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The UBA domain is a conserved sequence motif among proteins that can bind polyubiquitin. It is comprised of ~45 amino acids (13) . The amino acids 386-434 of p62, which bind polyubiquitin, has been shown to possess homology to other recently described UBA domains (9) . Interestingly, proteins with UBA domains are more likely to bind polyubiquitin chains over monoubiquitin, such as the yeast UBA protein Rad23, a highly conserved protein involved in nucleotide excision repair (13) . Recently, it has been shown that yeast cells lacking two UBA proteins (Dsk2 and Rad23) are deficient in protein degradation and that the UBA motif is essential for their function in proteolysis (14) .

In addition to the important role in recycling of amino acids from damaged or misfolded proteins, ubiquitin-protein conjugation also has functions unrelated to proteasomal targeting. For example, polyubiquitination is required for the internalization of several yeast and mammalian cell surface proteins into the endocytic pathway (15, 16) . Interestingly, p62 appears to sequester ubiquitinated substrates into a cytoplasmic structure referred to as a sequestosome, into which excess ubiquitinated proteins are segregated (17) . In addition, p62 is an immediate early response gene product for a variety of signals (18) . Thus, p62 appears to play a novel regulatory role for polyubiquitinated proteins and may have an essential function in cell proliferation and differentiation. We have developed a method that will enable identification of protein(s) that interact with p62's UBA domain.

Human adult brain library 10×96 well plates with 100 cDNAs per well and Gold TNT SP6

To search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system. The human adult brain library 96 well plates with 100 cDNAs per well was transcribed and translated employing the Gold TNT SP6 Express 96 plate and [ 35 S] methionine. The TNT Quick-coupled transcription-translation system contained a rabbit reticulocyte lysate pre-mixed with most of the reaction components necessary to carry out transcription/translation in the lysate, including all of the amino acids except methionine. [ 35 S] Methionine was used to label newly synthesized proteins. The reactions were set up according to the manufacturer's instructions. Rabbit reticulocyte lysate has been shown to be capable of carrying out ubiquitination of proteins that were translated in such an in vitro translation system (19, 20) . The reactions mixtures also contained ubiquitin so that the newly synthesized proteins could be ubiquitinated. The reactions were incubated at 30°C for 2 hours. The resulting proteins were assayed to determine their binding ability with p62's UBA domain. Potential positive "hits" were further subdivided and reassayed to link individual clones to the protein of interest (Fig. 1 ).

Each translated pool was resuspended in binding buffer (25 mM Tris pH 7.5, 125 mM NaCl, 0.1% NP-40) and used as a source of protein in p62 UBA pull down assays. Proteins that specifically interact with the UBA domain of p62 were isolated by interaction with agarose-immobilised p62-UBA peptide (amino acid 387-436 of p62) (5 µg) for 2 hours at 4ºC, then washed three times in washing buffer (25 mM Tris pH 7.6, 100 mM NaCl, 1% NP-40). Bound proteins were released by addition of SDS-sample buffer and separated by SDS-PAGE. The SDS-PAGE gels were fixed in 50% methanol, 10% acetic acid for 30 min, stained in 0.2% Commassie Brilliant Blue R-250, 45% methanol, 10% acetic acid for 15 min, destained in 10% acetic acid, 50% methanol overnight, and enhanced in autoradiography enhancer En 3 HANCE for 1 hr and exposed to X-Ray film.

By combining 4 pools as one mixed pool, 96 protein pools were divided into 24 mixed protein pools for use in p62 UBA pull down assays. Positive mixed protein pools were selected and individual pools were retested for its ability to bind p62's UBA domain. The individual cDNA pool from which the positive protein pool was generated was transformed into JM109 competent cells and plated on LB ampicillin plate. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNA was purified from the cell culture and used for TNT Quick coupled in vitro transcription/ translation. The individual protein synthesized from each plasmid DNA chosen was screened for its ability to bind p62's UBA domain.

To confirm the interaction with p62's UBA domain, the final resulting individual proteins were used in the coupled TNT/p62 UBA pull down assays. The cDNA inserts were sequenced in the Genomics Core Facility at Auburn University and the sequences were compared with known sequences in NCBI database by BLAST analysis.

Human embryonic kidney 293 (HEK 293) cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum and transfected with myc-tagged HSP70 plasmid using the Mammalian Cell Transfection Kit. Cells were harvested and lysed in 1 ml of SDS lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM NaF, 0.5% TX-100, 1 mM Na 3 VO 4 , 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM PMSF, 1% SDS) for 30 min on ice, followed by centrifugation at 14000 rpm for 15 min at 4°C to remove the insoluble fraction. The protein concentration of the supernatant was determined using the Bio-Rad DC protein assay reagent with bovine serum albumin (BSA) as standard. Equal amount of protein (750 µg) was immunoprecipitated with anti-myc and collected with agarose-coupled secondary antibody. To the agarose beads containing the immunoprecipitated HSP70, 50 µl of reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP) was added containing 100 ng E1, 200 ng E2 (UbcH7), and 100 µg of E3 (Flag-tagged TRAF6) along with 5 µg GST-WT-Ub, GST-K29R Ub, GST-K48R Ub, GST-K63R Ub, or K63 Ub. Control samples without HSP70, E1, E2, E3, or GST-WT-Ub were also included. Reactions were carried out by continuous shaking at 37°C for 2 hours and then washed three times with reaction buffer. The proteins were released by boiling for 2 min in SDS-PAGE sample buffer, separated on 7.5% SDS-PAGE and Western blotted for anti-ubiquitin.

To search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system from Promega (Cat. No. L6500). The human adult brain library 96 well plates with 100 cDNAs per well were transcribed and translated employing the Gold TNT SP6 Express 96 plate in the presence of [ 35 S] methionine and ubiquitin (25 µg/µl, Sigma). By combining 4 protein pools as one mixed pool, 96 protein pools were divided into 24 mixed pools ( Fig. 2A, 2B ). Each lane contained more than 100 proteins (theoretically 400) with different molecular weight. Therefore, each lane appeared as a smear, indicating that the in vitro transcription/translation system from Promega worked successfully. In order to examine whether proteins synthesized in the IVEC system are also ubiquitinated, Western blot analysis was performed by blotting the newly synthesized proteins (in the presence of cold methionine instead of 35 S methionine) with ubiquitin monoclonal antibody. In the mixed protein pools, each of the 24 lanes appeared as a smear, indicating that proteins synthesized by the IVEC system are also ubiquitinated (Fig. 3A) . Furthermore, the rabbit reticulocyte lysate in the IVEC system can utilize different lysine linkages of ubiquitin (i.e., Ub K29, Ub K48, and Ub K63) for ubiquitination (Fig. 3B) . In order to investigate whether the agarose-immobilised p62 UBA peptide has binding specificity, a mixed protein pool synthesized by IVEC system was tested in a pull down assay in the presence of agarose beads alone or in the presence of p62 UBA agarose beads (Fig. 3C ). Our results revealed that proteins that bound to p62's UBA domain could not be pulled down by agarose beads alone, indicating that the agarose-immobilised p62 UBA peptide had binding specificity.

In order to identify proteins that bind to p62's UBA domain, p62 UBA pull down assays were performed. Out of the 24 mixed protein pools, several pools contained [ 35 S] methionine-labeled bands in the primary p62 UBA pull down assays (Fig. 4) . We chose 6 pools (pool # 2, 4, 8, 14, 20, 21) because of their stronger signal to specifically identify which individual protein pool in the mixed pools has the ability to bind to p62's UBA domain. Therefore, a secondary screen was conducted on the 6 positive individual mixed pools (representing 24 individual protein pools) which bound with p62's UBA domain (Fig. 5) . Mixed protein pool # 2 generated a positive protein with molecular weight of 51 KDa (Fig. 4) , and only individual protein pool "c" out of the four protein pools (a, b, c, d) that comprised protein pool #2 had a protein with the same molecular weight (Fig. 5) . Depending on the size of the protein pulled down in the secondary screen compared to the primary screen (Fig. 4) , individual protein pools "c", "h", "i", "o", "t", and "v" were identified (Fig. 5) . To specifically identify which protein in the individual protein pool has the ability to bind p62's UBA domain, the cDNAs from the positive individual protein pools were then transformed into JM109 competent cells and plated out on LB ampicillin plates. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNAs were purified from the cell culture and used for TNT Quick coupled in vitro transcription/translation. The individual protein synthesized from each plasmid DNA was retested for its ability to bind p62's UBA domain. By synthesizing individual protein from individual plasmid using the Gold TNT Quick coupled in vitro transcription/translation system and subjecting them to p62 UBA pull-down assays, 11 positive clones were isolated from the 6 positive individual pools. It is not surprising that 5 more clones showed binding ability with p62's UBA domain since there are 100 cDNAs in each positive individual pool and some of them could have lower binding ability and therefore showed weak signal in the mixed protein pool. It is also possible that they are not as efficiently synthesized in the mixed TNT reaction as in the individual TNT reaction in which only one cDNA was used as template. The 11 positive plasmids were sequenced and compared with known cDNA sequences in NCBI database using BLAST analysis with results shown in Table 1 .

