwide school closures.38 5/3/2020 Public events banned The government bans all public events.39 9/3/2020 Lockdown The government closes all public places. People ordered have to stay at home except for essential travel.40 11/3/2020 A distance of more than 1m has to be kept and Social distancing any other form of alternative aggregation is to be encouraged excluded.40 9/3/2020 Case-based Advice to self—isolate if experiencing symptoms Italy measures and quarantine if tested positive.41 9/3/2020 Norwegian Directorate of Health closes all School closure educational institutions. Including childcare ordered facilities and all schools.42 13/3/2020 Public events The Directorate of Health bans all non-necessary banned social contact.42 12/3/2020 Lockdown Only people living together are allowed outside ordered together. Everyone has to keep a 2m distance.43 24/3/2020 Social distancing The Directorate of Health advises against all encouraged travelling and non-necessary social contacts.42 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Norway measures cough or fever symptoms.44 15/3/2020 ordered Nationwide school closures.45 13/3/2020 Public events banned Banning of all public events by lockdown.46 14/3/2020 Lockdown ordered Nationwide lockdown.43 14/3/2020 Social distancing Advice on social distancing and working remotely encouraged from home.47 9/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Spain measures cough or fever symptoms.47 17/3/2020 School closure ordered Colleges and upper secondary schools shut.48 18/3/2020 Public events banned The government bans events >500 people.49 12/3/2020 Lockdown ordered No lockdown occurred. NA People even with mild symptoms are told to limit Social distancing social contact, encouragement to work from encouraged home.50 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Sweden measures fever symptoms.51 10/3/2020 School closure ordered No in person teaching until 4th of April.52 14/3/2020 Public events banned The government bans events >100 people.52 13/3/2020 Lockdown ordered Gatherings of more than 5 people are banned.53 2020-03-20 Advice on keeping distance. All businesses where Social distancing this cannot be realised have been closed in all encouraged states (kantons).54 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Switzerland measures fever symptoms.55 2/3/2020 Nationwide school closure. Childminders, School closure nurseries and sixth forms are told to follow the ordered guidance.56 21/3/2020 Public events banned Implemented with lockdown.57 24/3/2020 Gatherings of more than 2 people not from the Lockdown same household are banned and police ordered enforceable.57 24/3/2020 Social distancing Advice to avoid pubs, clubs, theatres and other encouraged public institutions.58 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a UK measures cough or fever symptoms.59 12/3/2020 9 References 1. Li, R. et al. Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2). Science (2020) doi:10.1126/science.abb3221. 2. Zhang, J. et al. Patterns of human social contact and contact with animals in Shanghai, China. 5cLRep.9,1—11(2019) 3. Worldometers.info. Hong Kong: coronavirus cases. https://www.wo rldometers.info/co ronavirus/country/china-hong-kong-sar/. 4. Ferguson, N. et al. 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What is Spain's estimated mean percentage [95% credible interval] of total population infected as of 28th March?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

what baculovirus vector based immunization provided protection from lethal challenge?

Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840674/ SHA: ef38ed2f4cc96e16ce011623cc5d15d2d8ca58c3 Authors: Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik; Friis, Martin Barfred; Belsham, Graham J; Höper, Dirk; Reimann, Ilona; Beer, Martin Date: 2013-11-22 DOI: 10.1186/1471-2164-14-819 License: cc-by Abstract: BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses. Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs. BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates. Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual "foreign" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] . Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain "C-strain Riems". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain "Gifhorn") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed. Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV "C-strain Riems" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain "Gifhorn" [26] was used for amplification of the Gifhorn E2-coding sequence. Oligonucleotide primers used are listed in Additional file 1: Table S1 . The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the "C-strain Riems". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad). BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen). Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany). The Red/ET recombination involved three steps (i-iii). Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis. Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts. Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ). Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV "Gifhorn" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV "Gifhorn" RNA using two different primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR). For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below). BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days. BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters). Generation of a BAC containing full-length cDNA corresponding to the modified live vaccine "C-strain Riems" BACs containing the full-length cDNA corresponding to the parental vRiemser ("C-strain Riems") were constructed according to the method described previously for the "Paderborn" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with "pBelo" and rescued viruses with "v" (e.g. pBeloR26 and vR26). Cell culture passage no. of virus is indicated with "/P" (e.g. vR26/P-4). digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ). To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain "Gifhorn" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV "Gifhorn". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown). After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the "Gifhorn" E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells. The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences. The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process *Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA. was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ). To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12 passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ). In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain "Gifhorn" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results). Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for "C-strain Riems". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] . Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa

What is needed to direct genetic mutations in RNA viruses?

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580613/ SHA: ee5b43d20a640664510cb7a540caaae4a8e19933 Authors: Saksena, Sumeet; Fox, Jefferson; Epprecht, Michael; Tran, Chinh C.; Nong, Duong H.; Spencer, James H.; Nguyen, Lam; Finucane, Melissa L.; Tran, Vien D.; Wilcox, Bruce A. Date: 2015-09-23 DOI: 10.1371/journal.pone.0138138 License: cc-by Abstract: Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . Highly Pathogenic Avian Influenza (HPAI) (subtype H5N1) is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 [4] , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . Lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (CAS) [7, 8] . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by Saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . The 58 provinces in Vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, Red River Delta, and Mekong Delta) producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . Other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota (Indonesian for "village-town") [26] . McGee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . Saksena et al. [9] built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (LCLUC) [27, 28] . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers [36] . Large, enterprise-scale (15,000-100,000 birds) operations are still rare in Viet Nam (only 33 communes have such a facility). On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . In much of Asia there is often a mix of commercial and backyard farming at any one location [36] . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . Intensive systems allow for virus evolution (e.g. Low Pathogenic Avian Influenza to HPAI) and transformation, while extensive systems allow for environmental persistence and circulation [39] . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI (as reviewed in Gilbert and Pfeiffer [40] ). Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. [9] to define the urban character of each commune. The classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the Normalized Differentiated Vegetation Index (NDVI). The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index [41] . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class (complete homogeneity) the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity [42] . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density [11, 14-16, 18, 19, 44, 45] . • Poultry trade and market. Towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure [37, 47, 48] . So, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area [15, 19, 37, 49] . • Intermediate hosts. Duck and geese densities were calculated using total commune area [11, 19, 49] . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor [11, 17, 37] . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a) lake and b) river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam [10] . Compound Topographical Index (CTI, also known as Topographical Wetness Index) is a measure of the tendency for water to pool. Studies in Thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models (ENM) of HPAI H5N1 [55, 56] . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . Some studies have shown that at local scales, the slope of the terrain (a component of CTI) was significantly correlated with reservoir species dominance [58] . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln (A s / (tan (β)) where; A s = Area Value calculated as ((flow accumulation + 1) Ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . Though previous studies have indicated that Normalized Difference Vegetation Index (NDVI) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included NDVI [9] . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health [10] . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 [10] . We chose to study the first wave (Wave 1) that ended in February 2004 and the second wave (Wave 2) that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study [9] . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models (DEM) acquired from the Consortium for Spatial Information (CGIAR-CSI). Compound Topographical Index (CTI) data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software (Palisade Corporation). Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the Pearson correlation coefficient). When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and CTI), c) chicken density (only at national level, correlated with CTI), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI), e) annual temperature (correlated with elevation and CTI) and f) cropping intensity (correlated with percentage land under paddy). Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level Generalized Linear Mixed Model (GLMM) and 2) Boosted Regression trees (BRT) [63, 64] with an autoregressive term [65] . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance [65] . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . We used a two-level GLMM (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . It is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease [16, 51, [69] [70] [71] . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve (AUC) of the receiver operation characteristic (ROC) curve. AUC is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . We used the corrected Akaike Information Criteria (AICc) to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 (IBM Corp., New York, 2012) for GLMM and R version 3.1.0 (The R Foundation for Statistical Computing, 2014) for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled (see tables) were all found to be significantly associated with HPAI H5N1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( Table 1) . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 (Tables 2-7 ). The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 (AUC = 0.69) and Wave 2 (AUC = 0.77) by Gilbert et al. [11] . Both Gilbert et al. [11] and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China [51] and in the Red River Delta [53] . A global study of human cases also found occurrence to be higher under drier conditions [74] . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk (RR) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index (CTI) to be highest in peri-urban areas (Fig 1a-1e) . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (Fig 1f) . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). In the GLMM models we found the Akaike Information Criterion (AIC) using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. [17] found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam (Red River and Mekong), urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics (e.g. [75] [76] [77] . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). Mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in Viet Nam) is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta [61] . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present [80] . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

What is the effect of diversity of chicken flock on H5N1 disease?

Responding to the COVID-19 pandemic in complex humanitarian crises https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085188/ SHA: d013e42811c6442b184da3b9bbfd9e334031a975 Authors: Poole, Danielle N.; Escudero, Daniel J.; Gostin, Lawrence O.; Leblang, David; Talbot, Elizabeth A. Date: 2020-03-21 DOI: 10.1186/s12939-020-01162-y License: cc-by Abstract: nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 [1] . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale [2] . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene (WASH) tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations [3] . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing [4] . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% [5] . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production [6] . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance [7] . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines [5, 8] . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence [9] . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations [10] . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings [2] . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic.

What should be the priority of the national and international bodies trying to prevent the pandemic?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What is the status of MVA influenza vaccine?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What other viruses have been recently reported as contributing to acute exacerbations?

First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164/ SHA: ce358c18aac69fc83c7b2e9a7dca4a43b0f60e2e Authors: Spiteri, Gianfranco; Fielding, James; Diercke, Michaela; Campese, Christine; Enouf, Vincent; Gaymard, Alexandre; Bella, Antonino; Sognamiglio, Paola; Sierra Moros, Maria José; Riutort, Antonio Nicolau; Demina, Yulia V.; Mahieu, Romain; Broas, Markku; Bengnér, Malin; Buda, Silke; Schilling, Julia; Filleul, Laurent; Lepoutre, Agnès; Saura, Christine; Mailles, Alexandra; Levy-Bruhl, Daniel; Coignard, Bruno; Bernard-Stoecklin, Sibylle; Behillil, Sylvie; van der Werf, Sylvie; Valette, Martine; Lina, Bruno; Riccardo, Flavia; Nicastri, Emanuele; Casas, Inmaculada; Larrauri, Amparo; Salom Castell, Magdalena; Pozo, Francisco; Maksyutov, Rinat A.; Martin, Charlotte; Van Ranst, Marc; Bossuyt, Nathalie; Siira, Lotta; Sane, Jussi; Tegmark-Wisell, Karin; Palmérus, Maria; Broberg, Eeva K.; Beauté, Julien; Jorgensen, Pernille; Bundle, Nick; Pereyaslov, Dmitriy; Adlhoch, Cornelia; Pukkila, Jukka; Pebody, Richard; Olsen, Sonja; Ciancio, Bruno Christian Date: 2020-03-05 DOI: 10.2807/1560-7917.es.2020.25.9.2000178 License: cc-by Abstract: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters’ index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. Text: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. A cluster of pneumonia of unknown origin was identified in Wuhan, China, in December 2019 [1] . On 12 January 2020, Chinese authorities shared the sequence of a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from some clustered cases [2] . Since then, the disease caused by SARS-CoV-2 has been named coronavirus disease 2019 (COVID -19) . As at 21 February 2020, the virus had spread rapidly mostly within China but also to 28 other countries, including in the World Health Organization (WHO) European Region [3] [4] [5] . Here we describe the epidemiology of the first cases of COVID-19 in this region, excluding cases reported in the United Kingdom (UK), as at 21 February 2020. The study includes a comparison between cases detected among travellers from China and cases whose infection was acquired due to subsequent local transmission. On 27 January 2020, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe asked countries to complete a WHO standard COVID-19 case report form for all confirmed and probable cases according to WHO criteria [6] [7] [8] . The overall aim of surveillance at this time was to support the global strategy of containment of COVID-19 with rapid identification and follow-up of cases linked to affected countries in order to minimise onward transmission. The surveillance objectives were to: describe the key epidemiological and clinical characteristics of COVID-19 cases detected in Europe; inform country preparedness; and improve further case detection and management. Data collected included demographics, history of recent travel to affected areas, close contact with a probable or confirmed COVID-19 case, underlying conditions, signs and symptoms of disease at onset, type of specimens from which the virus was detected, and clinical outcome. The WHO case definition was adopted for surveillance: a confirmed case was a person with laboratory confirmation of SARS-CoV-2 infection (ECDC recommended two separate SARS-CoV-2 RT-PCR tests), irrespective of clinical signs and symptoms, whereas a probable case was a suspect case for whom testing for SARS-CoV-2 was inconclusive or positive using a pan-coronavirus assay [8] . By 31 January 2020, 47 laboratories in 31 countries, including 38 laboratories in 24 European Union and European Economic Area (EU/EEA) countries, had diagnostic capability for SARS-CoV-2 available (close to 60% of countries in the WHO European Region), with cross-border shipment arrangements in place for many of those lacking domestic testing capacity. The remaining six EU/EEA countries were expected to have diagnostic testing available by mid-February [9] . As at 09:00 on 21 February 2020, 47 confirmed cases of COVID-19 were reported in the WHO European Region and one of these cases had died [4] . Data on 38 of these cases (i.e. all except the nine reported in the UK) are included in this analysis. The first three cases detected were reported in France on 24 January 2020 and had onset of symptoms on 17, 19 and 23 January respectively [10] . The first death was reported on 15 February in France. As at 21 February, nine countries had reported cases ( Figure) : Belgium (1), Finland (1), France (12), Germany (16), Italy (3), Russia (2), Spain (2), Sweden (1) and the UK (9 -not included further). The place of infection (assessed at national level based on an incubation period presumed to be up to 14 days [11] , travel history and contact with probable or confirmed cases as per the case definition) was reported for 35 cases (missing for three cases), of whom 14 were infected in China (Hubei province: 10 cases; Shandong province: one case; province not reported for three cases). The remaining 21 cases were infected in Europe. Of these, 14 were linked to a cluster in Bavaria, Germany, and seven to a cluster in Haute-Savoie, France [12, 13] . Cases from the Bavarian cluster were reported from Germany and Spain, whereas cases from the Haute-Savoie cluster were reported from France All but two cases were hospitalised (35 of 37 where information on hospitalisation was reported), although it is likely that most were hospitalised to isolate the person rather than because of severe disease. The time from onset of symptoms to hospitalisation (and isolation) ranged between 0 and 10 days with a mean of 3.7 days (reported for 29 cases). The mean number of days to hospitalisation was 2.5 days for cases imported from China, but 4.6 days for those infected in Europe. This was mostly a result of delays in identifying the index cases of the two clusters in France and Germany. In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six took only a mean of 2 days to be hospitalised. Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc. were reported for any of the 38 cases. All hospitalised cases had a benign clinical evolution except four, two reported in Italy and two reported in France, all of whom developed viral pneumonia. All three cases who were aged 65 years or over were admitted to intensive care and required respiratory support and one French case died. The case who died was hospitalised for 21 days and required intensive care and mechanical ventilation for 19 days. The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] . As at 5 March 2020, there are 4,250 cases including 113 deaths reported among 38 countries in the WHO European region [19] . In our analysis of early cases, we observed transmission in two broad contexts: sporadic cases among travellers from China (14 cases) and cases who acquired infection due to subsequent local transmission in Europe (21 cases). Our analysis shows that the time from symptom onset to hospitalisation/case isolation was about 3 days longer for locally acquired cases than for imported cases. People returning from affected areas are likely to have a low threshold to seek care and be tested when symptomatic, however delays in identifying the index cases of the two clusters in France and Germany meant that locally acquired cases took longer to be detected and isolated. Once the exposure is determined and contacts identified and quarantined (171 contacts in France and 200 in Germany for the clusters in Haute-Savoie and Bavaria, respectively), further cases are likely to be rapidly detected and isolated when they develop symptoms [15, 20] . In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six were hospitalised after a mean of 2 days. Locally acquired cases require significant resources for contact tracing and quarantine, and countries should be prepared to allocate considerable public health resources during the containment phase, should local clusters emerge in their population. In addition, prompt sharing of information on cases and contacts through international notification systems such as the International Health Regulations (IHR) mechanism and the European Commission's European Early Warning and Response System is essential to contain international spread of infection. All of the imported cases had a history of travel to China. This was consistent with the epidemiological situation in Asia, and supported the recommendation for testing of suspected cases with travel history to China and potentially other areas of presumed ongoing community transmission. The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic. Hospital-based surveillance could help estimate the incidence of severe cases and identify risk factors for severity and death. Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2. This approach will be used globally to help identify evidence of widespread community transmission and, should the virus spread and containment no longer be deemed feasible, to monitor intensity of disease transmission, trends and its geographical spread. Additional research is needed to complement surveillance data to build knowledge on the infectious period, modes of transmission, basic and effective reproduction numbers, and effectiveness of prevention and case management options also in settings outside of China. Such special studies are being conducted globally, including a cohort study on citizens repatriated from China to Europe, with the aim to extrapolate disease incidence and risk factors for infection in areas with community transmission. Countries together with ECDC and WHO, should use all opportunities to address these questions in a coordinated fashion at the European and global level. provided input to the outline, multiple versions of the manuscript and gave approval to the final draft.

What was the duration of hospitalisation reported for 16 cases ?

Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review https://doi.org/10.3390/jcm9030623 SHA: 9b0c87f808b1b66f2937d7a7acb524a756b6113b Authors: Pang, Junxiong; Wang, Min Xian; Ang, Ian Yi Han; Tan, Sharon Hui Xuan; Lewis, Ruth Frances; Chen, Jacinta I. Pei; Gutierrez, Ramona A.; Gwee, Sylvia Xiao Wei; Chua, Pearleen Ee Yong; Yang, Qian; Ng, Xian Yi; Yap, Rowena K. S.; Tan, Hao Yi; Teo, Yik Ying; Tan, Chorh Chuan; Cook, Alex R.; Yap, Jason Chin-Huat; Hsu, Li Yang Date: 2020 DOI: 10.3390/jcm9030623 License: cc-by Abstract: Rapid diagnostics, vaccines and therapeutics are important interventions for the management of the 2019 novel coronavirus (2019-nCoV) outbreak. It is timely to systematically review the potential of these interventions, including those for Middle East respiratory syndrome-Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS)-CoV, to guide policymakers globally on their prioritization of resources for research and development. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Supplementary strategies through Google Search and personal communications were used. A total of 27 studies fulfilled the criteria for review. Several laboratory protocols for confirmation of suspected 2019-nCoV cases using real-time reverse transcription polymerase chain reaction (RT-PCR) have been published. A commercial RT-PCR kit developed by the Beijing Genomic Institute is currently widely used in China and likely in Asia. However, serological assays as well as point-of-care testing kits have not been developed but are likely in the near future. Several vaccine candidates are in the pipeline. The likely earliest Phase 1 vaccine trial is a synthetic DNA-based candidate. A number of novel compounds as well as therapeutics licensed for other conditions appear to have in vitro efficacy against the 2019-nCoV. Some are being tested in clinical trials against MERS-CoV and SARS-CoV, while others have been listed for clinical trials against 2019-nCoV. However, there are currently no effective specific antivirals or drug combinations supported by high-level evidence. Text: Since mid-December 2019 and as of early February 2020, the 2019 novel coronavirus (2019-nCoV) originating from Wuhan (Hubei Province, China) has infected over 25,000 laboratory-confirmed cases across 28 countries with about 500 deaths (a case-fatality rate of about 2%). More than 90% of the cases and deaths were in China [1] . Based on the initial reported surge of cases in Wuhan, the majority were males with a median age of 55 years and linked to the Huanan Seafood Wholesale Market [2] . Most of the reported cases had similar symptoms at the onset of illness such as fever, cough, and myalgia or fatigue. Most cases developed pneumonia and some severe and even fatal respiratory diseases such as acute respiratory distress syndrome [3] . The 2019 novel coronavirus (2019-nCoV), a betacoronavirus, forms a clade within the subgenus sarbecovirus of the Orthocoronavirinae subfamily [4] . The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also betacoronaviruses that are zoonotic in origin and have been linked to potential fatal illness during the outbreaks in 2003 and 2012, respectively [5, 6] . Based on current evidence, pathogenicity for 2019-nCoV is about 3%, which is significantly lower than SARS-CoV (10%) and MERS-CoV (40%) [7] . However, 2019-nCoV has potentially higher transmissibility (R0: 1.4-5.5) than both SARS-CoV (R0: [2] [3] [4] [5] and MERS-CoV (R0: <1) [7] . With the possible expansion of 2019-nCoV globally [8] and the declaration of the 2019-nCoV outbreak as a Public Health Emergency of International Concern by the World Health Organization, there is an urgent need for rapid diagnostics, vaccines and therapeutics to detect, prevent and contain 2019-nCoV promptly. There is however currently a lack of understanding of what is available in the early phase of 2019-nCoV outbreak. The systematic review describes and assesses the potential rapid diagnostics, vaccines and therapeutics for 2019-nCoV, based in part on the developments for MERS-CoV and SARS-CoV. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies examining the diagnosis, therapeutic drugs and vaccines for Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the 2019 novel coronavirus (2019-nCoV), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There were two independent reviewers each focusing on SARS, MERS, and 2019-nCoV, respectively. A third independent reviewer was engaged to resolve any conflicting article of interest. We used the key words "SARS", "coronavirus", "MERS", "2019 Novel coronavirus", "Wuhan virus" to identify the diseases in the search strategy. The systematic searches for diagnosis, therapeutic drugs and vaccines were carried out independently and the key words "drug", "therapy", "vaccine", "diagnosis", "point of care testing" and "rapid diagnostic test" were used in conjunction with the disease key words for the respective searches. Examples of search strings can be found in Table S1 . We searched for randomized controlled trials (RCTs) and validation trials (for diagnostics test) published in English, that measured (a) the sensitivity and/or specificity of a rapid diagnostic test or a point-of-care testing kit, (b) the impact of drug therapy or (c) vaccine efficacy against either of these diseases with no date restriction applied. For the 2019-nCoV, we searched for all in vitro, animal, or human studies published in English between 1 December 2019 and 6 February 2020, on the same outcomes of interest. In addition, we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial searches. Studies that examined the mechanisms of diagnostic tests, drug therapy or vaccine efficacy against SARS, MERS and 2019-nCoV were excluded. A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles. With the emergence of 2019-nCoV, real time RT-PCR remains the primary means for diagnosing the new virus strain among the many diagnostic platforms available ( [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ; Table S3 ). Among the 16 diagnostics studies selected, one study discussed the use of RT-PCR in diagnosing patients with 2019-nCoV [11] ( Table 1 ). The period and type of specimen collected for RT-PCR play an important role in the diagnosis of 2019-nCoV. It was found that the respiratory specimens were positive for the virus while serum was negative in the early period. It has also suggested that in the early days of illness, patients have high levels of virus despite the mild symptoms. Apart from the commonly used RT-PCR in diagnosing MERS-CoV, four studies identified various diagnostic methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), RT-insulated isothermal PCR (RT-iiPCR) and a one-step rRT-PCR assay based on specific TaqMan probes. RT-LAMP has similar sensitivity as real time RT-PCR. It is also highly specific and is used to detect MERS-CoV. It is comparable to the usual diagnostic tests and is rapid, simple and convenient. Likewise, RT-iiPCR and a one-step rRT-PCR assay have also shown similar sensitivity and high specificity for MER-CoV. Lastly, one study focused on the validation of the six commercial real RT-PCR kits, with high accuracy. Although real time RT-PCR is a primary method for diagnosing MERS-CoV, high levels of PCR inhibition may hinder PCR sensitivity (Table 1) . There are eleven studies that focus on SARS-CoV diagnostic testing (Table 1) . These papers described diagnostic methods to detect the virus with the majority of them using molecular testing for diagnosis. Comparison between the molecular test (i.e RT-PCR) and serological test (i.e., ELISA) showed that the molecular test has better sensitivity and specificity. Hence, enhancements to the current molecular test were conducted to improve the diagnosis. Studies looked at using nested PCR to include a pre-amplification step or incorporating N gene as an additional sensitive molecular marker to improve on the sensitivity (Table 1 ). In addition, there are seven potential rapid diagnostic kits (as of 24 January 2020; Table 2 ) available on the market for 2019-nCoV. Six of these are only for research purposes. Only one kit from Beijing Genome Institute (BGI) is approved for use in the clinical setting for rapid diagnosis. Most of the kits are for RT-PCR. There were two kits (BGI, China and Veredus, Singapore) with the capability to detect multiple pathogens using sequencing and microarray technologies, respectively. The limit of detection of the enhanced realtime PCR method was 10 2 -fold higher than the standard real-time PCR assay and 10 7fold higher than conventional PCR methods In the clinical aspect, the enhanced realtime PCR method was able to detect 6 cases of SARS-CoV positive samples that were not confirmed by any other assay [25] • The real time PCR has a threshold sensitivity of 10 genome equivalents per reaction and it has a good reproducibility with the inter-assay coefficients of variation of 1.73 to 2.72%. • 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. The real-time RT-PCR reaction was more sensitive than the nested PCR reaction, as the detection limit for the nested PCR reaction was about 10 3 genome equivalents in the standard cDNA control. [34] Real-time reverse-transcription PCR (rRT-PCR); RNA-dependent RNA polymerase (RdRp); open reading frame 1a (ORF1a); Loop-mediated isothermal amplification (LAMP); enzyme-linked immunosorbent assay (ELISA); immunofluorescent assay (IFA); immunochromatographic test (ICT); nasopharyngeal aspirate (NPA). With the emergence of 2019-nCoV, there are about 15 potential vaccine candidates in the pipeline globally (Table 3 ), in which a wide range of technology (such as messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle) was applied. It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening. The published clinical trials were mostly done in United States except for one on the SARS vaccine done in China [44] . All vaccine candidates for SARS and MERS were reported to be safe, well-tolerated and able to trigger the relevant and appropriate immune responses in the participants. In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60. [47] Molecules developed by the university scientists inhibit two coronavirus enzymes and prevent its replication. The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] . The trial conducted in the United States in 2017 demonstrated SAB-301 to be safe and well-tolerated at single doses. Another trial on MERS therapeutics was found on ClinicalTrials.gov-a phase 2/3 trial in the United States evaluating the safety, tolerability, pharmacokinetics (PK), and immunogenicity on coadministered MERS-CoV antibodies REGN3048 & REGN3051 [100]. Rapid diagnostics plays an important role in disease and outbreak management. The fast and accurate diagnosis of a specific viral infection enables prompt and accurate public health surveillance, prevention and control measures. Local transmission and clusters can be prevented or delayed by isolation of laboratory-confirmed cases and their close contacts quarantined and monitored at home. Rapid diagnostic also facilitates other specific public health interventions such as closure of high-risk facilities and areas associated with the confirmed cases for prompt infection control and environmental decontamination [11, 101] . Laboratory diagnosis can be performed by: (a) detecting the genetic material of the virus, (b) detecting the antibodies that neutralize the viral particles of interest, (c) detecting the viral epitopes of interest with antibodies (serological testing), or (d) culture and isolation of viable virus particles. The key limitations of genetic material detection are the lack of knowledge of the presence of viable virus, the potential cross-reactivity with non-specific genetic regions and the short timeframe for accurate detection during the acute infection phase. The key limitations of serological testing is the need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses. The limitation of virus culture and isolation is the long duration and the highly specialized skills required of the technicians to process the samples. All patients recovered. Significantly shorted time from the disease onset to the symptom improvement in treatment (5.10 ± 2.83 days) compared to control group (7.62 ± 2.27 days) (p < 0.05) No significant difference in blood routine improvement, pulmonary chest shadow in chest film improvement and corticosteroid usgae between the 2 groups. However, particularly in the respect of improving clinical symptoms, elevating quality of life, promoting immune function recovery, promoting absorption of pulmonary inflammation, reducing the dosage of cortisteroid and shortening the therapeutic course, treatment with integrative chinese and western medicine treatment had obvious superiority compared with using control treatment alone. Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well-tolerated in healthy participants. [46] Where the biological samples are taken from also play a role in the sensitivity of these tests. For SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates have higher and more prolonged levels of viral RNA because of the tropism of the virus. MERS-CoV viral loads are also higher for severe cases and have longer viral shedding compared to mild cases. Although upper respiratory tract specimens such as nasopharyngeal or oropharyngeal swabs can be used, they have potentially lower viral loads and may have higher risk of false-negatives among the mild MERS and SARS cases [102, 103] , and likely among the 2019-nCoV cases. The existing practices in detecting genetic material of coronaviruses such as SARS-CoV and MERS-CoV include (a) reverse transcription-polymerase chain reaction (RT-PCR), (b) real-time RT-PCR (rRT-PCR), (c) reverse transcription loop-mediated isothermal amplification (RT-LAMP) and (d) real-time RT-LAMP [104] . Nucleic amplification tests (NAAT) are usually preferred as in the case of MERS-CoV diagnosis as it has the highest sensitivity at the earliest time point in the acute phase of infection [102] . Chinese health authorities have recently posted the full genome of 2019-nCoV in the GenBank and in GISAID portal to facilitate in the detection of the virus [11] . Several laboratory assays have been developed to detect the novel coronavirus in Wuhan, as highlighted in WHO's interim guidance on nCoV laboratory testing of suspected cases. These include protocols from other countries such as Thailand, Japan and China [105] . The first validated diagnostic test was designed in Germany. Corman et al. had initially designed a candidate diagnostic RT-PCR assay based on the SARS or SARS-related coronavirus as it was suggested that circulating virus was SARS-like. Upon the release of the sequence, assays were selected based on the match against 2019-nCoV upon inspection of the sequence alignment. Two assays were used for the RNA dependent RNA polymerase (RdRP) gene and E gene where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing. All assays were highly sensitive and specific in that they did not cross-react with other coronavirus and also human clinical samples that contained respiratory viruses [11] . The Hong Kong University used two monoplex assays which were reactive with coronaviruses under the subgenus Sarbecovirus (consisting of 2019-nCoV, SARS-CoV and SARS-like coronavirus). Viral RNA extracted from SARS-CoV can be used as the positive control for the suggested protocol assuming that SARS has been eradicated. It is proposed that the N gene RT-PCR can be used as a screening assay while the Orf1b assay acts as a confirmatory test. However, this protocol has only been evaluated with a panel of controls with the only positive control SARS-CoV RNA. Synthetic oligonucleotide positive control or 2019-nCoV have yet to be tested [106] . The US CDC shared the protocol on the real time RT-PCR assay for the detection of the 2019-nCoV with the primers and probes designed for the universal detection of SARS-like coronavirus and the specific detection of 2019-nCoV. However, the protocol has not been validated on other platforms or chemistries apart from the protocol described. There are some limitations for the assay. Analysts engaged have to be trained and familiar with the testing procedure and result interpretation. False negative results may occur due to insufficient organisms in the specimen resulting from improper collection, transportation or handling. Also, RNA viruses may show substantial genetic variability. This could result in mismatch between the primer and probes with the target sequence which can diminish the assay performance or result in false negative results [107] . Point-of-care test kit can potentially minimize these limitations, which should be highly prioritized for research and development in the next few months. Serological testing such as ELISA, IIFT and neutralization tests are effective in determining the extent of infection, including estimating asymptomatic and attack rate. Compared to the detection of viral genome through molecular methods, serological testing detects antibodies and antigens. There would be a lag period as antibodies specifically targeting the virus would normally appear between 14 and 28 days after the illness onset [108] . Furthermore, studies suggest that low antibody titers in the second week or delayed antibody production could be associated with mortality with a high viral load. Hence, serological diagnoses are likely used when nucleic amplification tests (NAAT) are not available or accessible [102] . Vaccines can prevent and protect against infection and disease occurrence when exposed to the specific pathogen of interest, especially in vulnerable populations who are more prone to severe outcomes. In the context of the current 2019-nCoV outbreak, vaccines will help control and reduce disease transmission by creating herd immunity in addition to protecting healthy individuals from infection. This decreases the effective R0 value of the disease. Nonetheless, there are social, clinical and economic hurdles for vaccine and vaccination programmes, including (a) the willingness of the public to undergo vaccination with a novel vaccine, (b) the side effects and severe adverse reactions of vaccination, (c) the potential difference and/or low efficacy of the vaccine in populations different from the clinical trials' populations and (d) the accessibility of the vaccines to a given population (including the cost and availability of the vaccine). Vaccines against the 2019-nCoV are currently in development and none are in testing (at the time of writing). On 23 January 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) announced that they will fund vaccine development programmes with Inovio, The University of Queensland and Moderna, Inc respectively, with the aim to test the experimental vaccines clinically in 16 weeks (By June 2020). The vaccine candidates will be developed by the DNA, recombinant and mRNA vaccine platforms from these organizations [109] . Based on the most recent MERS-CoV outbreak, there are already a number of vaccine candidates being developed but most are still in the preclinical testing stage. The vaccines in development include viral vector-based vaccine, DNA vaccine, subunit vaccine, virus-like particles (VLPs)-based vaccine, inactivated whole-virus (IWV) vaccine and live attenuated vaccine. The latest findings for these vaccines arebased on the review by Yong et al. (2019) in August 2019 [110] . As of the date of reporting, there is only one published clinical study on the MERS-CoV vaccine by GeneOne Life Science & Inovio Pharmaceuticals [47] . There was one SARS vaccine trial conducted by the US National Institute of Allergy and Infectious Diseases. Both Phase I clinical trials reported positive results, but only one has announced plans to proceed to Phase 2 trial [111] . Due to the close genetic relatedness of SARS-CoV (79%) with 2019-nCoV [112] , there may be potential cross-protective effect of using a safe SARS-CoV vaccine while awaiting the 2019-nCoV vaccine. However, this would require small scale phase-by-phase implementation and close monitoring of vaccinees before any large scale implementation. Apart from the timely diagnosis of cases, the achievement of favorable clinical outcomes depends on the timely treatment administered. ACE2 has been reported to be the same cell entry receptor used by 2019-nCoV to infect humans as SARS-CoV [113] . Hence, clinical similarity between the two viruses is expected, particularly in severe cases. In addition, most of those who have died from MERS-CoV, SARS-CoV and 2019-nCoV were advance in age and had underlying health conditions such as hypertension, diabetes or cardiovascular disease that compromised their immune systems [114] . Coronaviruses have error-prone RNA-dependent RNA polymerases (RdRP), which result in frequent mutations and recombination events. This results in quasispecies diversity that is closely associated with adaptive evolution and the capacity to enhance viral-cell entry to cause disease over time in a specific population at-risk [115] . Since ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, coronaviruses are likely to infect the upper respiratory and gastrointestinal tract and this may influence the type of therapeutics against 2019-nCoV, similarly to SAR-CoV. However, in the years following two major coronavirus outbreaks SARS-CoV in 2003 and MERS-CoV in 2012, there remains no consensus on the optimal therapy for either disease [116, 117] . Well-designed clinical trials that provide the gold standard for assessing the therapeutic measures are scarce. No coronavirus protease inhibitors have successfully completed a preclinical development program despite large efforts exploring SARS-CoV inhibitors. The bulk of potential therapeutic strategies remain in the experimental phase, with only a handful crossing the in vitro hurdle. Stronger efforts are required in the research for treatment options for major coronaviruses given their pandemic potential. Effective treatment options are essential to maximize the restoration of affected populations to good health following infections. Clinical trials have commenced in China to identify effective treatments for 2019-nCoV based on the treatment evidence from SARS and MERS. There is currently no effective specific antiviral with high-level evidence; any specific antiviral therapy should be provided in the context of a clinical study/trial. Few treatments have shown real curative action against SARS and MERS and the literature generally describes isolated cases or small case series. Many interferons from the three classes have been tested for their antiviral activities against SARS-CoV both in vitro and in animal models. Interferon β has consistently been shown to be the most active, followed by interferon α. The use of corticosteroids with interferon alfacon-1 (synthetic interferon α) appeared to have improved oxygenation and faster resolution of chest radiograph abnormalities in observational studies with untreated controls. Interferon has been used in multiple observational studies to treat SARS-CoV and MERS-CoV patients [116, 117] . Interferons, with or without ribavirin, and lopinavir/ritonavir are most likely to be beneficial and are being trialed in China for 2019-nCoV. This drug treatment appears to be the most advanced. Timing of treatment is likely an important factor in effectiveness. A combination of ribavirin and lopinavir/ritonavir was used as a post-exposure prophylaxis in health care workers and may have reduced the risk of infection. Ribavirin alone is unlikely to have substantial antiviral activities at clinically used dosages. Hence, ribavirin with or without corticosteroids and with lopinavir and ritonavir are among the combinations employed. This was the most common agent reported in the available literature. Its efficacy has been assessed in observational studies, retrospective case series, retrospective cohort study, a prospective observational study, a prospective cohort study and randomized controlled trial ranging from seven to 229 participants [117] . Lopinavir/ritonavir (Kaletra) was the earliest protease inhibitor combination introduced for the treatment of SARS-CoV. Its efficacy was documented in several studies, causing notably lower incidence of adverse outcomes than with ribavirin alone. Combined usage with ribavirin was also associated with lower incidence of acute respiratory distress syndrome, nosocomial infection and death, amongst other favorable outcomes. Recent in vitro studies have shown another HIV protease inhibitor, nelfinavir, to have antiviral capacity against SARS-CoV, although it has yet to show favorable outcomes in animal studies [118] . Remdesivir (Gilead Sciences, GS-5734) nucleoside analogue in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several coronaviruses (CoVs), including highly pathogenic CoVs and potentially emergent BatCoVs. The use of remdesivir may be a good candidate as an investigational treatment. Improved mortality following receipt of convalescent plasma in various doses was consistently reported in several observational studies involving cases with severe acute respiratory infections (SARIs) of viral etiology. A significant reduction in the pooled odds of mortality following treatment of 0.25 compared to placebo or no therapy was observed [119] . Studies were however at moderate to high risk of bias given their small sample sizes, allocation of treatment based on the physician's discretion, and the availability of plasma. Factors like concomitant treatment may have also confounded the results. Associations between convalescent plasma and hospital length of stay, viral antibody levels, and viral load respectively were similarly inconsistent across available literature. Convalescent plasma, while promising, is likely not yet feasible, given the limited pool of potential donors and issues of scalability. Monoclonal antibody treatment is progressing. SARS-CoV enters host cells through the binding of their spike (S) protein to angiotensin converting enzyme 2 (ACE2) and CD209L [118] . Human monoclonal antibodies to the S protein have been shown to significantly reduce the severity of lung pathology in non-human primates following MERS-CoV infection [120] . Such neutralizing antibodies can be elicited by active or passive immunization using vaccines or convalescent plasma respectively. While such neutralizing antibodies can theoretically be harvested from individuals immunized with vaccines, there is uncertainty over the achievement of therapeutic levels of antibodies. Other therapeutic agents have also been reported. A known antimalarial agent, chloroquine, elicits antiviral effects against multiple viruses including HIV type 1, hepatitis B and HCoV-229E. Chloroquine is also immunomodulatory, capable of suppressing the production and release of factors which mediate the inflammatory complications of viral diseases (tumor necrosis factor and interleukin 6) [121] . It is postulated that chloroquine works by altering ACE2 glycosylation and endosomal pH. Its anti-inflammatory properties may be beneficial for the treatment of SARS. Niclosamide as a known drug used in antihelminthic treatment. The efficacy of niclosamide as an inhibitor of virus replication was proven in several assays. In both immunoblot analysis and immunofluorescence assays, niclosamide treatment was observed to completely inhibit viral antigen synthesis. Reduction of virus yield in infected cells was dose dependent. Niclosamide likely does not interfere in the early stages of virus attachment and entry into cells, nor does it function as a protease inhibitor. Mechanisms of niclosamide activity warrant further investigation [122] . Glycyrrhizin also reportedly inhibits virus adsorption and penetration in the early steps of virus replication. Glycyrrhizin was a significantly potent inhibitor with a low selectivity index when tested against several pathogenic flaviviruses. While preliminary results suggest production of nitrous oxide (which inhibits virus replication) through induction of nitrous oxide synthase, the mechanism of Glycyrrhizin against SARS-CoV remains unclear. The compound also has relatively lower toxicity compared to protease inhibitors like ribavirin [123] . Inhibitory activity was also detected in baicalin [124] , extracted from another herb used in the treatment of SARS in China and Hong Kong. Findings on these compounds are limited to in vitro studies [121] [122] [123] [124] . Due to the rapidly evolving situation of the 2019-nCoV, there will be potential limitations to the systematic review. The systematic review is likely to have publication bias as some developments have yet to be reported while for other developments there is no intention to report publicly (or in scientific platforms) due to confidentiality concerns. However, this may be limited to only a few developments for review as publicity does help in branding to some extent for the company and/or the funder. Furthermore, due to the rapid need to share the status of these developments, there may be reporting bias in some details provided by authors of the scientific articles or commentary articles in traditional media. Lastly, while it is not viable for any form of quality assessment and metaanalysis of the selected articles due to the limited data provided and the heterogeneous style of reporting by different articles, this paper has provided a comprehensive overview of the potential developments of these pharmaceutical interventions during the early phase of the outbreak. This systematic review would be useful for cross-check when the quality assessment and meta-analysis of these developments are performed as a follow-up study. Rapid diagnostics, vaccines and therapeutics are key pharmaceutical interventions to limit transmission of respiratory infectious diseases. Many potential developments on these pharmaceutical interventions for 2019-nCoV are ongoing in the containment phase of this outbreak, potentially due to better pandemic preparedness than before. However, lessons from MERS-CoV and SARS-CoV have shown that the journeys for these developments can still be challenging moving ahead. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1 : Example of full search strategy in Pubmed, Table S2 : Google Search: 2019-nCoV diagnostics, Table S3 : Summary of diagnostic assays developed for 2019-nCoV, Table S4