Interestingly, the proteins identified in the screen fall into three distinct categories. One set are proteins that are associated with Alzheimer's disease, including myelin basic protein, 14-3-3 protein, syntaxin binding protein munc18, transketolase, heat shock protein HSP70, reelin, and calcium/calmodulin kinase II (Table 1 ). Significant decrease in the amount of myelin basic protein has been reported in the white matter of Alzheimer's disease patients, accompanied by increased quantities of βamyloid peptides (21) . The presence of β-amyloid peptides containing senile plaques and neurofibrillary tangles are the two major pathological features in the brain of patients with Alzheimer's disease (22) . Interestingly, 14-3-3 proteins have also been demonstrated to be components of neurofibrillary tangles of Alzheimer's disease brains (23) . Syntaxin binding protein munc18 can powerfully regulate amyloid precursor protein metabolism and β-amyloid secretion through direct and indirect interactions with X11 proteins (24) . The activity of transketolase has been reported to be reduced in dementia of Alzheimer's type brain (25) . Heat shock protein HSP70 expression is significantly increased in the temporal cortex of patients with Alzheimer's disease (26) . Besides HSP70, other heat shock proteins are also linked with Alzheimer's disease. For example, increased synthesis of HSP27 has been suggested to play a role in preventing neuronal injury in AD (27) , and alpha-crystallin heat shock protein has a close relationship with neurofibrillary tangles of AD brains (28) . Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation (29) . Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimer's disease (30) . Enhanced activity of calcium/calmodulin kinase II has been suggested to contribute to phosphorylation of tau protein and lead to neurofibrillary tangle deposition and neuronal death in Alzheimer's disease (31) . Although the relationship between p62 and neurofibrillary tangles or neuritic plaques is unclear, both neurofibrillary tangles and dystrophic neuritis of neuritic plaques are associated with ubiquitin (32) , suggesting that dysfunction in ubiquitin-mediated proteolysis and the resulting accumulation of ubiquitinconjugated proteins may contribute to the origination of dystrophic neuritis and neurofibrillary tangles. Furthermore, p62 has been recently reported to accumulate early in neurofibrillary tangles in Alzheimer's disease (33) , suggesting that p62 may play an important role in Alzheimer's disease by interacting with those proteins through its UBA domain.

A second set of proteins identified in the screen that bind to p62's UBA domain are associated with brain development, including homeobox protein Meis2 and unc51 like kinase II (Table 1) . Although Meis proteins are not extensively studied in humans, these proteins have been shown to be required for hindbrain development in the zebrafish (34) . Unc51 like kinase II has been demonstrated to play a role in axonal elongation (35, 36) , which is needed for the formation of complicated neuronal networks. The third set of proteins that exhibit ability to bind p62's UBA domain are proteins that are linked with other neurodegenerative diseases, including FK506 binding proteins and nuclear receptor corepressor I (Table 1) . FK506 (tacrolimus) is a potent immunosuppressive drug used in the treatment of patients after organ transplantation and in selected autoimmune disorders (37) . FK506 is activated upon binding to members of the immunophilin family of proteins, which were designated as FK506 binding proteins (38) . Immunophilins are chaperone proteins and FK506 binding proteins have been suggested as therapeutics for neurological disorders (39, 40) . Nuclear receptor corepressor I has been suggested to play a role in Huntington's disease because it is able to interact with huntingtin (41) . The proteins identified here suggest that p62's UBA domain has the ability to interact with multiple proteins that play important roles in neurodegenerative diseases. Further screening from the whole genome-wide perspective will be necessary to define the important role that p62's UBA domain plays.

It has been reported that polyubiquitin chains assembled through lysine 48 of ubiquitin act as a signal for substrate proteolysis by the 26S proteasome (42) (43) (44) . In order to understand whether the proteins identified in our screen bind to the p62's UBA domain through lysine 48 (K48), polyubiquitin K48 chains were added to the p62 UBA pull down assay (Fig. 6) . Inclusion of polyubiquitin K48 chains in the assay should compete for the binding of substrate to the p62's UBA domain and reduce the interaction of those proteins with the p62's UBA domain if those proteins are assembled through K48 chains. An alternative interpretation for polyubiquitin K48 chain competition is that the ubiquitin chains are competing for the same binding site as the binding partners which are either ubiquitinated or non-ubiquitinated. We randomly chose five proteins out of the 11 binding partners for the competition pull down (Fig. 6) . Out of the five proteins, four proteins (# 2, 3, 4, and 5) showed reduced binding ability with p62's UBA domain when polyubiquitin K48 chains were included (Fig. 6A, 6B ). However, K48 chains failed to compete with HSP70, suggesting that p62's UBA domain binds to HSP70 through a ubiquitin lysine linkage other than K48. Interestingly, it has been reported that heat shock protein 70 cognate (HSP70) is ubiquitinated by CHIP (carboxyl terminus of Hsc70-interacting protein) via ubiquitin chain synthesis that uses either K29 or K63 (45) . In order to examine which lysine linkage utilized by HSP70 binds to p62's UBA domain, in vitro ubiquitination assay was performed by incubating lysates from HEK cells expressing HSP70 with E1, E2, and E3 in reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP). As control, the ubiquitination of HSP70 utilizing the rabbit reticulocyte lysate was also investigated by Western blot analysis. Our results revealed that HSP70 was ubiquitinated in the IVEC system (Fig. 7A, 7B) , and the rabbit reticulocyte lysate contained enzymes such as TRAF6 (E3) and UbcH7 (E2) for in vitro ubiquitination (Fig. 7C ). TRAF6 was chosen as an E3 in this in vitro ubiquitination assay due to its RING domain, a common feature of E3 ligases, and the observation that p62 is a scaffold for TRAF6 interaction (46) . Therefore, in vitro ubiquitination assays using the E1-E2-E3 system were performed in the presence of either ubiquitin wild type or ubiquitin mutants (K29R, K48R, and K63R). If one lysine mutant blocks the ubiquitination of HSP70, it would suggest that the ubiquitination of HSP70 utilizes that specific lysine linkage.