What was the purpose of the search?

Epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-nCoV) outbreak https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029449/ SHA: 90de2d957e1960b948b8c38c9877f9eca983f9eb Authors: Cowling, Benjamin J; Leung, Gabriel M Date: 2020-02-13 DOI: 10.2807/1560-7917.es.2020.25.6.2000110 License: cc-by Abstract: Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared.

when is viral shedding the highest?

Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.

What invention has made bacteriophage useful for research?

Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/ SHA: f2d842780b9928cc70f38a4458553f2431877603 Authors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian Date: 2011-08-03 DOI: 10.3390/molecules16086489 License: cc-by Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, "Asian Influenza (1957/H2N2)" and "Hong Kong Influenza (1968/H3N2)" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view.

What is Tamiflu?

Viruses and Evolution – Viruses First? A Personal Perspective https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/ SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1 Authors: Moelling, Karin; Broecker, Felix Date: 2019-03-19 DOI: 10.3389/fmicb.2019.00523 License: cc-by Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) . tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA (Flint, 2015) . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) . From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs. information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first." ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ). An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below). Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) . Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) . The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) . These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence.

How are the ribozymes able to replicate, join and create peptide bonds?

Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/ SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9 Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W. Date: 2012-11-15 DOI: 10.1371/journal.pone.0048702 License: cc-by Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production. Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] . The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] . The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated. In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis. In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ). We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination. Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus. We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold. Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes. We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression. Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] . Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] . STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] . YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] . ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] . Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] . CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] . ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] . To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold). In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) . Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF. Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] . Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression. Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) . DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) . Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] . Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro. The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function. We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag. Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] . Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis. HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability. Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection. Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] . HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules. We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined. The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach. The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours. While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention. Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore). The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter). Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology). For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] . Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap. The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown). The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%. Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] . The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific). To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective. The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key: (R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis. (DOCX)

What cellular process is the Tat protein essential to?

1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger" and David M. Morens1- The “Spanish" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population (or z500 million persons) were infected and had clinical- ly apparent illnesses (1,2) during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics (3,4). Total deaths were estimated at z50 million (577) and were arguably as high as 100 mil- lion (7). The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide (except- ing human infections from avian Viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses (now known to be H1N1 Viruses) were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic (8). Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 (“Asian flu”), the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer (9). They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage (so-called classic swine flu), and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains (prevalent from 1968 until the present). H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus (10). These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies (11717) and a number of reviews covering different aspects of the 1918 pandemic have recently been published ([8720) and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus (19) and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America (the first wave was best described in the United States in March 1918). Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus (21), and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context ([9). Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 (22). Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively (Figure 1). Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps (23), but a counter argument is that if a strain with a new hemagglutinin (HA) was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous (or nearly simultaneous) infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 (12,20). Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase (NA) surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before (12,15, 24). More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 (20). Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date (19). In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 (21). In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months (Figure 1). Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 (21). Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures (bene- ficial to thermolabile Viruses such as influenza), optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter (of the Northern Hemisphere), respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 (more than a year after the first pandemic appearance) and the third in early 1892 (21 ). As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients ([6), nothing can yet be said about whether the first (spring) wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source (17,19), as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods (24,25). For example, the 1918 nucleoprotein (NP) gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains (13,19). One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses (26); both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 (273) linked sugars (27729). Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 (2%) link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change (30), and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it (16). This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice (31). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (32). These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA (and possibly the NA) contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped (Figure 2), exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third (middle) distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years (35). Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic (33). A sharper perspective emerges when 1918 age-specific influenza morbidity rates (21) are used to adj ust the W- shaped mortality curve (Figure 3, panels, A, B, and C [35,37]). Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence (Figure 3, panel A). But even after adjusting age-specific deaths by age-specif— ic clinical attack rates (Figure 3, panel B), a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 (Figure 3, panel C). Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior Virus exposures [38]) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough (>35 years) to have been infected during that prior era (35). But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 (21). Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 (dashed line) and for the pandemic year 1918 (solid line) (33,34). Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia (P&l) (combined) age-specific incidence rates per 1,000 persons per age group (panel A), death rates per 1,000 persons, ill and well combined (panel B), and case-fatality rates (panel C, solid line), US Public Health Service house-to-house surveys, 8 states, 1918 (36). A more typical curve of age-specific influenza case-fatality (panel C, dotted line) is taken from US Public Health Service surveys during 1928—1929 (37). the 5 to 15-year-old group jumped to 25% of influenza cases (compatible with exposure to an antigenically novel Virus strain), while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum (Figure 2), a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 (>95% in most locales in industrialized nations) were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time (Figure 1). Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus (39), though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza (HPAI) Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation (e.g., receptor binding) are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the (alleged) pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second (fall) and third (winter) waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge (UK): Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A (H1N1) viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl(N-acetyllactosamine). Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

When did the first wave of the H1N1 swine flu (Spanish Influenza) occur?