Our results revealed that HSP70 utilizes K63 linkage to assemble polyubiquitin chains to bind to p62's UBA domain since only the K63R ubiquitin mutant blocked the ubiquitination of HSP70 (Fig. 8A) . A similar result was also observed when reactions were conducted with wild type ubiquitin or mutant ubiquitin with all lysines mutated to arginines except K63 and the ubiquitination of HSP70 occurred only in the reaction that has either intact K63 ubiquitin or wild type ubiquitin (Fig. 8B ). This finding is consistent with previous reports (45) , demonstrating that HSP70 is K63-polyubiquitinated. Furthermore, the in vivo interaction of HSP70 and p62 was confirmed by transfecting myc-tagged HSP70 into HEK 293 cells in the presence of the proteasome inhibitor MG132 and subjecting cell lysates to p62 immunoprecipitation and Western blot with anti-myc antibody (Fig. 8C) . The interaction between HSP70 and p62 in vivo took place only when MG132 was included, suggesting that the interaction in vivo is dependent upon the ubiquitination of HSP70. The specific type of polyubiquitin chain recognized by p62's UBA domain is not yet known and studies are underway lab to determine p62's interaction with specific polyubiquitin chains, however, our preliminary studies suggest that p62's UBA domain may recognize K63 linked polyubiquitin chains. Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin, resolved on 10% SDS-PAGE gels, transferred to nitrocellulose membrane and western blotted with ubiquitin monoclonal antibody. B: HSP70 Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin and 35 S-methionine, resolved on 10% SDS-PAGE and exposed to X-ray film. C: Western blot of rabbit reticulocyte lysate with TRAF6 (E3) and UbcH7 (E2). In summary, for the first time, we demonstrate a systematic approach to identify UBA domain binding proteins from a proteome wide perspective. This approach could be readily adapted to high throughput screening. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins in the human adult brain that interact with the UBA domain of p62, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer's disease. This is a very interesting finding since 9600 cDNAs have been screened and only 11 of them showed binding specificity with p62's UBA domain. Studies are underway to unfold the functional roles of p62 in the ubiquitin system. Our approach provides an easy route to the characterization of UBA domain binding proteins at the level of the whole proteome, its application will unfold the important roles that p62's UBA domain plays. This method could be easily adapted to identify proteins that interact with other UBA domains as well. Vaccinia virus infection disrupts microtubule organization and centrosome function We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein–dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function. Intracellular bacterial and viral pathogens have evolved numerous mechanisms to appropriate and exploit different systems of the host during their life cycles in order to facilitate their spread during entry and exit from the host (Cudmore et al., 1997; Finlay and Cossart, 1997; Dramsi and Cossart, 1998) . In the case of viruses, perhaps the best studied example is the exploitation of the actin cytoskeleton by vaccinia virus during its exit from infected cells (Cudmore et al., 1997) . Vaccinia virus is a large DNA virus with a genome of~191 kb encoding 260 open reading frames (ORFs) that is a close relative of variola virus, the causative agent of smallpox (Johnson et al., 1993; Massung et al., 1993) . Vaccinia virus morphogenesis is a complex process which occurs in the cytoplasm of infected cells and results in the formation of the intracellular mature virus (IMV) and the intracellular enveloped virus (IEV). IMV consist of a viral core of DNA and protein enveloped in a membrane cisterna derived from the intermediate compartment (Sodeik et al., 1993) . The IMV core contains ®ve major proteins, A3L, A4L, A10L, F17R and L4R (Vanslyke and Hruby, 1994; Jensen et al., 1996a) , while 12 proteins, A12L, A13L, A14L, A14.5L, A17L, A27L, D8L, G4L, G7L, H3L, I5L and L1R, are associated with the membranes around the virus particle (Jensen et al., 1996a; Betakova et al., 2000) . Depending on the virus strain and cell type, a proportion of IMV can become enwrapped by a membrane cisterna derived from the trans-Golgi apparatus to give rise to IEV particles (Schmelz et al., 1994) . To date, six IEV-speci®c proteins, A33R (Roper et al., 1996) , A34R (Duncan and Smith, 1992) , A36R (Parkinson and Smith, 1994) , A56R (Payne and Norrby, 1976; Shida, 1986) , B5R (Engelstad et al., 1992; Isaacs et al., 1992) and F13L (Hirt et al., 1986) , have been identi®ed. Studies using recombinant viruses have shown that A33R, A34R, B5R and F13L play an important role in IEV assembly (Blasco and Moss, 1991; Engelstad and Smith, 1993; Wolffe et al., 1993 Wolffe et al., , 1997 Roper et al., 1998; Sanderson et al., 1998a; Ro Èttger et al., 1999) . Vaccinia virus is thought to leave the cell by fusion of the outer IEV membrane with the plasma membrane, to give rise to the extracellular enveloped virus (EEV) (Morgan, 1976; Payne, 1980; Blasco and Moss, 1991) or the cell-associated enveloped viruses (CEV) which remain associated with the outer surface of the plasma membrane (Blasco and Moss, 1992) .

During the complex vaccinia infection process, the actin cytoskeleton is dramatically reorganized and numerous actin comet-like tails are induced by IEV particles (Cudmore et al., 1995; Ro Èttger et al., 1999) . Using actin polymerization as the driving force, IEV particles are propelled on actin tails until they contact the plasma membrane and extend outwards, thereby facilitating infection of neighbouring cells (Cudmore et al., 1995) . In addition, vaccinia infection results in stimulation of cell motility, loss of contact inhibition and changes in cell adhesion (Sanderson and Smith, 1998; Sanderson et al., 1998b) . Vaccinia virus-induced cell motility can be subdivided further into cell migration and extension of neurite-like projections, the latter of which is dependent on microtubules (Sanderson et al., 1998b) . The dependence of neurite-like projection formation on microtubules suggests that the microtubule cytoskeleton may also play a role during the life cycle of vaccinia virus. Indeed, recently, the vaccinia A27L protein and microtubules have been shown to be required for ef®cient IMV dispersion (Sanderson et al., 2000) . Furthermore, in the absence of vaccinia actin-based motility, cell to cell spread still occurs although it is less ef®cient (Wolffe et al., 1997 Sanderson et al., 1998a) , suggesting that additional transport mechanisms must exist.

Given these observations, we wondered whether the microtubule cytoskeleton has a function during the life Vaccinia virus infection disrupts microtubule organization and centrosome function The EMBO Journal Vol. 19 No. 15 pp. 3932±3944, 2000 cycle of vaccinia virus. We now report that the microtubule cytoskeleton and the dynein±dynactin complex play an important role during the early stages of vaccinia infection. However, later during the infection cycle, loss of centrosome function and accumulation of viral-encoded microtubule-associated proteins (MAPs) result in a dramatic rearrangement of the microtubule cytoskeleton.

Vaccinia localization in the vicinity of the MTOC depends on microtubules and the dynein±dynactin complex Indirect immuno¯uorescence labelling shows that by 6 h post-infection the majority of vaccinia virus particles are concentrated in the area coinciding with the centre of the microtubule aster ( Figure 1A and C). To examine whether this localization is indeed microtubule dependent, we infected cells pre-treated with nocodazole to depolymerize microtubules. In the absence of microtubules, virus particles were distributed throughout the cytoplasm ( Figure 1B and D) . The accumulation of virus particles in the area around the centre of the microtubule aster suggested that a microtubule minus end-directed motor may be involved in establishing the position of the virus in this location. To examine this possibility, we infected cells overexpressing p50/dynamitin which acts as a dominantnegative for dynein±dynactin function (Echeverri et al., 1996) . We found in cells overexpressing p50/dynamitin that virus particles did not accumulate at the centre of the microtubule aster but rather throughout the cytoplasm, as occurs in the absence of microtubules (compare Figure 2B with Figure 1D ).

As vaccinia morphogenesis involves wrapping by host membranes, it was possible that the effects of nocodazole and p50/dynamitin on virus localization were in fact due to disruption of the intermediate compartment and Golgi apparatus by these reagents (Burkhardt et al., 1997) . However, two independent experiments showed that this is not the case. First, in cells infected in the absence of microtubules, the Golgi apparatus as well as vaccinia virus particles are dispersed throughout the cytoplasm but do not co-localize ( Figure 3F and O). Secondly, vaccinia particles remain in the vicinity of the microtubule-organizing centre (MTOC) when the Golgi but not the microtubules was disrupted by treatment with brefeldin A ( Figure 3G and P). Similar results were obtained using other markers: A17L for vaccinia, galactosyltransferase for the Golgi or ERGIC53 for the intermediate compartment (data not shown). Taken together, our data indicate that the microtubule cytoskeleton is required for the localization of newly assembled virus particles in the vicinity of the MTOC during vaccinia infection.

Formation of functional IEV, but not IMV, is microtubule dependent Given the requirement for microtubules in vaccinia localization, we subsequently examined whether this localization has a role in morphogenesis of the two different intracellular forms of vaccinia virus, IMV and IEV. From electron microscopic examination of cells infected in the presence of nocodazole, it became clear that IMV particles which are morphologically indistinguishable from controls are formed ( Figure 4 ). Although IMV particles are assembled in the absence of microtubules, we wondered whether their number is reduced and whether those that are formed are infectious, since the integrity of the intermediate compartment depends on microtubules (Burkhardt et al., 1997) . To address this question, three independent virus stocks were prepared in the presence or absence of nocodazole. To simplify the interpretation of the data, we used the recombinant vaccinia virus mutant DF13L, which is unable to form IEV (Blasco and Moss, 1991) . The ®nal concentration of virus particles produced, Vaccinia uses and abuses the microtubule cytoskeleton as determined by the method of Joklik (1962) , was 30.2 6 5.2 3 10 10 particles/ml in the presence of microtubules and 9.0 6 6.7 3 10 10 particles/ml in the absence of microtubules. Although there is a 3-fold decrease in the number of virus particles formed in the absence of microtubules, the particles that are formed are infectious (data not shown).

While infectious IMV are formed in the absence of microtubules, we found no evidence for IEV formation, based on electron microscope examination of cells infected in the presence of nocodazole ( Figure 4 ). We did, however, observe IMV particles partially wrapped in trans-Golgi membranes most probably in the process of abortive IEV formation ( Figure 4D ). Given these data, we examined by indirect immuno¯uorescence whether low amounts of IEV particles are formed in the absence of microtubules. However, we could ®nd no evidence for colocalization of the IEV protein markers A36R, A34R or A33R with vaccinia particles formed in the presence of nocodazole ( Figure 5F ). We also found no evidence for IEV formation, based on their ability to nucleate actin tails ( Figure 5O ). As IEV particle assembly involves wrapping by the Golgi apparatus (Schmelz et al., 1994) , we examined the effects of only disrupting this membrane compartment using brefeldin A. We could ®nd no evidence for IEV formation, based on co-localization of IEV protein markers with virus particles and actin tails in cells infected in the presence of brefeldin A ( Figure 5G±I and P±R). Indeed, in brefeldin A-treated cells, the IEV membrane proteins required for assembly were observed in the endoplasmic reticulum and not the trans-Golgi ( Figure 5H ). In summary, our data indicate that the microtubule cytoskeleton is required for ef®cient IMV assembly and is essential for IEV formation.