Responding to the COVID-19 pandemic in complex humanitarian crises https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085188/ SHA: d013e42811c6442b184da3b9bbfd9e334031a975 Authors: Poole, Danielle N.; Escudero, Daniel J.; Gostin, Lawrence O.; Leblang, David; Talbot, Elizabeth A. Date: 2020-03-21 DOI: 10.1186/s12939-020-01162-y License: cc-by Abstract: nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 [1] . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale [2] . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene (WASH) tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations [3] . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing [4] . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% [5] . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production [6] . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance [7] . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines [5, 8] . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence [9] . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations [10] . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings [2] . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic.

For what there is evidence for the efficacy of hand washing?

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459876/ SHA: f0b1fa4036434b57c8307d43c39a4193f7e8053a Authors: Morokutti-Kurz, Martina; König-Schuster, Marielle; Koller, Christiane; Graf, Christine; Graf, Philipp; Kirchoff, Norman; Reutterer, Benjamin; Seifert, Jan-Marcus; Unger, Hermann; Grassauer, Andreas; Prieschl-Grassauer, Eva; Nakowitsch, Sabine Date: 2015-06-08 DOI: 10.1371/journal.pone.0128794 License: cc-by Abstract: BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as "carrageenan"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted We investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)

What are common concamitant infections during the course of influenza infection?

Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/ SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4 Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John Date: 2018-10-19 DOI: 10.1038/s41598-018-33605-6 License: cc-by Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down"). A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ). In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare). To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02). All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/).

What cells are the main target of the influenza A virus in the lungs?

Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/ SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4 Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John Date: 2018-10-19 DOI: 10.1038/s41598-018-33605-6 License: cc-by Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down"). A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ). In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare). To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02). All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/).

Where is CEACAM1 expressed in the body?

A super-spreading ewe infects hundreds with Q fever at a farmers' market in Germany https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618839/ SHA: ee1b5a9618dcc4080ed100486cedd0969e80fa4d Authors: Porten, Klaudia; Rissland, Jürgen; Tigges, Almira; Broll, Susanne; Hopp, Wilfried; Lunemann, Mechthild; van Treeck, Ulrich; Kimmig, Peter; Brockmann, Stefan O; Wagner-Wiening, Christiane; Hellenbrand, Wiebke; Buchholz, Udo Date: 2006-10-06 DOI: 10.1186/1471-2334-6-147 License: cc-by Abstract: BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study (cases were Q fever patients, controls were randomly selected Soest citizens) and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii (C. burnetii). RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3(rd )trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii (C. burnetii), a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats [1] . High concentrations of C. burnetii can be found in birth products of infected mammals [2] . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool [1] . Because the infectious dose is very low [3] and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances [4] [5] [6] . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare [1] . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases [2] . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis [1] . Children develop clinical disease less frequently [7, 8] . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay (IFA) is considered to be the reference method, complement fixation (CF), ELISA and microagglutination (MA) can also be used [9] . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections [1] . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year [10] . In 2001 and 2002, 293 and 191 cases were notified, respectively [11, 12] . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory (NCL) for Coxiellae and the Robert Koch-Institute (RKI), the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens (ELISA or IFA), a 4-fold increase in anti-phase II IgG antibody titer (ELISA or IFA) or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection [13, 14] we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case (CCS1 case) as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 (AFE) as (OR-1)/OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context ("notified case") was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition (as used in the vendors' study (s.b.)) identified through random sampling of the Soest population (within CCS2 (s.b.)) as well as in two cohorts (vendors' study and the 9 sailor friends (see below)). The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market (see below "Estimation of the overall attack rate") and gave an estimate of the proportion of clinically ill cases that were hospitalized (s.a.). In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit (approximately 3 meters). Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose (360°) in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east ( Figure 1 ). For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen (plus minus 45°). Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen (in stand units or meters) and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition (as defined in the cohort study on vendors). The overall attack rate among adults was estimated based on the following sources: (1) Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * (2) Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; (3) Results of the small cohort of 9 sailor friends (s.a.); (4) Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever (s.a. "determination of outbreak size and descriptive epidemiology") interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in (1), (3) and (4) we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children (numerator). For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions (number of visitors, proportion of adult visitors and clinical attack rate among adults) we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks (70 animals). Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 (CDC, Atlanta, USA). Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 (65%) of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. (Figure 2 ). If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases (mean +/-2 standard deviations) had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 (53%) were male and 140 (47%) were female. Of the notified cases, 189 (63%) were from the county of Soest, 104 (35%) were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state (Northrhine Westphalia) and 6 (2%) were from five other federal states in Germany (Figure 3 ). Only eight (3%) cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively ( Figure 4 ). 75 (25%) of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 (21%; 95% CI = 6-46%; (1/5 (CCS2), 2/11 (vendors study) and 1/3 (sailor friends)) clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 (56%) outbreak cases; 66 (22%) were confirmed by an increase in anti-phase II antibody titer (CF), 89 (30%) had IgM antibodies against phase II antigens, 11 (4%) were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 (44%) cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two (11%) of 18 cardiological patients and four (36%) of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman (case 3; Table 1 ). Serological follow-up of the mothers detected chronic infection in the same woman (case 3) 12 weeks after delivery. One year follow-up of two newborn children (of cases 1 and 3) identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender (OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23). Seventeen (85%) of 20 cases indicated that they had seen the cow (that also was on display at the market next to the sheep) compared to 7 (32%) of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 (32%) of 25 controls (OR = 5.0; 95%CI = 1.2-22.3; p = 0.03). Touching the sheep was also significantly more common among cases (5/20 (25%) CCS2 cases vs. 0/22 (0%) controls; OR undefined; lower 95% CI = 1.1; p = 0.02). 17 (85%) of 20 CCS2 cases, but only 6 (25%) of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" (OR = 17.0; 95%CI = 3.0-112.5; p < 0.01). Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 (86%) of 87 vendors, and received second hand information about 7 more (overall response rate: 94%). Fourty-five (56%) were male and 35 (44%) were female. 13 (16%) met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 (55%) became cases compared to only 7 (10%) of 70 persons who worked in a stand at a greater distance (relative risk (RR) = 5.5 (95%CI = 2.3-13.2; p = 0.002); Figure 1 ). Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen (section 4, Figure 1 ), 4 (9%) of 44 vendors became cases, compared to 2 (13%) of 15 persons who worked in section 1 (p = 0.6). Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 (32%) became cases. (Table 3 ). In all scenarios the AR among adults was significantly higher than that among children ( Figure 5 ). In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births (57 (86%)) had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 (25%) tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% (8%; 12%; 12%; 16%; 24%). We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market [15] . More recently a large outbreak occurred in Jena (Thuringia) in 2005 with 322 reported cases [16] associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases (source 5) were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] . An additional interview with the two cases with early onset (2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" (see Table 3 ). Displayed are attack rates and 95% confidence intervals. respectively) could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high [17] . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to (within 6 meters) the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% (8/ 191) [7] , 5% [1] and 10% (4/39) [5] ), and there was at least one study with a much higher proportion (63% (10/ 16)) [18] . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep (n = 1000-2000) [5, 7] the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep [19] . In the most severely affected village, the clinical attack rate was 16% (estimated from the data provided) [19] . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic (50% (3/6)) is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease [7] . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness [7, 8] few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock [20] . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks [7, 20, 21] , was 40% compared to 1% in sheep flocks not linked to human outbreaks [20] . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that (in such a setting) can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred [20] , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of (farm) animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents [20, [22] [23] [24] . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations [25] which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author(s) declare that they have no competing interests.

What important risk factors to infection were found during the second case-controlled study?

Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/ SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9 Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W. Date: 2012-11-15 DOI: 10.1371/journal.pone.0048702 License: cc-by Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production. Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] . The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] . The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated. In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis. In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ). We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination. Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus. We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold. Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes. We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression. Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] . Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] . STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] . YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] . ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] . Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] . CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] . ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] . To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold). In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) . Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF. Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] . Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression. Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) . DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) . Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] . Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro. The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function. We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag. Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] . Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis. HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability. Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection. Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] . HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules. We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined. The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach. The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours. While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention. Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore). The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter). Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology). For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] . Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap. The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown). The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%. Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] . The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific). To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective. The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key: (R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis. (DOCX)

What is the significance of this study?

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459876/ SHA: f0b1fa4036434b57c8307d43c39a4193f7e8053a Authors: Morokutti-Kurz, Martina; König-Schuster, Marielle; Koller, Christiane; Graf, Christine; Graf, Philipp; Kirchoff, Norman; Reutterer, Benjamin; Seifert, Jan-Marcus; Unger, Hermann; Grassauer, Andreas; Prieschl-Grassauer, Eva; Nakowitsch, Sabine Date: 2015-06-08 DOI: 10.1371/journal.pone.0128794 License: cc-by Abstract: BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as "carrageenan"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted We investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)

What is the effect of intranasal Zanamivir on laboratory confirmed infleunza infection?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What is recommended that patients with chronic airway inflammatory disease?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

Which are the type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier?

A novel anti-mycobacterial function of mitogen-activated protein kinase phosphatase-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2804704/ SHA: f6ed1f1e9999e57793addb1c9c54f61c7861a995 Authors: Cheung, Benny KW; Yim, Howard CH; Lee, Norris CM; Lau, Allan SY Date: 2009-12-17 DOI: 10.1186/1471-2172-10-64 License: cc-by Abstract: BACKGROUND: Mycobacterium tuberculosis (MTB) is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α (TNF-α), which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin (BCG), a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase (MAPK) in human blood monocytes. Since MAPK phosphatase-1 (MKP-1) is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK (p38 MAPK and ERK1/2) and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis (TB) remains a major cause of morbidity and mortality in the world, especially in the developing countries [1] . The disease is caused by Mycobacterium tuberculosis (MTB) and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization (WHO) estimated that there are 9.2 million new TB cases around the world in 2006 [1] . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity [2] . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) have been implicated as important cellular signaling molecules activated by mycobacteria [3] . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv [4] . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes (PBMo) leading to cytokine expressions via the interaction with PKR [5] . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases (DUSPs). Some DUSPs are also known as MAPK phosphatases (MKPs) [6] [7] [8] . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout (KO) macrophages in response to lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment [9] . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity [10] . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-β (TRIF) [10] , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities [10, 11] . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus (S. aureus), a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus [12] . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus [12] . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down [13] . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts [13] . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E (IgE) receptor (FcεRI) and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 [13] . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs [13] . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK [13] . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts [6] . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction (QPCR) method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys (TLR2 agonist), poly IC (TLR3 agonist), or LPS (TLR4 agonist) for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment ( Figure 1A ). These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys ( Figure 1A ). In contrast, poly IC did not induce MKP-1 ( Figure 1A ). The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro [9, 12] . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression (Figure 1B) . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG (MOI = 1 CFU/cell), there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 ( Figure 2A ). The effects lasted for at least 6 hours ( Figure 2A ). To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours ( Figure 2B ). The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages [14] . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 (inhibitor for MAP kinase kinase [MEK] or ERK1/2), SB203580 (inhibitor for p38 MAPK), SP600125 (inhibitor for JNK), and CAPE (inhibitor for NF-κB) for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced ( Figure 3 ). In addition, using higher dose of SB203580, we showed that the inhibition is increased further (data not shown). On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG ( Figure 3 ). These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression (lanes 1 and 2). In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased (lanes 2 and 4). The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA ( Figure 4B , lanes 1-3). However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA ( Figure 4B , lanes 4-6). Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature [9] , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported ( [5] and data not shown). Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA ( Figure 4D ). With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways [7] . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 [7] . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes (data not shown) and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA ( Figure 5 ). Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation ( Figure 5 ). The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response (Figure 2) . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK ( Figure 3 ). Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 ( Figures 4D and 5) . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK [14] . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation [15] . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors (for p38 MAPK and ERK1/ 2) was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects ( Figure 6 ), the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α [9] . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS (Figure 4C ). There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 [13] . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST [6] . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members [13] . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts [13] . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs [13] . It was shown that there are interactions between JNK and ERK1/2 pathways [16] . Here, we showed that the sustained activation of JNK blocks ERK activation ( Figure 6 ). In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 [13] . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings (Left). In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated (Middle), since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 (Right) may be explained by events similar to the DUSP2 effects. In this case (Right), there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA (data not shown). Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 ( Figure 6 ) may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction ( Figure 6 ). In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment ( Figure 6 ). Taken together, the role of MAPK crosstalks need further exploration. (3) TNF-α, 30 cycles (TM = 56°C), upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ (Invitrogen, USA) was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH (as internal control) were assayed by the gene-specific Assays-on-Demand reagent kits (Applied Biosystems, USA). All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold (C T ) method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample (ΔC T ). The C T value of the treated cells was compared with that of the untreated or mock-treated cells (ΔΔCT). The relative gene expression of the targeted genes (fold induction) was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit (Pierce, IL, USA). Western blot was done as described [20] . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes (Schleicher & Schuell), and followed by probing with specific antibod-ies for Actin, MKP-1 (Santa Cruz Biotech., USA), phospho-p38 MAPK, phospho-ERK1/2 (Cell Signaling, USA). After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech) as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described [21] . MKP-1 siRNA included (i) MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; (ii) MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and (iii) MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi (ratio = 1:1:1, 200 nM, Invitrogen, USA). Stealth™ RNAi Negative Control Duplex (200 nM) was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent (Polyplus Transfection, USA) according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05.

What is lipopolysaccharide?

A super-spreading ewe infects hundreds with Q fever at a farmers' market in Germany https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618839/ SHA: ee1b5a9618dcc4080ed100486cedd0969e80fa4d Authors: Porten, Klaudia; Rissland, Jürgen; Tigges, Almira; Broll, Susanne; Hopp, Wilfried; Lunemann, Mechthild; van Treeck, Ulrich; Kimmig, Peter; Brockmann, Stefan O; Wagner-Wiening, Christiane; Hellenbrand, Wiebke; Buchholz, Udo Date: 2006-10-06 DOI: 10.1186/1471-2334-6-147 License: cc-by Abstract: BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study (cases were Q fever patients, controls were randomly selected Soest citizens) and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii (C. burnetii). RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3(rd )trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii (C. burnetii), a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats [1] . High concentrations of C. burnetii can be found in birth products of infected mammals [2] . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool [1] . Because the infectious dose is very low [3] and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances [4] [5] [6] . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare [1] . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases [2] . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis [1] . Children develop clinical disease less frequently [7, 8] . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay (IFA) is considered to be the reference method, complement fixation (CF), ELISA and microagglutination (MA) can also be used [9] . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections [1] . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year [10] . In 2001 and 2002, 293 and 191 cases were notified, respectively [11, 12] . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory (NCL) for Coxiellae and the Robert Koch-Institute (RKI), the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens (ELISA or IFA), a 4-fold increase in anti-phase II IgG antibody titer (ELISA or IFA) or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection [13, 14] we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case (CCS1 case) as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 (AFE) as (OR-1)/OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context ("notified case") was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition (as used in the vendors' study (s.b.)) identified through random sampling of the Soest population (within CCS2 (s.b.)) as well as in two cohorts (vendors' study and the 9 sailor friends (see below)). The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market (see below "Estimation of the overall attack rate") and gave an estimate of the proportion of clinically ill cases that were hospitalized (s.a.). In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit (approximately 3 meters). Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose (360°) in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east ( Figure 1 ). For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen (plus minus 45°). Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen (in stand units or meters) and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition (as defined in the cohort study on vendors). The overall attack rate among adults was estimated based on the following sources: (1) Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * (2) Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; (3) Results of the small cohort of 9 sailor friends (s.a.); (4) Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever (s.a. "determination of outbreak size and descriptive epidemiology") interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in (1), (3) and (4) we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children (numerator). For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions (number of visitors, proportion of adult visitors and clinical attack rate among adults) we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks (70 animals). Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 (CDC, Atlanta, USA). Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 (65%) of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. (Figure 2 ). If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases (mean +/-2 standard deviations) had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 (53%) were male and 140 (47%) were female. Of the notified cases, 189 (63%) were from the county of Soest, 104 (35%) were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state (Northrhine Westphalia) and 6 (2%) were from five other federal states in Germany (Figure 3 ). Only eight (3%) cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively ( Figure 4 ). 75 (25%) of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 (21%; 95% CI = 6-46%; (1/5 (CCS2), 2/11 (vendors study) and 1/3 (sailor friends)) clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 (56%) outbreak cases; 66 (22%) were confirmed by an increase in anti-phase II antibody titer (CF), 89 (30%) had IgM antibodies against phase II antigens, 11 (4%) were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 (44%) cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two (11%) of 18 cardiological patients and four (36%) of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman (case 3; Table 1 ). Serological follow-up of the mothers detected chronic infection in the same woman (case 3) 12 weeks after delivery. One year follow-up of two newborn children (of cases 1 and 3) identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender (OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23). Seventeen (85%) of 20 cases indicated that they had seen the cow (that also was on display at the market next to the sheep) compared to 7 (32%) of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 (32%) of 25 controls (OR = 5.0; 95%CI = 1.2-22.3; p = 0.03). Touching the sheep was also significantly more common among cases (5/20 (25%) CCS2 cases vs. 0/22 (0%) controls; OR undefined; lower 95% CI = 1.1; p = 0.02). 17 (85%) of 20 CCS2 cases, but only 6 (25%) of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" (OR = 17.0; 95%CI = 3.0-112.5; p < 0.01). Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 (86%) of 87 vendors, and received second hand information about 7 more (overall response rate: 94%). Fourty-five (56%) were male and 35 (44%) were female. 13 (16%) met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 (55%) became cases compared to only 7 (10%) of 70 persons who worked in a stand at a greater distance (relative risk (RR) = 5.5 (95%CI = 2.3-13.2; p = 0.002); Figure 1 ). Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen (section 4, Figure 1 ), 4 (9%) of 44 vendors became cases, compared to 2 (13%) of 15 persons who worked in section 1 (p = 0.6). Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 (32%) became cases. (Table 3 ). In all scenarios the AR among adults was significantly higher than that among children ( Figure 5 ). In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births (57 (86%)) had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 (25%) tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% (8%; 12%; 12%; 16%; 24%). We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market [15] . More recently a large outbreak occurred in Jena (Thuringia) in 2005 with 322 reported cases [16] associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases (source 5) were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] . An additional interview with the two cases with early onset (2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" (see Table 3 ). Displayed are attack rates and 95% confidence intervals. respectively) could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high [17] . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to (within 6 meters) the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% (8/ 191) [7] , 5% [1] and 10% (4/39) [5] ), and there was at least one study with a much higher proportion (63% (10/ 16)) [18] . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep (n = 1000-2000) [5, 7] the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep [19] . In the most severely affected village, the clinical attack rate was 16% (estimated from the data provided) [19] . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic (50% (3/6)) is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease [7] . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness [7, 8] few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock [20] . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks [7, 20, 21] , was 40% compared to 1% in sheep flocks not linked to human outbreaks [20] . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that (in such a setting) can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred [20] , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of (farm) animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents [20, [22] [23] [24] . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations [25] which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author(s) declare that they have no competing interests.

What was the median seropresence of C. burnetti in sheep flocks not linked to human outbreaks?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

Which is the major surface and attachment glycoprotein on influenza virus?

MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The

Why has MERS had a constant presence in the Arabian Peninsula?

Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566411/ SHA: ee48061797d29eeef5a9e606841bf8ab04b1d75b Authors: Beier, Kevin T.; Saunders, Arpiar B.; Oldenburg, Ian A.; Sabatini, Bernardo L.; Cepko, Constance L. Date: 2013-02-07 DOI: 10.3389/fncir.2013.00011 License: cc-by Abstract: Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer. Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task. Recombinant viruses engineered to trace synaptic connections and express transgenes promise to enable higher-throughput mapping of connections among neurons than other methods, e.g., serial reconstruction from electron micrographs (Bock et al., 2011; Briggman et al., 2011) . The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) . RABV was recently modified by Wickersham and colleagues such that it can travel across only one synapse, allowing for a straightforward definition of monosynaptic connections (Wickersham et al., 2007b) . This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell ("starter cell"), without the need for electrophysiology. Moreover, the starter cell could be defined through the expression of a specific viral receptor that limited the initial infection. Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) . Vesicular stomatitis virus (VSV), a virus related to RABV, with its own glycoprotein (G) gene (VSV-G), or with a G from the unrelated lymphocytic choriomeningitis virus (LCMV-G), spreads in the anterograde direction across synapses. VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) . Whether the virus is a monosynaptic or polysynaptic tracer is determined by the method of delivery of the G gene ( Figure 1A) . Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells. It is also being developed as a vaccine vector, often using a G of another virus as the immunogen, as well as being developed as a cytocidal agent that will target tumor cells in humans (Balachandran and Barber, 2000; Stojdl et al., 2000 Stojdl et al., , 2003 . Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) . In addition, it has been shown that RABV, as well as lentiviruses with RABV-G in their envelope, travel retrogradely from an injection site (Mazarakis et al., 2001; Wickersham et al., 2007a) . We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV. Our initial characterization of rVSV with RABV-G showed that indeed FIGURE 1 | Synaptic tracing strategies using VSV. (A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP. The initially infected cell is indicated by an asterisk. VSV encoding a glycoprotein (G) within its genome can spread polysynaptically. The direction of the spread depends on the identity of the glycoprotein. Infected neurons are shown in green. In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein). The TVA-expressing neurons can then be specifically infected by rVSV G with the EnvA/RABV-G (A/RG) glycoprotein (Wickersham et al., 2007b) on the virion surface [rVSV G(A/RG)]. These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression. For monosynaptic tracing, the G protein is expressed in trans in the TVA-expressing cell, and thus complements rVSV G to allow transmission in a specific direction. (B) Genomic diagrams of rVSV vectors. All VSVs contain four essential proteins: N, P, M, and L. Some viruses encode a G gene in their genome, which allows them to spread polysynaptically. rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position. (C) Morphological characterization of rVSV-infected neurons in several locations within the mouse brain. (i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange. (iii) Labeled neurons of the CA1 region of the hippocampus are shown at 5 dpi following injection into the hippocampus of rVSV(VSV-G) encoding Venus. (iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi. Inset in (iv) is a high magnification of the neuron in panel (iv), highlighting labeling of dendritic spines. (vi) Multiple viruses can be co-injected into the same animal. Here, individual rVSV G(VSV-G) viruses encoding CFP, GFP , Venus, Korange, and mCherry were used to infect the cortex. Scale bars = 50 µm. www.frontiersin.org February 2013 | Volume 7 | Article 11 | 2 it could be taken up as a retrograde tracer (Beier et al., 2011) . To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations. In addition, we performed co-infections of rVSV with RABV-G and the anterograde form of rVSV in order to exploit the differences in the directionality of transmission of these two viruses in mapping circuits. Schematics of viruses created and used throughout this study are shown in Figure 1 . We created rVSV vector plasmids carrying different transgenes in either the first or fifth genomic positions ( Figure 1B) . After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ). All rVSV vectors drove robust fluorophore expression 1 or 2 days post-infection (hpi) ( Figure 1C ) (van den Pol et al., 2009) . In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv). To characterize the physiological properties of cells infected with rVSV, we tested a replication-competent rVSV encoding GFP, with RABV-G in the genome in place of VSV-G [hereafter designated rVSV(RABV-G)]. van den Pol et al. reported that hippocampal neurons infected with replication-incompetent (G-deleted or " G") rVSV were physiologically healthy at 12-14 hpi, but were less so by 1 day post-infection (dpi) (van den Pol et al., 2009) . Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G). Between 12 and 18 hpi, the membrane capacitance, input resistance, resting membrane potential, and current-to-action potential firing relationship were indistinguishable between infected and uninfected neurons (Figure 2) . However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made. The speed and strength of the expression of transgenes encoded by VSV depends upon the gene's genomic position (van den Pol et al., 2009; Beier et al., 2011) . Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand. When GFP was inserted into the first position of VSV, GFP fluorescence was first detectable at approximately 1 hpi in cultured cells (van den Pol et al., 2009) . In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) . This was a G virus which had the RABV-G supplied in trans during the preparation of the virus stock [referred to as rVSV G(RABV-G)]. Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ). Similar results were obtained with a virus encoding GFP in the first genomic position rather than mCherry (i.e., Figure 1B ) (N = 3). We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication. To distinguish between these two mechanisms and to extend the previous analyses, we conducted further experiments in the mammalian visual system (Figures 4A-G) . As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication. Following a V1 injection with rVSV(RABV-G), GFP-positive RGCs were observed in the retina by 3 dpi (N = 3; Figure 4G ). Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi. These included the LGN ( Figure 4D ) and the hypothalamus (Figure 4E) , two areas known to project directly to V1 (Kandel, 2000) . Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) . Labeling was also observed in the nucleus basalis, which projects to the cortex, as well as many components of the basal ganglia circuit, which provide input to the thalamus [such as the caudate-putamen (CP), globus pallidus (GP), and the subthalamic nucleus (STn)]. The amygdala, which projects to the hypothalamus, was also labeled. Consistent with a lack of widespread viral transmission, animals did not exhibit signs of disease at 7 dpi. These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction. Directional transsynaptic specificity can only be definitively addressed using a unidirectional circuit. We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) . Injections of rVSV(RABV-G) into M1 should not label neurons in the CP if the virus can only label cells across synapses in the retrograde direction. Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ). Only axons from cortical cells were observed in the CP, with no GFP-labeled cell bodies present in the CP (Figure 4J) , consistent with lack of anterograde transsynaptic spread. By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown). A particular advantage of retrograde viral tracers is the ability to label CNS neurons projecting to peripheral sites. This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) . To test if rVSV(RABV-G) could also perform this function, we examined the innervation of the dura surface by neurons of the trigeminal ganglion, a neuronal circuit thought to be involved in migraine headaches (Penfield and McNaughton, 1940; Mayberg et al., 1984) . These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem. Therefore, the only way trigeminal neurons could become labeled from viral application to the dura is through retrograde uptake of the virus. We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ). At the earliest time point examined, 3 dpi, we observed axons traveling along the dura, but little other evidence of infection ( Figure 4L) . No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface. In contrast, we did find labeled cell bodies in the trigeminal ganglion ( Figure 4M ). No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals). To further characterize patterns and kinetics of viral transmission and directional specificity of transsynaptic spread, injections of rVSV(RABV-G) were made into the CP ( Figure 5A ). In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi). Cells labeled by rVSV G(RABV-G) were observed in the CP, GP, substantia nigra (SN), thalamus, and layers 3 and 5 of the cortex, consistent with infection at the axon terminal and retrograde labeling of cell bodies of neurons known to project directly to the CP ( Figure 5C ) (Albin et al., 1995) . Areas labeled by CP injection are indicated in Figure 5B . The patterns of spread for the replication-competent rVSV(RABV-G) were characterized over the course of 1-5 dpi ( Figures 5D-H) . During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ). In addition, more cortical cells were labeled in clusters near cortical pyramidal neurons, both ipsilateral and contralateral to the injected side, including neurogliaform cells (data not shown). These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) . At 3 dpi following rVSV(VSV-G) injection into the CP, the cerebral cortex was not labeled, but regions receiving projections from the CP, such as the STn, GP, and SN, were labeled (Beier et al., 2011) . In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G). The nucleus basalis was labeled by 2 dpi (Figures 5E-H) , consistent with at least a single transsynaptic jump, as this area does not directly project to the CP. The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) . Labeling remained well-restricted to the expected corticostriatal circuits at 5 dpi, suggesting that viral spread becomes less efficient after crossing one or two connections, consistent with injections into V1 (Figure 4) . While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed. One advantage of having both anterograde and retrograde forms of the same virus is that they can be used in parallel, or in tandem, to trace circuitry to and from a single or multiple sites of injection, with each virus having similar kinetics of spread and gene expression. In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ). This is most straightforward if there are no cells at the injection site that are initially infected by both viruses. Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection. In order to determine whether two viruses would allow simultaneous anterograde and retrograde transsynaptic tracing from a single injection site, a rVSV(VSV-G) expressing Venus and a rVSV(RABV-G) expressing mCherry were injected individually (Figures 6B-D) or co-injected (Figures 6E-G) into the motor cortex, and brains were examined 3 dpi. The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4). The initial co-infection rate is dependent upon The presence or absence of labeling is indicated by (+) and (−), respectively. The extent of labeling is indicated by the number of (+). Some animals were infected with G viruses to determine which areas were labeled by direct uptake of the virions, rather than by replication and transmission. These were sacrificed at 3 dpi. (C) Parasaggital section of a brain infected with VSV[greek delta]G(RABV-G). The injection site is marked by a red arrow. Several areas that project directly to the CP were labeled due to direct uptake of the virions, including the cortex, thalamus, and GP (arrowheads), 3 dpi. the dose of the initial inocula. When injecting 3 × 10 3 focus forming units (ffu) rVSV(VSV-G) and 3 × 10 4 ffu rVSV(RABV-G), no co-infection was observed at the injection site. Thus, co-infection of the same brain region, without co-infection of the same cells, does not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G). One example of how this dual retrograde and anterograde transsynaptic tracing system can be used is to determine if three Frontiers in Neural Circuits www.frontiersin.org February 2013 | Volume 7 | Article 11 | 8 distinct regions are connected and the directionality of any connections. For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ). To test this possibility, rVSV(VSV-G) was injected into the motor cortex, rVSV(RABV-G) was injected into the substantia nigra pars reticulara (SNr), and animals were sacrificed at 3 dpi. We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) . These results suggest that the anterograde connections from the cells infected with rVSV(VSV-G) in the M1 were with CP MSNs that did not project to the region of the SNr injected with rVSV(RABV-G) (N = 3 animals). In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) . With RABV, this was achieved in vivo by first infecting with an adeno-associated virus (AAV) expressing TVA, a receptor for an avian retrovirus, and RABV-G (Wall et al., 2010) . This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA. A similar strategy was used to test rVSV's ability to monosynaptically trace retrogradely connected neurons in vivo. Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test. These neurons primarily receive input from the cortex and the thalamus (Thomas et al., 2000; Bloomfield et al., 2007) (Figure 7A) . In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) . Six-week-old mice from this cross were injected in the CP with two AAV vectors: one expressing a Cre-conditional ("floxed") TVA-mCherry fusion protein, and another expressing a floxed RABV-G. Two weeks later, the mice were injected in the same coordinates with rVSV G with the EnvA/RABV-G chimeric glycoprotein on the virion surface [rVSV G(A/RG)] (Beier et al., 2011) . Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface. Such starter cells should also express tdTomato and GFP. If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP. Mice injected with these AAV and rVSV viruses were sacrificed 5 days after rVSV infection, and brains analyzed for fluorescence. As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) . Outside of the CP, small numbers of GFP+ neurons that were not mCherry+ were observed in the cortex (Figures 7C,D) and thalamus (Figure 7E) , consistent with retrograde spread. Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition). Here, we report on the use of rVSV as a retrograde transsynaptic tracer for CNS circuitry. VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer. Alternatively, if the virus has the G gene deleted from its genome and RABV-G is provided in trans, it behaves as a monosynaptic tracer (Beier et al., 2011) . Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein. Since native VSV does not have these retrograde transsynaptic properties (van den Pol et al., 2002; Beier et al., 2011) , and the only alteration to the VSV genome was the substitution of the VSV G gene with the G gene of RABV, it is clear that the RABV glycoprotein is responsible for retrograde direction of viral transmission across synapses, at least in the case of rVSV. The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants. In addition, more than one transgene can be encoded in the viral genome without the need of a 2A or IRES element. The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) . This leads to rational predictions of expression levels so that one can choose the position of insertion of a transgene, or transgenes, according to this gradient and the desired level of expression. The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) . The fact that VSV can be made to spread anterogradely (Beier et al., 2011) or retrogradely across synapses with the change of a single gene affords several advantages over viral tracers that heretofore have not shown such flexibility in the directionality of tracing. In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus. One example that employs the simultaneous infection with an anterograde and retrograde form of VSV is demonstrated in Figure 6 . This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN. Although a block of superinfection by the virus may preclude infection of the same cell with multiple rVSVs, adjacent cells could still become labeled by different viruses (Whitaker-Dowling et al., 1983) . The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias. Alternatively, it could indicate that the cortical neurons in the injected region largely do not label the MSNs that project to the area of the SN injected with the retrograde virus. One further possibility is that too little virus was used to observe co-labeling of a given region. However, given the density of infection (i.e., Figures 6I,J) , the latter possibility seems unlikely. Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread. This is quite fortuitous, as if spread were to continue, it would lead to widespread infection and lethality. In addition, reconstruction of connectivity would be more difficult. This reduced efficiency appears to also hold for the monosynaptic form of VSV complemented with RABV-G, as the efficiency of transmission appeared lower than the comparable experiment with RABV (Watabe-Uchida et al., 2012) . This is likely due to viral attenuation when VSV-G is replaced with RABV-G. We were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent. Laboratory workers using VSV have not contracted any diseases, and natural VSV infections among human populations in Central America and the southwestern United States (Rodríguez, 2002) occur without evident pathology (Johnson et al., 1966; Brody et al., 1967) . VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles. Both replicationcompetent and incompetent forms of VSV are in use under Biosafety Level 2 containment. Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) . Differences in pathogenicity between VSV and RABV are likely due to the ability of RABV to evade the innate immune system, particularly interferon (Hangartner et al., 2006; Junt et al., 2007; Lyles and Rupprecht, 2007; Rieder and Conzelmann, 2009; Iannacone et al., 2010) . VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) . In fact, VSV is being pursued as a vaccine for other viruses, including RABV (Lichty et al., 2004; Publicover et al., 2004; Kapadia et al., 2005; Schwartz et al., 2007; Iyer et al., 2009; Geisbert and Feldmann, 2011) . VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) . This likely is the cause of the minor or absent symptoms in humans and animals infected in nature. Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors. We have tested this prediction by injecting a series of mice in the footpads and hind leg muscles with rVSV(RABV-G), with the result that no injected animals showed any evidence of morbidity or mortality (Beier, Goz et al., in preparation) . While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) . Toxicity is due to suppression of cellular transcription and a block in the export of cellular RNAs from the nucleus to the cytoplasm (Black and Lyles, 1992; Her et al., 1997; Ahmed and Lyles, 1998; Petersen et al., 2000; von Kobbe et al., 2000) , as well as inhibition of the translation of cellular mRNAs (Francoeur et al., 1987; Jayakar et al., 2000; Kopecky et al., Frontiers in Neural Circuits www.frontiersin.org February 2013 | Volume 7 | Article 11 | 10 2001). VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection. One aspect of VSV that can be exploited in the future to ameliorate the speed of toxicity is the use of VSV mutants and variants. One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) . We are in the process of examining the transmission properties of this mutant in vivo, as well as the effects of other mutations or viral variants on prolonging the health of neurons after infection. rVSV vectors can be used to study the connectivity of neuronal circuitry. In addition to combinations of replication-competent forms of VSV, the replication-incompetent, monosynaptic forms of the virus can be easily combined, without the need to change viruses (Beier et al., 2011) . This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population. This can be done with the same viral genome, with the only change needed being the glycoprotein, for the selection of the direction of transmission. This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS. All rVSV clones were cloned from the rVSV G backbone (Chandran et al., 2005) . mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites. Genes for fluorescent proteins were obtained from Clontech. Viruses were rescued as previously described (Whelan et al., 1995) . At 95% confluency, eight 10 cm plates of BSR cells were infected at an MOI of 0.01. Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume. For titering, concentrated viral stocks were applied in a dilution series to 100% confluent BSR cells and plates were examined at 12 hpi. Viral stocks were stored at −80 • C. For G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry. Viral supernatants were collected for the subsequent 4 days at 24 h intervals. Virus preparations are now available from the Salk GT3 viral core (http://vectorcore.salk.edu/). All plasmids are available from Addgene (http://www.addgene.org/). AAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School. ChAT-Cre (B6;129S6-Chat tm1(cre)Lowl /J) and Ai9 (B6.Cg-Gt(ROSA)26Sor<tm9(CAG-tdTomato)Hze>/J) mice were obtained from the Jackson Laboratory (Madisen et al., 2010) . Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat. No: 5-000-2005) , using coordinates from The Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 1997) . Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions. For CP injections, 100 nL of rVSV(RABV-G) or rVSV(VSV-G) at 3 × 10 7 ffu/mL was injected at a rate of 100 nL/min. For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected. In the motor cortex, 100 nL of 1 × 10 7 ffu/mL rVSV(RABV-G) was injected, and mice harvested 2 dpi. For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi. For infections of the dura mater, 1 µL of 3 × 10 10 ffu/mL rVSV(RABV-G) was applied to the surface of the dura. The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured. For co-injections of virus into the same animal, 100 nL of a combination of 3 × 10 7 ffu/mL rVSV(VSV-G) and 3 × 10 8 ffu/mL rVSV(RABV-G) were co-injected into the motor cortex, and brains examined 3 dpi. For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi. A lower titer of rVSV(VSV-G) was used, as rVSV(RABV-G) is attenuated. All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee. Recordings were made from cortical pyramidal neurons in slices taken from postnatal day 12-18 mice, inoculated in the CP 12-18 h prior with rVSV(RABV-G). Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 . Slices were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , and 25 glucose, bubbled with 95% O 2 and 5% CO 2 . After an incubation period Frontiers in Neural Circuits www.frontiersin.org February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature. All experiments were conducted at room temperature (25 • C). In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively. All chemicals were from Sigma or Tocris. Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination. With the deep layers of the cortex, 2-photon laser scanning microscopy (2PLSM) was used to confirm the cell types based on morphology. Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines. Glass electrodes (2-4 M ) were filled with internal solution containing (in mM): 135 KMeSO 4 , 5 KCl, 5 HEPES, 4 MgATP, 0.3 NaGTP, 10 Na 2 HPO 4 , 1 EGTA, and 0.01 Alexa Fluor-594 (to image neuronal morphology) adjusted to pH 7.4 with KOH. Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier. Data was filtered at 5 kHz and digitized at 10 kHz. Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) . Resting membrane potential was determined by the average of three 5-s sweeps with no injected current. Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV. The current-firing relationship was determined in current clamp with 1-s periods of injected current from 100 to 500 pA. The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) . Slices were infected after 7 days in vitro, and images were acquired on a two-photon microscope.