In the course of our experiments, it became obvious that the Golgi apparatus becomes progressively dispersed during infection co-concominantly with disruption of the microtubule network ( Figure 6 ). Further analysis showed that during infection the normal morphology of the microtubule cytoskeleton is replaced by morphologically aberrant microtubule forms, which vary among each other but have in common the absence of a discrete MTOC ( Figure 7 ). These aberrant forms can be broadly classi®ed into three types: (i) cells with a disorganized microtubule network where microtubules seem randomly oriented ( Figure 7E ); (ii) cells in which microtubules form rings around the nucleus and throughout the cytoplasm ( Figure 7H ); or (iii) cells with long projections consisting of microtubule bundles ( Figure 7K ). We quanti®ed the appearance of the different morphological forms in ®ve independent infection experiments, in which 200 cells were counted for each time point for each experiment ( Figure 7C , F, I and L). Small compact cells, representing 20.7 6 2.6, 21.8 6 12.4 and 29.7 6 15.2% for 5, 8 and 24 h post-infection, respectively, in which the microtubule cytoskeleton morphology was not evident were not included in the analysis. Already by 5 h post-infection, when virus particle assembly has occurred, the normal aster microtubule con®guration has been disrupted and replaced in the majority of cells by microtubules without obvious organization from the MTOC ( Figure 7F ). Furthermore,~10% of cells have microtubule rings and 5% of cells have long projections by this time point ( Figure 7I and L). As the infection proceeds, microtubules become progressively more disrupted and bundled ( Figure 7I and L).

From our observations, there seems to be no obvious connection between the disruption and changes in the actin and the microtubule cytoskeletons ( Figure 7) . Moreover, the same reorganization of the microtubule network occurs in cells infected with the vaccinia deletion mutants DF13L and DA36R which do not make actin tails (data not shown). The effects of vaccinia virus infection on the reorganization of the microtubule cytoskeleton were also observed in all cell lines we examined (BHK-21, C 2 C 12 , PtK2, RK 13 and Swiss 3T3) to varying degrees (data not shown). Our data show that vaccinia infection results in severe disruption of the normal morphology of the microtubule cytoskeleton.

The formation of microtubule bundles and the loss of organization from the MTOC in vaccinia-infected cells is strongly reminiscent of the phenotype observed in cells overexpressing a MAP (Weisshaar et al., 1992; Togel et al., 1998) . As overexpression of MAPs stabilizes microtubules, we examined whether the microtubule cytoskeleton in infected cells was more resistant to depolymerization by nocodazole or cold treatment ( Figure 8 ). This was indeed the case, suggesting that the virus genome may encode viral proteins with MAP-like properties. To identify viral proteins which exhibit microtubule-binding properties, we performed microtubule co-sedimentation assays using extracts prepared from uninfected and vaccinia-infected cells (Figure 9 ). Initial experiments, however, revealed that intact virus particles in the extracts were prone to pellet even in the absence of microtubules, making identi®cation of viral MAPs impossible. To avoid this problem, we prepared extracts from cells infected in the presence of rifampicin, a drug that inhibits vaccinia virus particle assembly but does not affect viral protein expression (Moss et al., 1969; Tan and McAuslan, 1970) . The morphological effects of vaccinia infection on the microtubule cytoskeleton were the same in the presence or absence of rifampicin (data not shown). Comparison of the proteins present in pellets from microtubule co-sedimentation assays reveals that a number of additional prominent and minor bands are present in extracts prepared from vaccinia-infected but not from uninfected cells (Figure 9 ). Co-sedimentation assays Vaccinia uses and abuses the microtubule cytoskeleton performed in the presence of nocodazole or with coldtreated extracts reveal that the majority of these additional bands disappear in the absence of microtubules. To identify the viral proteins co-sedimenting with microtubules, we performed in-gel protease digestion followed by analysis of the resulting peptides by MALDI mass spectrometry. Using this approach, we identi®ed a number of potential vaccinia-encoded MAPs: A10L (a structural protein), I1L and L4R (which are DNA-binding proteins), all of which are associated with viral cores (Vanslyke and Hruby, 1994; Jensen et al., 1996a; Klemperer et al., 1997) , and A6L which is conserved in all poxvirus genomes but is of unknown function (Figure 9 ).

A10L and L4R associate with microtubules in vivo and mediate binding of viral cores to microtubules in vitro Using available antibodies, we examined the localization of A10L, L4R and I1L in infected cells to see whether they associate with microtubules in vivo, in addition to their essential role in the virus core (Vanslyke and Hruby, 1994; Jensen et al., 1996a) . As a negative control, we also examined the localization of the A3L core protein which was identi®ed as the prominent 70 kDa protein pelleting in the absence of microtubules (Figure 9 ). Indirect immunouorescence analysis showed that A10L and L4R are associated with microtubules, in both the presence and absence of rifampicin ( Figure 10 ). As expected, A10L and L4R were also associated with viral particles (data not shown). In contrast, I1L and A3L were never observed in association with microtubules, regardless of the ®xation conditions, but were localized to viral factories and viral particles, respectively (data not shown). Interestingly, A10L and L4R were not associated with all microtubules but were co-localized with a subset of acetylated microtubules ( Figure 10) .

The association of A10L and L4R with virus particles and microtubules raises the question of whether there is a role for this microtubule-binding activity during infection. We wondered whether these two proteins mediate the interaction of incoming viral cores with microtubules at the beginning of infection, as cores and not virus particles are released in the cytoplasm at the start of the infection cycle (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) . To examine this possibility, we investigated whether puri®ed viral cores would bind microtubules in vitro. We found that viral cores were able to bind microtubules, while protease-treated cores showed no association ( Figure 11A and B) . Pre-incubation of puri®ed viral cores with antibodies against A10L and L4R speci®cally inhibited the interaction of viral cores with microtubules ( Figure 11C and D); in contrast, IgG or antibodies against A3L had no inhibitory effect ( Figure 11E and F). Taken together, our data suggest that A10L and L4R have MAP-like properties and may play a role in mediating interactions of incoming viral cores with microtubules.

The dramatic rearrangement of the microtubule cytoskeleton which occurs during vaccinia infection is unlikely to be attributed exclusively to the action of A10L and L4R since they only associate with a subset of microtubules ( Figure 10) . Furthermore, the loss of microtubule organization precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. We therefore wondered whether vaccinia infection disrupts centrosome function, given the loss of microtubule aster con®guration during infection (Figure 7 ). Since microtubules are nucleated by the centrosome in animal cells, we examined whether vaccinia infection affects g-tubulin, which is critically required for this process (Stearns and Kirschner, 1994) . We observed that g-tubulin labelling of the centrosome is greatly reduced from as early as 2 h post-infection ( Figure 12 ). The same result was obtained when we infected PtK1 cells stably expressing green¯uorescent protein (GFP)-labelled g-tubulin (Khodjakov and Rieder, 1999) . In addition, the centrosomal and centriolar components pericentrin, C-Nap 1, Nek 2 and centrin are reduced by immuno¯uorescence in the centrosomes/centrioles of vaccinia-infected cells ( Figure 12) . Furthermore, the reduction of centrosomal markers requires viral protein synthesis as their levels are not affected when cells are infected in the presence of cycloheximide (data not shown).

The dramatic reduction of g-tubulin from the centrosome implies that vaccinia infection perturbs centrosome Vaccinia uses and abuses the microtubule cytoskeleton function. To test this hypothesis, we examined whether the centrosome in vaccinia-infected cells could re-nucleate microtubules, following their depolymerization by nocodazole. We found that by 2 h post-infection, when we already see a reduction in g-tubulin, microtubule nucleation from the centrosome was very inef®cient, as compared with uninfected controls, indicating that vaccinia has disrupted`normal' centrosome function (Figure 13 ). At later times post-infection, microtubule re-nucleation ef®ciency from the centrosome was even lower (data not shown). However, following nocodazole washout, microtubules eventually are repolymerized throughout the cytoplasm of infected cells but do not display any organization from the MTOC, as do controls (compare Figure 13I and K).