What types of viruses can be used to study the connectivity of neuronal circuitry?

No credible evidence supporting claims of the laboratory engineering of SARS-CoV-2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054935/ SHA: 5a9154aee79901dd8fecd58b7bcd9b7351102d24 Authors: Liu, Shan-Lu; Saif, Linda J.; Weiss, Susan R.; Su, Lishan Date: 2020-02-26 DOI: 10.1080/22221751.2020.1733440 License: cc-by Abstract: nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) (https://globalbiodefense. com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (https:// www.nature.com/articles/d41586-020-00364-2). Another claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy (https://www.nih.gov/about-nih/who-weare/nih-director/statements/nih-lifts-funding-pausegain-function-research). The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss http://orcid.org/0000-0002-8155-4528

What was done to test if an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. T

Accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064339/ SHA: f2cc0d63ff2c4aaa127c4caae21d8f3a0067e3d5 Authors: Brook, Cara E; Boots, Mike; Chandran, Kartik; Dobson, Andrew P; Drosten, Christian; Graham, Andrea L; Grenfell, Bryan T; Müller, Marcel A; Ng, Melinda; Wang, Lin-Fa; van Leeuwen, Anieke Date: 2020-02-03 DOI: 10.7554/elife.48401 License: cc-by Abstract: Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations: We defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of " > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and "; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of ": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2): Pathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model: At equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of " that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of ", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + ") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and " were fixed at 0 while b was estimated; under induced immune assumptions, " was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, ", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition ("). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, ", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with ") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of ", any parameter tests of " values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from " prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation: where P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium: Because and " both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and ", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and " derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for ", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, ". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 To derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and " by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and " across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and " (" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (" = r = 0) version of the theoretical model and, subsequently, an induced immunity (" = 0; r >0) and constitutive immunity (" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and ") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and " by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare

How were the spread of GFP-expressing virus-infected cells tracked?

Development of an ELISA-array for simultaneous detection of five encephalitis viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/ SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122 Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui Date: 2012-02-27 DOI: 10.1186/1743-422x-9-56 License: cc-by Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown). The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use.

What kind of antibodies were used in the ELISA-array assay?

Epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-nCoV) outbreak https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029449/ SHA: 90de2d957e1960b948b8c38c9877f9eca983f9eb Authors: Cowling, Benjamin J; Leung, Gabriel M Date: 2020-02-13 DOI: 10.2807/1560-7917.es.2020.25.6.2000110 License: cc-by Abstract: Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared.

What should have reduced the basic reproduction number in January?

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459876/ SHA: f0b1fa4036434b57c8307d43c39a4193f7e8053a Authors: Morokutti-Kurz, Martina; König-Schuster, Marielle; Koller, Christiane; Graf, Christine; Graf, Philipp; Kirchoff, Norman; Reutterer, Benjamin; Seifert, Jan-Marcus; Unger, Hermann; Grassauer, Andreas; Prieschl-Grassauer, Eva; Nakowitsch, Sabine Date: 2015-06-08 DOI: 10.1371/journal.pone.0128794 License: cc-by Abstract: BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as "carrageenan"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted We investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)

Do carageenan and Zanamivir delivered intranasally have a benefit when taken for influenza subtype H1N1 infection?

Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review https://doi.org/10.3390/jcm9030623 SHA: 9b0c87f808b1b66f2937d7a7acb524a756b6113b Authors: Pang, Junxiong; Wang, Min Xian; Ang, Ian Yi Han; Tan, Sharon Hui Xuan; Lewis, Ruth Frances; Chen, Jacinta I. Pei; Gutierrez, Ramona A.; Gwee, Sylvia Xiao Wei; Chua, Pearleen Ee Yong; Yang, Qian; Ng, Xian Yi; Yap, Rowena K. S.; Tan, Hao Yi; Teo, Yik Ying; Tan, Chorh Chuan; Cook, Alex R.; Yap, Jason Chin-Huat; Hsu, Li Yang Date: 2020 DOI: 10.3390/jcm9030623 License: cc-by Abstract: Rapid diagnostics, vaccines and therapeutics are important interventions for the management of the 2019 novel coronavirus (2019-nCoV) outbreak. It is timely to systematically review the potential of these interventions, including those for Middle East respiratory syndrome-Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS)-CoV, to guide policymakers globally on their prioritization of resources for research and development. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Supplementary strategies through Google Search and personal communications were used. A total of 27 studies fulfilled the criteria for review. Several laboratory protocols for confirmation of suspected 2019-nCoV cases using real-time reverse transcription polymerase chain reaction (RT-PCR) have been published. A commercial RT-PCR kit developed by the Beijing Genomic Institute is currently widely used in China and likely in Asia. However, serological assays as well as point-of-care testing kits have not been developed but are likely in the near future. Several vaccine candidates are in the pipeline. The likely earliest Phase 1 vaccine trial is a synthetic DNA-based candidate. A number of novel compounds as well as therapeutics licensed for other conditions appear to have in vitro efficacy against the 2019-nCoV. Some are being tested in clinical trials against MERS-CoV and SARS-CoV, while others have been listed for clinical trials against 2019-nCoV. However, there are currently no effective specific antivirals or drug combinations supported by high-level evidence. Text: Since mid-December 2019 and as of early February 2020, the 2019 novel coronavirus (2019-nCoV) originating from Wuhan (Hubei Province, China) has infected over 25,000 laboratory-confirmed cases across 28 countries with about 500 deaths (a case-fatality rate of about 2%). More than 90% of the cases and deaths were in China [1] . Based on the initial reported surge of cases in Wuhan, the majority were males with a median age of 55 years and linked to the Huanan Seafood Wholesale Market [2] . Most of the reported cases had similar symptoms at the onset of illness such as fever, cough, and myalgia or fatigue. Most cases developed pneumonia and some severe and even fatal respiratory diseases such as acute respiratory distress syndrome [3] . The 2019 novel coronavirus (2019-nCoV), a betacoronavirus, forms a clade within the subgenus sarbecovirus of the Orthocoronavirinae subfamily [4] . The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also betacoronaviruses that are zoonotic in origin and have been linked to potential fatal illness during the outbreaks in 2003 and 2012, respectively [5, 6] . Based on current evidence, pathogenicity for 2019-nCoV is about 3%, which is significantly lower than SARS-CoV (10%) and MERS-CoV (40%) [7] . However, 2019-nCoV has potentially higher transmissibility (R0: 1.4-5.5) than both SARS-CoV (R0: [2] [3] [4] [5] and MERS-CoV (R0: <1) [7] . With the possible expansion of 2019-nCoV globally [8] and the declaration of the 2019-nCoV outbreak as a Public Health Emergency of International Concern by the World Health Organization, there is an urgent need for rapid diagnostics, vaccines and therapeutics to detect, prevent and contain 2019-nCoV promptly. There is however currently a lack of understanding of what is available in the early phase of 2019-nCoV outbreak. The systematic review describes and assesses the potential rapid diagnostics, vaccines and therapeutics for 2019-nCoV, based in part on the developments for MERS-CoV and SARS-CoV. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies examining the diagnosis, therapeutic drugs and vaccines for Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the 2019 novel coronavirus (2019-nCoV), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There were two independent reviewers each focusing on SARS, MERS, and 2019-nCoV, respectively. A third independent reviewer was engaged to resolve any conflicting article of interest. We used the key words "SARS", "coronavirus", "MERS", "2019 Novel coronavirus", "Wuhan virus" to identify the diseases in the search strategy. The systematic searches for diagnosis, therapeutic drugs and vaccines were carried out independently and the key words "drug", "therapy", "vaccine", "diagnosis", "point of care testing" and "rapid diagnostic test" were used in conjunction with the disease key words for the respective searches. Examples of search strings can be found in Table S1 . We searched for randomized controlled trials (RCTs) and validation trials (for diagnostics test) published in English, that measured (a) the sensitivity and/or specificity of a rapid diagnostic test or a point-of-care testing kit, (b) the impact of drug therapy or (c) vaccine efficacy against either of these diseases with no date restriction applied. For the 2019-nCoV, we searched for all in vitro, animal, or human studies published in English between 1 December 2019 and 6 February 2020, on the same outcomes of interest. In addition, we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial searches. Studies that examined the mechanisms of diagnostic tests, drug therapy or vaccine efficacy against SARS, MERS and 2019-nCoV were excluded. A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles. With the emergence of 2019-nCoV, real time RT-PCR remains the primary means for diagnosing the new virus strain among the many diagnostic platforms available ( [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ; Table S3 ). Among the 16 diagnostics studies selected, one study discussed the use of RT-PCR in diagnosing patients with 2019-nCoV [11] ( Table 1 ). The period and type of specimen collected for RT-PCR play an important role in the diagnosis of 2019-nCoV. It was found that the respiratory specimens were positive for the virus while serum was negative in the early period. It has also suggested that in the early days of illness, patients have high levels of virus despite the mild symptoms. Apart from the commonly used RT-PCR in diagnosing MERS-CoV, four studies identified various diagnostic methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), RT-insulated isothermal PCR (RT-iiPCR) and a one-step rRT-PCR assay based on specific TaqMan probes. RT-LAMP has similar sensitivity as real time RT-PCR. It is also highly specific and is used to detect MERS-CoV. It is comparable to the usual diagnostic tests and is rapid, simple and convenient. Likewise, RT-iiPCR and a one-step rRT-PCR assay have also shown similar sensitivity and high specificity for MER-CoV. Lastly, one study focused on the validation of the six commercial real RT-PCR kits, with high accuracy. Although real time RT-PCR is a primary method for diagnosing MERS-CoV, high levels of PCR inhibition may hinder PCR sensitivity (Table 1) . There are eleven studies that focus on SARS-CoV diagnostic testing (Table 1) . These papers described diagnostic methods to detect the virus with the majority of them using molecular testing for diagnosis. Comparison between the molecular test (i.e RT-PCR) and serological test (i.e., ELISA) showed that the molecular test has better sensitivity and specificity. Hence, enhancements to the current molecular test were conducted to improve the diagnosis. Studies looked at using nested PCR to include a pre-amplification step or incorporating N gene as an additional sensitive molecular marker to improve on the sensitivity (Table 1 ). In addition, there are seven potential rapid diagnostic kits (as of 24 January 2020; Table 2 ) available on the market for 2019-nCoV. Six of these are only for research purposes. Only one kit from Beijing Genome Institute (BGI) is approved for use in the clinical setting for rapid diagnosis. Most of the kits are for RT-PCR. There were two kits (BGI, China and Veredus, Singapore) with the capability to detect multiple pathogens using sequencing and microarray technologies, respectively. The limit of detection of the enhanced realtime PCR method was 10 2 -fold higher than the standard real-time PCR assay and 10 7fold higher than conventional PCR methods In the clinical aspect, the enhanced realtime PCR method was able to detect 6 cases of SARS-CoV positive samples that were not confirmed by any other assay [25] • The real time PCR has a threshold sensitivity of 10 genome equivalents per reaction and it has a good reproducibility with the inter-assay coefficients of variation of 1.73 to 2.72%. • 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. The real-time RT-PCR reaction was more sensitive than the nested PCR reaction, as the detection limit for the nested PCR reaction was about 10 3 genome equivalents in the standard cDNA control. [34] Real-time reverse-transcription PCR (rRT-PCR); RNA-dependent RNA polymerase (RdRp); open reading frame 1a (ORF1a); Loop-mediated isothermal amplification (LAMP); enzyme-linked immunosorbent assay (ELISA); immunofluorescent assay (IFA); immunochromatographic test (ICT); nasopharyngeal aspirate (NPA). With the emergence of 2019-nCoV, there are about 15 potential vaccine candidates in the pipeline globally (Table 3 ), in which a wide range of technology (such as messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle) was applied. It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening. The published clinical trials were mostly done in United States except for one on the SARS vaccine done in China [44] . All vaccine candidates for SARS and MERS were reported to be safe, well-tolerated and able to trigger the relevant and appropriate immune responses in the participants. In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60. [47] Molecules developed by the university scientists inhibit two coronavirus enzymes and prevent its replication. The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] . The trial conducted in the United States in 2017 demonstrated SAB-301 to be safe and well-tolerated at single doses. Another trial on MERS therapeutics was found on ClinicalTrials.gov-a phase 2/3 trial in the United States evaluating the safety, tolerability, pharmacokinetics (PK), and immunogenicity on coadministered MERS-CoV antibodies REGN3048 & REGN3051 [100]. Rapid diagnostics plays an important role in disease and outbreak management. The fast and accurate diagnosis of a specific viral infection enables prompt and accurate public health surveillance, prevention and control measures. Local transmission and clusters can be prevented or delayed by isolation of laboratory-confirmed cases and their close contacts quarantined and monitored at home. Rapid diagnostic also facilitates other specific public health interventions such as closure of high-risk facilities and areas associated with the confirmed cases for prompt infection control and environmental decontamination [11, 101] . Laboratory diagnosis can be performed by: (a) detecting the genetic material of the virus, (b) detecting the antibodies that neutralize the viral particles of interest, (c) detecting the viral epitopes of interest with antibodies (serological testing), or (d) culture and isolation of viable virus particles. The key limitations of genetic material detection are the lack of knowledge of the presence of viable virus, the potential cross-reactivity with non-specific genetic regions and the short timeframe for accurate detection during the acute infection phase. The key limitations of serological testing is the need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses. The limitation of virus culture and isolation is the long duration and the highly specialized skills required of the technicians to process the samples. All patients recovered. Significantly shorted time from the disease onset to the symptom improvement in treatment (5.10 ± 2.83 days) compared to control group (7.62 ± 2.27 days) (p < 0.05) No significant difference in blood routine improvement, pulmonary chest shadow in chest film improvement and corticosteroid usgae between the 2 groups. However, particularly in the respect of improving clinical symptoms, elevating quality of life, promoting immune function recovery, promoting absorption of pulmonary inflammation, reducing the dosage of cortisteroid and shortening the therapeutic course, treatment with integrative chinese and western medicine treatment had obvious superiority compared with using control treatment alone. Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well-tolerated in healthy participants. [46] Where the biological samples are taken from also play a role in the sensitivity of these tests. For SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates have higher and more prolonged levels of viral RNA because of the tropism of the virus. MERS-CoV viral loads are also higher for severe cases and have longer viral shedding compared to mild cases. Although upper respiratory tract specimens such as nasopharyngeal or oropharyngeal swabs can be used, they have potentially lower viral loads and may have higher risk of false-negatives among the mild MERS and SARS cases [102, 103] , and likely among the 2019-nCoV cases. The existing practices in detecting genetic material of coronaviruses such as SARS-CoV and MERS-CoV include (a) reverse transcription-polymerase chain reaction (RT-PCR), (b) real-time RT-PCR (rRT-PCR), (c) reverse transcription loop-mediated isothermal amplification (RT-LAMP) and (d) real-time RT-LAMP [104] . Nucleic amplification tests (NAAT) are usually preferred as in the case of MERS-CoV diagnosis as it has the highest sensitivity at the earliest time point in the acute phase of infection [102] . Chinese health authorities have recently posted the full genome of 2019-nCoV in the GenBank and in GISAID portal to facilitate in the detection of the virus [11] . Several laboratory assays have been developed to detect the novel coronavirus in Wuhan, as highlighted in WHO's interim guidance on nCoV laboratory testing of suspected cases. These include protocols from other countries such as Thailand, Japan and China [105] . The first validated diagnostic test was designed in Germany. Corman et al. had initially designed a candidate diagnostic RT-PCR assay based on the SARS or SARS-related coronavirus as it was suggested that circulating virus was SARS-like. Upon the release of the sequence, assays were selected based on the match against 2019-nCoV upon inspection of the sequence alignment. Two assays were used for the RNA dependent RNA polymerase (RdRP) gene and E gene where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing. All assays were highly sensitive and specific in that they did not cross-react with other coronavirus and also human clinical samples that contained respiratory viruses [11] . The Hong Kong University used two monoplex assays which were reactive with coronaviruses under the subgenus Sarbecovirus (consisting of 2019-nCoV, SARS-CoV and SARS-like coronavirus). Viral RNA extracted from SARS-CoV can be used as the positive control for the suggested protocol assuming that SARS has been eradicated. It is proposed that the N gene RT-PCR can be used as a screening assay while the Orf1b assay acts as a confirmatory test. However, this protocol has only been evaluated with a panel of controls with the only positive control SARS-CoV RNA. Synthetic oligonucleotide positive control or 2019-nCoV have yet to be tested [106] . The US CDC shared the protocol on the real time RT-PCR assay for the detection of the 2019-nCoV with the primers and probes designed for the universal detection of SARS-like coronavirus and the specific detection of 2019-nCoV. However, the protocol has not been validated on other platforms or chemistries apart from the protocol described. There are some limitations for the assay. Analysts engaged have to be trained and familiar with the testing procedure and result interpretation. False negative results may occur due to insufficient organisms in the specimen resulting from improper collection, transportation or handling. Also, RNA viruses may show substantial genetic variability. This could result in mismatch between the primer and probes with the target sequence which can diminish the assay performance or result in false negative results [107] . Point-of-care test kit can potentially minimize these limitations, which should be highly prioritized for research and development in the next few months. Serological testing such as ELISA, IIFT and neutralization tests are effective in determining the extent of infection, including estimating asymptomatic and attack rate. Compared to the detection of viral genome through molecular methods, serological testing detects antibodies and antigens. There would be a lag period as antibodies specifically targeting the virus would normally appear between 14 and 28 days after the illness onset [108] . Furthermore, studies suggest that low antibody titers in the second week or delayed antibody production could be associated with mortality with a high viral load. Hence, serological diagnoses are likely used when nucleic amplification tests (NAAT) are not available or accessible [102] . Vaccines can prevent and protect against infection and disease occurrence when exposed to the specific pathogen of interest, especially in vulnerable populations who are more prone to severe outcomes. In the context of the current 2019-nCoV outbreak, vaccines will help control and reduce disease transmission by creating herd immunity in addition to protecting healthy individuals from infection. This decreases the effective R0 value of the disease. Nonetheless, there are social, clinical and economic hurdles for vaccine and vaccination programmes, including (a) the willingness of the public to undergo vaccination with a novel vaccine, (b) the side effects and severe adverse reactions of vaccination, (c) the potential difference and/or low efficacy of the vaccine in populations different from the clinical trials' populations and (d) the accessibility of the vaccines to a given population (including the cost and availability of the vaccine). Vaccines against the 2019-nCoV are currently in development and none are in testing (at the time of writing). On 23 January 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) announced that they will fund vaccine development programmes with Inovio, The University of Queensland and Moderna, Inc respectively, with the aim to test the experimental vaccines clinically in 16 weeks (By June 2020). The vaccine candidates will be developed by the DNA, recombinant and mRNA vaccine platforms from these organizations [109] . Based on the most recent MERS-CoV outbreak, there are already a number of vaccine candidates being developed but most are still in the preclinical testing stage. The vaccines in development include viral vector-based vaccine, DNA vaccine, subunit vaccine, virus-like particles (VLPs)-based vaccine, inactivated whole-virus (IWV) vaccine and live attenuated vaccine. The latest findings for these vaccines arebased on the review by Yong et al. (2019) in August 2019 [110] . As of the date of reporting, there is only one published clinical study on the MERS-CoV vaccine by GeneOne Life Science & Inovio Pharmaceuticals [47] . There was one SARS vaccine trial conducted by the US National Institute of Allergy and Infectious Diseases. Both Phase I clinical trials reported positive results, but only one has announced plans to proceed to Phase 2 trial [111] . Due to the close genetic relatedness of SARS-CoV (79%) with 2019-nCoV [112] , there may be potential cross-protective effect of using a safe SARS-CoV vaccine while awaiting the 2019-nCoV vaccine. However, this would require small scale phase-by-phase implementation and close monitoring of vaccinees before any large scale implementation. Apart from the timely diagnosis of cases, the achievement of favorable clinical outcomes depends on the timely treatment administered. ACE2 has been reported to be the same cell entry receptor used by 2019-nCoV to infect humans as SARS-CoV [113] . Hence, clinical similarity between the two viruses is expected, particularly in severe cases. In addition, most of those who have died from MERS-CoV, SARS-CoV and 2019-nCoV were advance in age and had underlying health conditions such as hypertension, diabetes or cardiovascular disease that compromised their immune systems [114] . Coronaviruses have error-prone RNA-dependent RNA polymerases (RdRP), which result in frequent mutations and recombination events. This results in quasispecies diversity that is closely associated with adaptive evolution and the capacity to enhance viral-cell entry to cause disease over time in a specific population at-risk [115] . Since ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, coronaviruses are likely to infect the upper respiratory and gastrointestinal tract and this may influence the type of therapeutics against 2019-nCoV, similarly to SAR-CoV. However, in the years following two major coronavirus outbreaks SARS-CoV in 2003 and MERS-CoV in 2012, there remains no consensus on the optimal therapy for either disease [116, 117] . Well-designed clinical trials that provide the gold standard for assessing the therapeutic measures are scarce. No coronavirus protease inhibitors have successfully completed a preclinical development program despite large efforts exploring SARS-CoV inhibitors. The bulk of potential therapeutic strategies remain in the experimental phase, with only a handful crossing the in vitro hurdle. Stronger efforts are required in the research for treatment options for major coronaviruses given their pandemic potential. Effective treatment options are essential to maximize the restoration of affected populations to good health following infections. Clinical trials have commenced in China to identify effective treatments for 2019-nCoV based on the treatment evidence from SARS and MERS. There is currently no effective specific antiviral with high-level evidence; any specific antiviral therapy should be provided in the context of a clinical study/trial. Few treatments have shown real curative action against SARS and MERS and the literature generally describes isolated cases or small case series. Many interferons from the three classes have been tested for their antiviral activities against SARS-CoV both in vitro and in animal models. Interferon β has consistently been shown to be the most active, followed by interferon α. The use of corticosteroids with interferon alfacon-1 (synthetic interferon α) appeared to have improved oxygenation and faster resolution of chest radiograph abnormalities in observational studies with untreated controls. Interferon has been used in multiple observational studies to treat SARS-CoV and MERS-CoV patients [116, 117] . Interferons, with or without ribavirin, and lopinavir/ritonavir are most likely to be beneficial and are being trialed in China for 2019-nCoV. This drug treatment appears to be the most advanced. Timing of treatment is likely an important factor in effectiveness. A combination of ribavirin and lopinavir/ritonavir was used as a post-exposure prophylaxis in health care workers and may have reduced the risk of infection. Ribavirin alone is unlikely to have substantial antiviral activities at clinically used dosages. Hence, ribavirin with or without corticosteroids and with lopinavir and ritonavir are among the combinations employed. This was the most common agent reported in the available literature. Its efficacy has been assessed in observational studies, retrospective case series, retrospective cohort study, a prospective observational study, a prospective cohort study and randomized controlled trial ranging from seven to 229 participants [117] . Lopinavir/ritonavir (Kaletra) was the earliest protease inhibitor combination introduced for the treatment of SARS-CoV. Its efficacy was documented in several studies, causing notably lower incidence of adverse outcomes than with ribavirin alone. Combined usage with ribavirin was also associated with lower incidence of acute respiratory distress syndrome, nosocomial infection and death, amongst other favorable outcomes. Recent in vitro studies have shown another HIV protease inhibitor, nelfinavir, to have antiviral capacity against SARS-CoV, although it has yet to show favorable outcomes in animal studies [118] . Remdesivir (Gilead Sciences, GS-5734) nucleoside analogue in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several coronaviruses (CoVs), including highly pathogenic CoVs and potentially emergent BatCoVs. The use of remdesivir may be a good candidate as an investigational treatment. Improved mortality following receipt of convalescent plasma in various doses was consistently reported in several observational studies involving cases with severe acute respiratory infections (SARIs) of viral etiology. A significant reduction in the pooled odds of mortality following treatment of 0.25 compared to placebo or no therapy was observed [119] . Studies were however at moderate to high risk of bias given their small sample sizes, allocation of treatment based on the physician's discretion, and the availability of plasma. Factors like concomitant treatment may have also confounded the results. Associations between convalescent plasma and hospital length of stay, viral antibody levels, and viral load respectively were similarly inconsistent across available literature. Convalescent plasma, while promising, is likely not yet feasible, given the limited pool of potential donors and issues of scalability. Monoclonal antibody treatment is progressing. SARS-CoV enters host cells through the binding of their spike (S) protein to angiotensin converting enzyme 2 (ACE2) and CD209L [118] . Human monoclonal antibodies to the S protein have been shown to significantly reduce the severity of lung pathology in non-human primates following MERS-CoV infection [120] . Such neutralizing antibodies can be elicited by active or passive immunization using vaccines or convalescent plasma respectively. While such neutralizing antibodies can theoretically be harvested from individuals immunized with vaccines, there is uncertainty over the achievement of therapeutic levels of antibodies. Other therapeutic agents have also been reported. A known antimalarial agent, chloroquine, elicits antiviral effects against multiple viruses including HIV type 1, hepatitis B and HCoV-229E. Chloroquine is also immunomodulatory, capable of suppressing the production and release of factors which mediate the inflammatory complications of viral diseases (tumor necrosis factor and interleukin 6) [121] . It is postulated that chloroquine works by altering ACE2 glycosylation and endosomal pH. Its anti-inflammatory properties may be beneficial for the treatment of SARS. Niclosamide as a known drug used in antihelminthic treatment. The efficacy of niclosamide as an inhibitor of virus replication was proven in several assays. In both immunoblot analysis and immunofluorescence assays, niclosamide treatment was observed to completely inhibit viral antigen synthesis. Reduction of virus yield in infected cells was dose dependent. Niclosamide likely does not interfere in the early stages of virus attachment and entry into cells, nor does it function as a protease inhibitor. Mechanisms of niclosamide activity warrant further investigation [122] . Glycyrrhizin also reportedly inhibits virus adsorption and penetration in the early steps of virus replication. Glycyrrhizin was a significantly potent inhibitor with a low selectivity index when tested against several pathogenic flaviviruses. While preliminary results suggest production of nitrous oxide (which inhibits virus replication) through induction of nitrous oxide synthase, the mechanism of Glycyrrhizin against SARS-CoV remains unclear. The compound also has relatively lower toxicity compared to protease inhibitors like ribavirin [123] . Inhibitory activity was also detected in baicalin [124] , extracted from another herb used in the treatment of SARS in China and Hong Kong. Findings on these compounds are limited to in vitro studies [121] [122] [123] [124] . Due to the rapidly evolving situation of the 2019-nCoV, there will be potential limitations to the systematic review. The systematic review is likely to have publication bias as some developments have yet to be reported while for other developments there is no intention to report publicly (or in scientific platforms) due to confidentiality concerns. However, this may be limited to only a few developments for review as publicity does help in branding to some extent for the company and/or the funder. Furthermore, due to the rapid need to share the status of these developments, there may be reporting bias in some details provided by authors of the scientific articles or commentary articles in traditional media. Lastly, while it is not viable for any form of quality assessment and metaanalysis of the selected articles due to the limited data provided and the heterogeneous style of reporting by different articles, this paper has provided a comprehensive overview of the potential developments of these pharmaceutical interventions during the early phase of the outbreak. This systematic review would be useful for cross-check when the quality assessment and meta-analysis of these developments are performed as a follow-up study. Rapid diagnostics, vaccines and therapeutics are key pharmaceutical interventions to limit transmission of respiratory infectious diseases. Many potential developments on these pharmaceutical interventions for 2019-nCoV are ongoing in the containment phase of this outbreak, potentially due to better pandemic preparedness than before. However, lessons from MERS-CoV and SARS-CoV have shown that the journeys for these developments can still be challenging moving ahead. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1 : Example of full search strategy in Pubmed, Table S2 : Google Search: 2019-nCoV diagnostics, Table S3 : Summary of diagnostic assays developed for 2019-nCoV, Table S4