The size of virus particles is such that they are unlikely to move within and between cells by diffusion alone, suggesting that their movements will require interactions with the host cytoskeleton. Previous data have shown that vaccinia virus both disrupts and hijacks the actin cytoskeleton to facilitate movement of the intracellular enveloped form of vaccinia virus (Cudmore et al., 1995; Ro Èttger et al., 1999) and of the infected cell itself (Sanderson and Smith, 1998; Sanderson et al., 1998b) . The data described here now show that vaccinia also uses and subsequently disrupts the microtubule cytoskeleton during its infection cycle. It is clear from our experiments and the previous observations of Ulaeto et al. (1995) that microtubules are required to maintain the integrity of the Golgi apparatus which is in turn required for IMV wrapping to form IEV (Schmelz et al., 1994) . In contrast, IMV are assembled in the absence of microtubules, albeit at reduced levels. While microtubules are not required for IMV assembly, they are required together with the dynein±dynactin complex for virion accumulation in the vicinity of the microtubule aster. One can envisage that minus enddirected microtubule-dependent movements of IMV particles from their site of assembly in the viral factory towards the MTOC, by the dynein±dynactin complex, would enhance the possibility of wrapping with the Golgi apparatus and subsequent IEV formation. Recently it has been shown that the IMV protein A27L and microtubules are required for ef®cient IMV dispersion from the viral factories (Sanderson et al., 2000) . In the absence of A27L, mature IMV particles accumulate at the periphery of the virus factory but do not subsequently wrap to form IEV, presumably because they are unable to move on microtubules (Sanderson et al., 2000) .

The microtubule-and dynein±dynactin-dependent accumulation of vaccinia in the vicinity of the MTOC is analogous to the microtubule-dependent movements required for herpes simplex virus 1 (HSV-1) and adenovirus to reach their site of replication in the nucleus (Sodeik et al., 1997; Suomalainen et al., 1999; Leopold et al., 2000) . In the case of HSV-1, the UL34 protein, 9 . Vaccinia encodes proteins that co-sediment with microtubules. Analysis of pellets from in vitro microtubule co-sedimentation assays performed with protein extracts from vaccinia-infected (inf.) and uninfected (uninf.) cells. Twice the amount of pellet has been loaded in control assays performed in the absence of microtubules (nocodazole or 4°C). Proteins co-sedimenting with microtubules that were only present in extracts from infected cells are indicated by an asterisk. The identity of proteins determined by in-gel proteolysis MALDI mass spectrometry is indicated (arrowheads).

which is associated with the incoming nucleocapsids, interacts with the intermediate chain of cytoplasmic dynein (IC-1a) (Ye et al., 2000) . It has also been reported that incoming nucleocapsids of pseudorabies virus, an alphaherpes virus closely related to HSV-1, are associated with and dependent on microtubules for their movement to the nucleus (Kaelin et al., 2000) . This interaction may be mediated by the UL25 protein, a minor but essential component of the capsid, which co-localizes with microtubules and accumulates at the MTOC (Kaelin et al., 2000) . The accumulation of UL25 at the MTOC is consistent with a possible interaction with the dynein± dynactin motor complex which is known to be localized at the MTOC (Echeverri et al., 1996) . It would not be surprising, based on observations with HSV-1 and pseudorabies, if microtubules and dynein±dynactin were also involved in establishing the infection cycle of cytomegalovirus (CMV), Epstein±Barr virus and varicella-zoster virus, all of which are herpes viruses. The other clear example of microtubule-dependent virus movements during the establishment of infection is that of incoming human foamy virus (HFV) which is dependent on microtubules and presumably a minus end-directed microtubule motor to get to its nuclear replication site (Saib et al., 1997) . In the absence of protein expression, HFV Gag proteins, which are associated with the viral genome, accumulate at the centrosome in a microtubuledependent fashion prior to nuclear import (Saib et al., 1997) . The centrosomal accumulation of Gag proteins of HFV, however, appears to be unique for this class of retroviruses as no similar localization has been reported for human immunode®ciency virus (HIV) or other retroviruses. On the other hand, the Gag protein of murine leukaemia virus and HIV has been shown to interact with KIF4, a microtubule plus end-directed kinesin motor, both in vitro and in vivo (Kim et al., 1998; Tang et al., 1999) , suggesting that additional roles may exist for microtubules and motors during the outward movement of virus particles. Indeed, vaccinia virus particles are able to reach the cell periphery in the absence of actin-based motility (see images in Wolffe et al., 1997; Sanderson et al., 1998a Sanderson et al., , 2000 Ro Èttger et al., 1999) , suggesting that viral particles can also move out on microtubules (Sanderson et al., 2000) . Microtubule-dependent motordriven movements of virus particles represent an ef®cient mechanism to achieve a peri-nuclear localization, required to facilitate entry into the nucleus during establishment of infection. They also provide an excellent way for newly assembled virus particles to reach the cell periphery, facilitating the continued spread of infection.

Our data show that although vaccinia virus uses the microtubule cytoskeleton to achieve a peri-nuclear localization, microtubule and Golgi organization becomes disrupted later during the infection process. Interestingly, HSV-1 and CMV have also been reported to disrupt the microtubule cytoskeleton and Golgi organization in their infection cycles (Avitabile et al., 1995; Fish et al., 1996) . While disruption of the microtubule network might at ®rst sight not appear to be bene®cial to the virus, it may not actually hinder viral spread but could enhance it. First, extensive virus assembly and spread to the cell periphery have already occurred by the time the microtubule cytoskeleton and Golgi organization are disrupted. Secondly, disruption of microtubule organization may overcome potential microtubule motor anchoring effects at the MTOC, thus allowing viral spread to the periphery to occur more easily. Lastly, the formation of long projections of up to 200 mm supported by extensive microtubule bundles provides a means to achieve long range spread of virus particles (Sanderson et al., 1998b) .

It is clear that disruption and reorganization of the microtubule cytoskeleton by vaccinia virus is mediated by the combined effects of viral proteins with MAP-like properties and loss of microtubule-organizing function from the MTOC. The same may also be true for HSV-1, although disruption of centrosome function remains to be established, as late in infection microtubules are organized in bundles around the nucleus and do not show MTOCorchestrated organization (Avitabile et al., 1995) . The identi®cation of viral proteins with MAP-like properties is not unique to vaccinia virus. The VP22 tegument protein from HSV-1 co-localizes with microtubules in infected cells and induces microtubule bundles when expressed in uninfected cells (Elliott and O'Hare, 1998) . Other examples of viral MAPs based on their in vivo localization Fig. 11 . Vaccinia cores bind directly to microtubules in vitro. Puri®ed viral cores labelled by DAPI (green) bind to rhodamine-labelled microtubules (red) in the absence of ®xation (A). Binding to microtubules is not observed if cores are pre-treated with protease (B) or pre-incubated with antibodies against the A10L (C) or L4R (D) proteins. In contrast, pre-incubation of puri®ed viral cores with control IgG (E) or antibody against the A3L protein (F) does not inhibit their interaction with microtubules. Scale bar = 5 mm.

Vaccinia uses and abuses the microtubule cytoskeleton or in vitro association with microtubules are the N protein from murine coronavirus (Kalicharran and Dales, 1996) , the movement protein from tobamovirus (Heinlein et al., 1995) , the aphid transmission factor from cauli¯ower mosaic virus (Blanc et al., 1996) , the UL25 protein from pseudorabies virus (Kaelin et al., 2000) , the VP4 spike protein from rotavirus (Nejmeddine et al., 2000) and the M protein of vesicular stomatitis virus (VSV) (Melki et al., 1994) . The identi®cation of A10L and L4R, two viral core proteins, as MAP-like proteins was, however, unexpected given their previously characterized role in viral morphogenesis (Vanslyke and Hruby, 1994) . The interaction of A10L and L4R with microtubules in vivo, together with the in vitro microtubule-binding data, suggest a potential mechanism for the association of viral cores with microtubules. One could envisage that viral cores which are released into the cytoplasm at the beginning of infection (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) bind directly to microtubules in a manner analogous to adenovirus or HSV-1 nucleocapsids. Further work is required to determine whether incoming cores do in fact move towards the MTOC by the dynein± dynactin complex and/or use the complex for anchoring on microtubules.

The loss of centrosome function must enhance disruption of the microtubule cytoskeleton during infection. Indeed, the loss of microtubule organization from the MTOC precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. Vaccinia-induced loss of centrosomal proteins is inhibited by cycloheximide, indicating that viral protein expression is required for disruption of the centrosome microtubule nucleation activity. To our knowledge, vaccinia virus infection represents the ®rst example of virus-induced disruption of centrosome function, although we would predict that HSV-1 may have a similar effect. The mechanism by which vaccinia virus disrupts the centrosome requires further study; nevertheless, it is clear that understanding the molecular basis of this disruption will provide important insights into the regulation and stability of centrosome function which currently is the subject of intense research (Ohta et al., 1993; Lane and Nigg, 1997; Karsenti, 1999) .