Why is RT-PCR not the best method sometimes?

A super-spreading ewe infects hundreds with Q fever at a farmers' market in Germany https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618839/ SHA: ee1b5a9618dcc4080ed100486cedd0969e80fa4d Authors: Porten, Klaudia; Rissland, Jürgen; Tigges, Almira; Broll, Susanne; Hopp, Wilfried; Lunemann, Mechthild; van Treeck, Ulrich; Kimmig, Peter; Brockmann, Stefan O; Wagner-Wiening, Christiane; Hellenbrand, Wiebke; Buchholz, Udo Date: 2006-10-06 DOI: 10.1186/1471-2334-6-147 License: cc-by Abstract: BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study (cases were Q fever patients, controls were randomly selected Soest citizens) and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii (C. burnetii). RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3(rd )trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii (C. burnetii), a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats [1] . High concentrations of C. burnetii can be found in birth products of infected mammals [2] . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool [1] . Because the infectious dose is very low [3] and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances [4] [5] [6] . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare [1] . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases [2] . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis [1] . Children develop clinical disease less frequently [7, 8] . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay (IFA) is considered to be the reference method, complement fixation (CF), ELISA and microagglutination (MA) can also be used [9] . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections [1] . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year [10] . In 2001 and 2002, 293 and 191 cases were notified, respectively [11, 12] . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory (NCL) for Coxiellae and the Robert Koch-Institute (RKI), the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens (ELISA or IFA), a 4-fold increase in anti-phase II IgG antibody titer (ELISA or IFA) or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection [13, 14] we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case (CCS1 case) as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 (AFE) as (OR-1)/OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context ("notified case") was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition (as used in the vendors' study (s.b.)) identified through random sampling of the Soest population (within CCS2 (s.b.)) as well as in two cohorts (vendors' study and the 9 sailor friends (see below)). The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market (see below "Estimation of the overall attack rate") and gave an estimate of the proportion of clinically ill cases that were hospitalized (s.a.). In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit (approximately 3 meters). Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose (360°) in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east ( Figure 1 ). For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen (plus minus 45°). Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen (in stand units or meters) and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition (as defined in the cohort study on vendors). The overall attack rate among adults was estimated based on the following sources: (1) Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * (2) Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; (3) Results of the small cohort of 9 sailor friends (s.a.); (4) Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever (s.a. "determination of outbreak size and descriptive epidemiology") interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in (1), (3) and (4) we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children (numerator). For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions (number of visitors, proportion of adult visitors and clinical attack rate among adults) we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks (70 animals). Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 (CDC, Atlanta, USA). Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 (65%) of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. (Figure 2 ). If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases (mean +/-2 standard deviations) had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 (53%) were male and 140 (47%) were female. Of the notified cases, 189 (63%) were from the county of Soest, 104 (35%) were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state (Northrhine Westphalia) and 6 (2%) were from five other federal states in Germany (Figure 3 ). Only eight (3%) cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively ( Figure 4 ). 75 (25%) of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 (21%; 95% CI = 6-46%; (1/5 (CCS2), 2/11 (vendors study) and 1/3 (sailor friends)) clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 (56%) outbreak cases; 66 (22%) were confirmed by an increase in anti-phase II antibody titer (CF), 89 (30%) had IgM antibodies against phase II antigens, 11 (4%) were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 (44%) cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two (11%) of 18 cardiological patients and four (36%) of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman (case 3; Table 1 ). Serological follow-up of the mothers detected chronic infection in the same woman (case 3) 12 weeks after delivery. One year follow-up of two newborn children (of cases 1 and 3) identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender (OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23). Seventeen (85%) of 20 cases indicated that they had seen the cow (that also was on display at the market next to the sheep) compared to 7 (32%) of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 (32%) of 25 controls (OR = 5.0; 95%CI = 1.2-22.3; p = 0.03). Touching the sheep was also significantly more common among cases (5/20 (25%) CCS2 cases vs. 0/22 (0%) controls; OR undefined; lower 95% CI = 1.1; p = 0.02). 17 (85%) of 20 CCS2 cases, but only 6 (25%) of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" (OR = 17.0; 95%CI = 3.0-112.5; p < 0.01). Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 (86%) of 87 vendors, and received second hand information about 7 more (overall response rate: 94%). Fourty-five (56%) were male and 35 (44%) were female. 13 (16%) met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 (55%) became cases compared to only 7 (10%) of 70 persons who worked in a stand at a greater distance (relative risk (RR) = 5.5 (95%CI = 2.3-13.2; p = 0.002); Figure 1 ). Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen (section 4, Figure 1 ), 4 (9%) of 44 vendors became cases, compared to 2 (13%) of 15 persons who worked in section 1 (p = 0.6). Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 (32%) became cases. (Table 3 ). In all scenarios the AR among adults was significantly higher than that among children ( Figure 5 ). In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births (57 (86%)) had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 (25%) tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% (8%; 12%; 12%; 16%; 24%). We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market [15] . More recently a large outbreak occurred in Jena (Thuringia) in 2005 with 322 reported cases [16] associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases (source 5) were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] . An additional interview with the two cases with early onset (2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" (see Table 3 ). Displayed are attack rates and 95% confidence intervals. respectively) could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high [17] . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to (within 6 meters) the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% (8/ 191) [7] , 5% [1] and 10% (4/39) [5] ), and there was at least one study with a much higher proportion (63% (10/ 16)) [18] . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep (n = 1000-2000) [5, 7] the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep [19] . In the most severely affected village, the clinical attack rate was 16% (estimated from the data provided) [19] . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic (50% (3/6)) is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease [7] . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness [7, 8] few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock [20] . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks [7, 20, 21] , was 40% compared to 1% in sheep flocks not linked to human outbreaks [20] . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that (in such a setting) can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred [20] , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of (farm) animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents [20, [22] [23] [24] . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations [25] which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author(s) declare that they have no competing interests.

What is the primary reservoir for Coxiella burnetii?

SARS to novel coronavirus – old lessons and new lessons https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/ SHA: 5d254ed178c092d3639ce70ae9653593acc471f9 Authors: McCloskey, Brian; Heymann, David L. Date: 2020-02-05 DOI: 10.1017/s0950268820000254 License: cc-by Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future. Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days. By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal. This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable? According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date. This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR). One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified. Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014. On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO. Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic. While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety. To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems. The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations. Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk. A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] . The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China. The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response. The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact. At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners. There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event. The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less. The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared. The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful. A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic. Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged. Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future. However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure? These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event. This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available. In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves. Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult. In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce.

What are the risks of health workers failing to wash hands?

Epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-nCoV) outbreak https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029449/ SHA: 90de2d957e1960b948b8c38c9877f9eca983f9eb Authors: Cowling, Benjamin J; Leung, Gabriel M Date: 2020-02-13 DOI: 10.2807/1560-7917.es.2020.25.6.2000110 License: cc-by Abstract: Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared.

What do mild clinical presentations of 2019-nCOV indicate?

Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.

Which are some phage based contraceptive vaccines for animals?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What is the appealing quality of the NDV vector?

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067204/ SHA: c097a8a9a543d69c34f10e5c3fd78019e560026a Authors: Chan, Jasper Fuk-Woo; Kok, Kin-Hang; Zhu, Zheng; Chu, Hin; To, Kelvin Kai-Wang; Yuan, Shuofeng; Yuen, Kwok-Yung Date: 2020-01-28 DOI: 10.1080/22221751.2020.1719902 License: cc-by Abstract: A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Deltacoronavirus (δCoV), and Gammacoronavirus (γCoV) [1] . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV (SARS-CoV) which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV (MERS-CoV) which has caused a persistent epidemic in the Arabian Peninsula since 2012 [2, 3] . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host [the Himalayan palm civet (Paguma larvata) for SARS-CoV and the dromedary camel (Camelus dromedarius) for MERS-CoV] before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV (HCoV-229E and HKU-NL63) and 4 βCoV (HCoV-OC43 [ HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly [4] . In contrast, SARS-CoV (lineage B βCoV) and MERS-CoV (lineage C βCoV) may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively [5, 6] . On 31 December 2019, the World Health Organization (WHO) was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China [7] . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan [8, 9] . The earliest date of symptom onset was 1 December 2019 [10] . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 (32%) required intensive care and 6 (15%) died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans [10] . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan [11] . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank (accession no. MN975262) ( Table 1 ). The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees [12] . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [13] . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site [14] . All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X [15] . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction (PSIPRED) [16] . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). Secondary structure prediction in the 5 ′ -untranslated region (UTR) and 3 ′ -UTR was performed using the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/ RNAWebSuite/RNAfold.cgi) with minimum free energy (MFE) and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other (Table 2 ). There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV (Table 3) . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain (NTD) and receptor-binding domain (RBD), while the S2 subunit contains conserved fusion peptide (FP), heptad repeat (HR) 1 and 2, transmembrane domain (TM), and cytoplasmic domain (CP). We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs (SL-CoV ZXC21 and ZC45) and human SARS-CoV (Figure 2 ). Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials [18] . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV (Figure 3(A) ), the core domain of RBD (excluding the external subdomain) are highly conserved (Figure 3(B) ). Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain (RBD) when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b ( Figure 4 ). It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types [19] , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points [20] . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling [21] . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV [22] . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 [23] . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients [24] . In addition, we have previously identified two bat SARS-related-CoV (Bat-CoV YNLF_31C and YNLF_34C) and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV [25] . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 (human SARS-CoV GZ02), the mid-and late-phase SARS-CoV that includes the split orf8b (human SARS-CoV Tor2), civet SARS-CoV (paguma SARS-CoV), two bat SARS-related-CoV containing full-length orf8 (bat-CoV YNLF_31C and YNLF_34C), 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45), and bat SARS-related-CoV HKU3-1 ( Figure 5(A) ). As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5(B) ) which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes [26] , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet(s) containing six strands ( Figure 5(C) ). The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 (MG772934.1), and 82% with human SARS-CoV BJ01 2003 (AY278488) and human SARS-CoV Tor2 (AY274119). The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes (S, E, M, and N) were shown (Figure 6(A-E) ). For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 (A-C), the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif [27] . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b (marked in red), which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR (Figure 7(D-F) ). The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs [27] . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV.

What are four generas?

Hantaviruses in the Americas and Their Role as Emerging Pathogens https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/ SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101 Authors: Hjelle, Brian; Torres-Pérez, Fernando Date: 2010-11-25 DOI: 10.3390/v2122559 License: cc-by Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] . The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖). Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: (1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] . (2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] . (3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil). Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] . Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] . Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease.

What potential mechanism, could be presumed to underlie the pathogenesis of HCPS?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What are the risk factors in childhood pneumonia?

Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.

What demonstrate the potential of phage in applications for nanomaterials?