HeLa cells (ATCC CCL2) were infected with the wild-type vaccinia virus strain Western Reserve (WR) or with the vaccinia deletion mutants DF13L (vRB12) (Blasco and Moss, 1991) or DA36R (Parkinson and Smith, 1994) at a multiplicity of infection of 1 p.f.u. (plaque-forming unit) per cell, as described previously (Ro Èttger et al., 1999) . Nocodazole dissolved in dimethyl sulfoxide (DMSO) and brefeldin A dissolved in ethanol were added to the culture medium to ®nal concentration of 10 mM and 5 mg/ml, respectively unless otherwise stated. In non-treated controls, an equal volume of DMSO or ethanol was added. Cells transfected with a myc-tagged p50/dynamitin expression construct (Echeverri et al., 1996) were infected 24 h later with WR and subsequently ®xed 6 h postinfection. All experiments described have been repeated 3±10 times.

The following antibodies were kindly provided: anti-a-tubulin by Dr E.Karsenti, anti-centrin (20H5) by Professor J.L.Salisbury (Sanders and Salisbury, 1994; Paoletti et al., 1996) , anti-Nek2 and anti-C-Nap1 by Professor E.Nigg (Fry et al., 1998a,b) , anti-myc and anti-gp27 by Dr T.Nilsson (Fu Èllekrug et al., 1999) and antibodies against the corresponding vaccinia proteins: A3L, A10L and L4R by Professor D.Hruby (Vanslyke and Hruby, 1994) , I1L by Professor P.Traktman (Klemperer et al., 1997) , A27L (C3) by Dr M.Esteban (Rodriguez et al., 1985) , A33R, A34R and A36R (Ro Èttger et al., 1999) . In addition, the following antibodies were obtained from commercial sources: anti-a-tubulin (N356) (Amersham International, UK), anti-acetylated a-tubulin (6-11B-1) (Sigma, USA), anti-g-tubulin (GTU-88; Sigma), anti-pericentrin and anti-TGN46 (BAbCO, USA), and rabbit IgG (Sigma). Actin was visualized with¯uorescently labelled phalloidin derivatives (Molecular Probes, USA).

Cells were ®xed in ±20°C methanol or in 5% paraformaldehyde in BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl 2 , 1 mM EGTA) followed by 0.1% Triton X-100 permeabilization. Fixed cells were processed for immuno¯uorescence, viewed and images recorded as described previously (Ro Èttger et al., 1999) .

HeLa cells were pre-incubated with 25 mM nocodazole in the medium for 1 h to depolymerize microtubules, prior to infection with vaccinia DF13L at 1 p.f.u./cell. Nocodazole was kept in the medium throughout the infection, while an equal volume of DMSO was added to the controls. At 24 h post-infection, the cells were scraped from the¯asks into the medium and sedimented by centrifugation (300 g, 7 min, 4°C). The cell membranes were disrupted and the nuclei were removed by centrifugation. The resulting post-nuclear supernatant was centrifuged through a 36% sucrose cushion (76 000 g, 30 min, 4°C). The virus pellet was resuspended in 10 mM Tris pH 9; the virus was collected by centrifugation (76 000 g, 30 min, 4°C), resuspended in 10 mM Tris pH 9 and stored at ±80°C. The concentration of the virus (elemental bodies) was determined by OD 260 measurement (Joklik, 1962) . Fig. 13 . Vaccinia infection reduces centrosome microtubule nucleation ef®ciency. In uninfected cells, microtubules (A, E and I) nucleate from centrosomes (B, F and J) after nocodazole washout for the times indicated. In contrast, 2 h after infection with vaccinia, microtubules (C, G and K) are nucleated inef®ciently from centrosomes (D, H and L). All images were collected with identical camera settings, to allow comparison of uorescence intensity between centrosomes. Inserts (B, D, F, H, J and L) are adjusted as in Figure 12 to facilitate visualization of the weak g-tubulin centrosomal labelling. Arrowheads indicate the position of the centrosome. Scale bar = 10 mm.

In vitro microtubule binding assays Puri®ed EEV particles were prepared as described previously (Ro Èttger et al., 1999) and subsequently were used to prepare virus cores following the method of Cudmore et al. (1996) . Rhodamine-labelled microtubules were prepared according to Hyman et al. (1991) . Vaccinia virus cores were incubated with rhodamine-labelled microtubules in BRB80 buffer containing 10 mM taxol for 5 min at room temperature. 4¢,6-diamidino-2phenylindole (DAPI) was added subsequently to a ®nal concentration of 0.1 mg/ml to label the virus cores. Finally, the mixture was diluted 1:1± 1:10 with antifade solution (0.1 mg/ml catalase, 0.1 mg/ml glucose oxidase, 10 mM glucose) and viewed without ®xation. Proteinase K or trypsin treatment of core particles prior to incubation with microtubules was performed as described previously (Roos et al., 1996) . Anti-A3L, A10L, L4R or control IgG antibodies were incubated with puri®ed cores for 1 h at room temperature prior to incubation with microtubules.

Cell extracts and microtubule co-sedimentation assay Extracts from HeLa cells infected for 24 h or uninfected controls maintained in the presence of 0.1 mg/ml rifampicin were prepared as described previously (Ro Èttger et al., 1999) . The extract was clari®ed by centrifugation at 150 000 g for 20 min at 4°C and cytochalasin D added to a ®nal concentration of 1 mg/ml to depolymerize actin ®laments. Endogenous tubulin in the extract was polymerized in a two-step procedure. First, the extract supernatant was supplemented with protease inhibitors, 2 mM MgGTP and 5 mM taxol and incubated for 5 min at room temperature; subsequently, an additional 15 mM taxol was added to the mix and the reaction incubated at 33°C for 30 min. For controls, no taxol was added at any stage and microtubule polymerization was inhibited either by the addition of nocodazole to a ®nal concentration of 40 mM or by maintaining the extract at 4°C throughout the experiment. Following microtubule assembly, each 400 mg extract reaction was diluted 5-fold in BRB80 buffer (containing protease inhibitors and 20 mM taxol) and centrifuged through a 10% sucrose cushion containing protease inhibitors and 20 mM taxol at 165 000 g for 20 min at 25°C. The microtubule pellet was solubilized in SDS±PAGE sample buffer and analysed by SDS± PAGE.

In-gel proteolytic cleavage was performed automatically in the`Progest' as described (Houthaeve et al., 1997) [Genomic Solutions Cambridge (http://www.genomicsolutions.com)] and the peptides obtained were analysed on a Bruker REFLEX MALDI mass spectrometer (Bruker Analytik, Germany) (Jensen et al., 1996b) . Proteins were identi®ed by peptide mass ®ngerprinting (Jensen et al., 1997) using the program PeptideSearch (http://www.narrador.embl-heidelberg.de/Services/ PeptideSearch/PeptideSearchIntro.html.

At 1 h post-infection, nocodazole was added to the culture medium to a ®nal concentration of 25 mM to depolymerize microtubules. At 2 h postinfection, the cells were washed 3±4 times in warm medium to remove nocodazole. Washed cells were incubated in medium without nocodazole for the indicated time at 37°C to re-initiate microtubule polymerization; they were then washed brie¯y in warm phosphate-buffered saline (PBS) and immediately ®xed. In parallel, samples were also removed at the same time point, brie¯y rinsed in ice-cold PBS, ®xed and processed for immuno¯uorescence to con®rm complete microtubule depolymerization before initiation of microtubule assembly. Uninfected control HeLa cells were treated and processed in an identical fashion. The same numbers of images were integrated using identical camera settings to allow direct comparison between infected and uninfected samples from the same experiment. The site of origin of the 1918 influenza pandemic and its public health implications The 1918-1919 influenza pandemic killed more people than any other outbreak of disease in human history. The lowest estimate of the death toll is 21 million, while recent scholarship estimates from 50 to 100 million dead. World population was then only 28% what is today, and most deaths occurred in a sixteen week period, from mid-September to mid-December of 1918.

It has never been clear, however, where this pandemic began. Since influenza is an endemic disease, not simply an epidemic one, it is impossible to answer this question with absolute certainty. Nonetheless, in seven years of work on a history of the pandemic, this author conducted an extensive survey of contemporary medical and lay literature searching for epidemiological evidence -the only evidence available. That review suggests that the most likely site of origin was Haskell County, Kansas, an isolated and sparsely populated county in the southwest corner of the state, in January 1918 [1] . If this hypothesis is correct, it has public policy implications.

But before presenting the evidence for Haskell County it is useful to review other hypotheses of the site of origin. Some medical historians and epidemiologists have theorized that the 1918 pandemic began in Asia, citing a lethal outbreak of pulmonary disease in China as the forerunner of the pandemic. Others have speculated the virus was spread by Chinese or Vietnamese laborers either crossing the United States or working in France.

More recently, British scientist J.S. Oxford has hypothesized that the 1918 pandemic originated in a British Army post in France, where a disease British physicians called "purulent bronchitis" erupted in 1916. Autopsy reports of soldiers killed by this outbreak -today we would classify the cause of death as ARDS -bear a striking resemblance to those killed by influenza in 1918 [2] .

But these alternative hypotheses have problems. After the 1918-1919 pandemic, many investigators searched for the source of the disease. The American Medical Association sponsored what is generally considered the best of several comprehensive international studies of the pandemic conducted by Dr. Edwin Jordan, editor of The Journal of Infectious Disease. He spent years reviewing evidence from all over the world; the AMA published his work in 1927.

Since several influenza pandemics in preceding centuries were already well-known and had come from the orient, Jordan first considered Asia as the source. But he found no evidence. Influenza did surface in early 1918 in China, but the outbreaks were minor, did not spread, and contemporary Chinese scientists, trained by Rockefeller Institute for Medical Research (now Rockefeller University) investigators, stated they believed these outbreaks were endemic disease unrelated to the pandemic [3] . Jordan also looked at the lethal pulmonary disease cited by some historians as influenza, but this was diagnosed by contemporary scientists as pneumonic plague. By 1918 the plague bacillus could be easily and conclusively identified in the laboratory [3] . So after tracing all known outbreaks of respiratory disease in China, Jordan concluded that none of them "could be reasonably regarded as the true forerunner" of the pandemic [3] .

Jordan also considered Oxford's theory that the "purulent bronchitis" in British Army camps in 1916 and 1917 was the source. He rejected it for several reasons. The disease had flared up, true, but had not spread rapidly or widely outside the affected bases; instead, it seemed to disappear [3] . As we now know a mutation in an existing influenza virus can account for a virulent flare-up. In the summer of 2002, for example, an influenza epidemic erupted in parts of Madagascar with an extremely high mortality and morbidity; in some towns it sickened an outright majority -in one instance sixty-seven percent -of the population. But the virus causing this epidemic was an H3N2 virus that normally caused mild disease. In fact, the epidemic affected only thirteen of 111 health districts in Madagascar before fading away [4]. Something similar may have happened in the British base.

Jordan considered other possible origins of the pandemic in early 1918 in France and India. He concluded that it was highly unlikely that the pandemic began in any of them [3] .

That left the United States. Jordan looked at a series of spring outbreaks there. The evidence seemed far stronger. One could see influenza jumping from Army camp to camp, then into cities, and traveling with troops to Europe. His conclusion: the United States was the site of origin.

A later equally comprehensive, multi-volume British study of the pandemic agreed with Jordan. It too found no evidence for the influenza's origin in the Orient, it too rejected the 1916 outbreak among British troops, and it too concluded, "The disease was probably carried from the United States to Europe [5] ." Australian Nobel laureate MacFarlane Burnet spent most of his scientific career working on influenza and studied the pandemic closely. He too concluded that the evidence was "strongly suggestive" that the disease started in the United States and spread with "the arrival of American troops in France [6] ."

Before dismissing the conclusions of these contemporary investigators who lived through and studied the pandemic, one must remember how good many of them were. They were very good indeed.

The Rockefeller Institute, whose investigators were intimately involved in the problem, alone included extraordinary people. By 1912 its head Simon Flexner -his brother wrote the "Flexner report" that revolutionized American medical education -used immune serum to bring the mortality rate for meningococcal meningitis down from over 80% to 18%; by contrast, in the 1990s at Massachusetts General Hospital a study found a 25% mortality rate for bacterial meningitis. Peyton Rous won the Nobel Prize in 1966 for work he did at the institute in 1911; he was that far ahead of the scientific consensus. By 1918 Oswald Avery and others at Rockefeller Institute had already produced both an effective curative serum and a vaccine for the most common pneumococcal pneumonias. At least partly because of the pandemic, Avery would spend the rest of his career studying pneumonia. That work led directly to his discovery of the "transforming principle"his discovery that DNA carries the genetic code.

The observations of investigators of this quality cannot be dismissed lightly. Jordan was of this quality.

More evidence against Oxford's hypothesis comes from Dr. Jeffrey Taubenberger, well-known for his work extracting samples of the 1918 virus from preserved tissue and sequencing its genome. He initially believed, based on statistical analysis of the rate of mutation of the virus that it existed for two or three years prior to the pandemic. But further work convinced him that the virus emerged only a few months prior to the pandemic (personal communication with the author from J Taubenberger, June 5 th 2003).

So if the contemporary observers were correct, if American troops carried the virus to Europe, where in the United States did it begin?

Both contemporary epidemiological studies and lay histories of the pandemic have identified the first known outbreak of epidemic influenza as occurring at Camp Funston, now Ft. Riley, in Kansas. But there was one place where a previously unknown -and remarkable -epidemic of influenza occurred.

Haskell County, Kansas, lay three hundred miles to the west of Funston. There the smell of manure meant civilization. People raised grains, poultry, cattle, and hogs. Sod-houses were so common that even one of the county's few post offices was located in a dug-out sod home. In 1918 the population was just 1,720, spread over 578 square miles. But primitive and raw as life could be there, science had penetrated the county in the form of Dr. Loring Miner. Enamored of ancient Greece -he periodically reread the classics in Greek -he epitomized William Welch's comment that "the results [of medical education] were better than the system." His son was also a doctor, trained in fully scientific ways, serving in the Navy in Boston.

In late January and early February 1918 Miner was suddenly faced with an epidemic of influenza, but an influenza unlike any he had ever seen before. Soon dozens of his patients -the strongest, the healthiest, the most robust people in the county -were being struck down as suddenly as if they had been shot. Then one patient pro-gressed to pneumonia. Then another. And they began to die. The epidemic got worse. Then, as abruptly as it came, it disappeared. Men and women returned to work. Children returned to school. And the war regained its hold on people's thoughts.

The disease did not, however, slip from Miner's thoughts. Influenza was neither a reportable disease, nor a disease that any state or federal public health agency tracked. Yet Miner considered this incarnation of the disease so dangerous that he warned national public health officials about it. Public Health Reports (now Morbidity and Mortality Weekly Report), a weekly journal produced by the U.S. Public Health Service to alert health officials to outbreaks of communicable diseases throughout the world, published his warning. In the first six months of 1918, this would be the only reference in that journal to influenza anywhere in the world.

Historians and epidemiologists have previously ignored Haskell most likely because his report was not published until April and it referred to deaths on March 30, after influenza outbreaks elsewhere. In actuality, by then the county was free of influenza. Haskell County, Kansas, is the first recorded instance anywhere in the world of an outbreak of influenza so unusual that a physician warned public health officials. It remains the first recorded instance suggesting that a new virus was adapting, violently, to man.

If the virus did not originate in Haskell, there is no good explanation for how it arrived there. There were no other known outbreaks anywhere in the United States from which someone could have carried the disease to Haskell, and no suggestions of influenza outbreaks in either newspapers or reflected in vital statistics anywhere else in the region. And unlike the 1916 outbreak in France, one can trace with perfect definiteness the route of the virus from Haskell to the outside world.

All Army personnel from the county reported to Funston for training. Friends and family visited them at Funston. Soldiers came home on leave, then returned to Funston.

The Monitor reported in late February, "Most everybody over the country is having lagrippe or pneumonia (Santa Fe Monitor, February 21 st 1918)." It also noted, "Dean Nilson surprised his friends by arriving at home from Camp Funston on a five days furlough. Dean looks like soldier life agrees with him." He soon returned to the camp. Ernest Elliot left to visit his brother at Funston as his child fell ill. On February 28, John Bottom left for Funston. "We predict John will make an ideal soldier," said the paper (Santa Fe Monitor February 28 th , 1918).

These men, and probably others unnamed by the paper, were exposed to influenza and would have arrived in Funston between February 26 and March 2. On March 4 the first soldier at the camp reported ill with influenza at sick call. The camp held an average of 56,222 troops. Within three weeks more than eleven hundred others were sick enough to require hospitalization, and thousands morethe precise number was not recorded -needed treatment at infirmaries scattered around the base.

Whether or not the Haskell virus did spread across the world, the timing of the Funston explosion strongly suggests that the influenza outbreak there did come from Haskell. Meanwhile Funston fed a constant stream of men to other American locations and to Europe, men whose business was killing. They would be more proficient at it than they knew.

Soldiers moved uninterrupted between Funston and the outside world, especially to other Army bases and France. On March 18, Camps Forrest and Greenleaf in Georgia saw their first cases of influenza and by the end of April twenty-four of the thirty-six main Army camps suffered an influenza epidemic [3] . Thirty of the fifty largest cities in the country also had an April spike in excess mortality from influenza and pneumonia [7] . Although this spring wave was generally mild -the killing second wave struck in the fall -there were still some disturbing findings. A subsequent Army study said, "At this time the fulminating pneumonia, with wet hemorrhagic lungs, fatal in from 24 to 48 hours, was first observed [8] ." (Pathology reports suggest what we now call ARDS.) The first recorded autopsy in Chicago of an influenza victim was conducted in early April. The pathologist noted, "The lungs were full of hemorrhages." He found this unusual enough to ask the then-editor of The Journal of Infectious Diseases "to look over it as a new disease" [3] .

By then, influenza was erupting in France, first at Brest, the single largest port of disembarkation for American troops. By then, as MacFarlane Burnet later said, "It is convenient to follow the story of influenza at this period mainly in regard to the army experiences in America and Europe [6] ."

The fact that the 1918 pandemic likely began in the United States matters because it tells investigators where to look for a new virus. They must look everywhere.

In recent years the World Health Organization and local public health authorities have intervened several times when new influenza viruses have infected man. These interventions have prevented the viruses from adapting to man and igniting a new pandemic. But only 83 countries in the world -less than half -participate in WHO's surveillance system (WHO's flunet website http:// rhone.b3e.jussieu.fr/flunet/www/docs.html). While some monitoring occurs even in those countries not formally affiliated with WHO's surveillance system, it is hardly adequate. If the virus did cross into man in a sparsely populated region of Kansas, and not in a densely populated region of Asia, then such an animal-to-man cross-over can happen anywhere. And unless WHO gets more resources and political leaders move aggressively on the diplomatic front, then a new pandemic really is all too inevitable. Multi-faceted, multi-versatile microarray: simultaneous detection of many viruses and their expression profiles There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients. There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients.

Microarray technology has been proven to be a powerful tool with great potential for biological and medical uses. In this technique, recombinant DNA fragments or synthesized oligonucleotides affixed on the surface of glass slides or nylon membranes are used for detecting complementary nucleic acid sequences (frequently representing a few hundred to >10,000 genes/expressed sequence tags) as well as for genotyping microorganisms and for profiling the gene-expression patterns in cells from higher organisms [1].

A new report by Ghedin, et al. [2] in Retrovirology describes the successful use of a multi-virus array (termed multivi-rus-chip) to detect multiple viral co-infections in cultured cells as well as to study viral gene expression and promoter activities (Figure 1 ). Ghedin's multivirus-chip contains genes from eight human viruses including human immunodeficiency virus type 1 (HIV-1). Conceptually, this chip can be used to detect viral co-infections in AIDS patients who are frequently rendered susceptible to additional opportunistic infections. In developing their multivirus-chip, Ghedin, et al. tested more than 250 ORFs from HIV-1, human T cell leukemia virus types 1 (HTLV-1) and 2 (HTLV-2), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human herpesvirus 6A (HHV6A) and 6B (HHV6B), and Kaposi's sarcoma-associated herpesvirus (KSHV) which were PCR-amplified and spotted on glass slides. They then hybridized their slides with Cy3-or Cy5labeled genomic DNA or cDNAs derived from various virus-infected cells. Their multivirus-chip was found to be highly specific and sensitive for detecting different viral genomic sequences in cell lines. Moreover, the chip could also detect the effect of various drugs on viral gene expression. In such instance, cell lines latently infected with HIV-1 and KSHV were used to generate profiles of viral gene expression in the presence of cyclin-dependent Schematic drawing of the multivirus-chip that possesses multiple functions kinase inhibitor (CKI), Roscovitine, which was applied to cells to suppress the reactivation of latently infected viruses.

Ghedin, et al. [2] also studied the role of cellular chromatin structure on viral gene expression using their multivirus-chip.

They employed the chromatin immunoprecipitation technique (ChIP) [3] to isolate cellular DNA fragments that were bound to phosphorylated histone H3 (P-H3). These DNA fragments were hybridized to the viral ORFs contained on the multivirus-chip to investigate the role of phospho-H3 on viral gene expression. They showed that whether transcriptionally active or silent the chromatin state played a role in regulating the expression of KSHV genes under the different cellular context. Current routine clinical diagnostics employ PCR, Southern blotting, Northern blotting, DNA sequencing and microarray hybridization to detect and characterize genes of interest in biomedicine. PCR is generally regarded as the most sensitive diagnostic method. However, Iyer, et al. [4] have shown that the sensitivity of cDNA-chip hybridization is comparable to that of TaqMan-driven quantitative PCR assay, and that the microarray hybridization technique is less likely to be complicated by high false positive rates due to carry-over contaminations. Furthermore, using microarrays, the viral gene transcripts in infected cells can be easily detected by hybridization without any prior amplification steps, and the microarray technique requires much less experimental material when compared to Southern or Northern blotting and can provide high sensitivity in the setting of large throughput.

In view of the above, the multivirus-chip described in Retrovirology [2] holds several advantages over other more commonly used techniques (e.g. PCR, DNA sequencing) for the diagnosis of viral infections. First, this chip provides a more accurate diagnosis of viral infection by simultaneously evaluating the transcription of all viral genes, and can use such cumulative data to correlate infection with clinical disease manifestations. Second, the high throughput and flexible synthesis nature of DNA microarray construction can allow scientists to tailor-make and rapidly alter arrays to match evolving emergence of new pathogens. The SARS genome chip made by the US NIAID, NIH is a good example [5] of how diagnostic arrays can be developed quickly and be used in a timely manner.

Finally, the most novel application described by Ghedin, et al. is their use of microarrays to correlate the cellular "histone code" [6] with the promoter activity of KSHV. Usually the transcription of a gene located on chromosomal DNA is influenced not only by the cis-acting ele-ments (or DNA-binding motifs), but also by the structure of chromatin. The latter can be vary depending on the post-translational modifications of histone proteins. Methylation, acetylation, and/or phosphorylation of certain amino acid residues at the amino terminal "tails" of histone H3 and/or H4 can indeed influence chromatin structure. Thus accumulating evidence has shown that chromatin-associated proteins and their modifications play vital roles in many physiological processes such as growth, differentiation, and development in mammals, plants and fungi [6, 7] . Many studies have used DNA array technology to investigate viral gene expression or to genotype viral isolates; however, none has used this technique to study the influence of cellular chromatin structure on viral gene expression [1]. Ghedin, et al. [2] demonstrated that only DNA fragments derived from ChIP of latent BCBL-1 cell genomic DNA captured using phospho-H3 antibody bound specifically to the KSHV ORF on the multivirus-chip. This result suggests that latent KSHV genome in BCBL-1 cells is packed into a nucleosomal structure and that histone H3 proteins near the viral promoter can be phosphorylated at serines to make the DNA at the promoter region less tightly packed with histones and more easily accessible to transcription factors.

In conclusion, the multivirus-chip improvements developed by Ghedin, et al. [2] provide versatile clinical and basic uses. In the near future, such chips are likely to be used to detect viral co-infections in many different clinical settings. Herpes simplex virus type 1 and normal protein permeability in the lungs of critically ill patients: a case for low pathogenicity? INTRODUCTION: The pathogenicity of late respiratory infections with herpes simplex virus type 1 (HSV-1) in the critically ill is unclear. METHODS: In four critically ill patients with persistent pulmonary infiltrates of unknown origin and isolation of HSV-1 from tracheal aspirate or bronchoalveolar lavage fluid, at 7 (1–11) days after start of mechanical ventilatory support, a pulmonary leak index (PLI) for (67)Gallium ((67)Ga)-transferrin (upper limit of normal 14.1 × 10(-3)/min) was measured. RESULTS: The PLI ranged between 7.5 and 14.0 × 10(-3)/min in the study patients. Two patients received a course of acyclovir and all survived. CONCLUSIONS: The normal capillary permeability observed in the lungs argues against pathogenicity of HSV-1 in the critically ill, and favors that isolation of the virus reflects reactivation in the course of serious illness and immunodepresssion, rather than primary or superimposed infection in the lungs. In some critically ill patients herpes simplex virus (HSV)-1 is isolated from the upper or lower respiratory tract [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . Immunodepressed patients may be susceptible to transmission and acquisition of viral diseases; alternatively, viral reactivation may occur and may contribute relatively little to morbidity and mortality. Indeed, reactivation of human herpesvirus-6 is common in critically ill patients and does not worsen outcome [16, 17] . In immunocompetent patients, however, isolation of HSV-1 may be associated with viral pneumonia, even if reactivation rather than primary infection is responsible [6, 8, 18] . HSV-1 has been associated with acute respiratory distress syndrome (ARDS) and ventilator-associated pneumonia in the critically ill [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] , as either a primary or a superimposed infection. However, there are few reports of the virus eliciting an infectious host response, as demonstrated by a rise in serum antibodies, by bronchoscopic airway disease, by 'typical' findings on computed tomography of the lungs, or by the presence of giant cells or nuclear inclusion bodies on cytology or biopsy of the lower respiratory tract [3, 5, 9, 10, 18] . Indeed, Tuxen and coworkers [4] observed that prophylactic antiviral therapy in ARDS prevented respiratory HSV-1 emergence but it had no impact on duration of mechanical ventilation or on patient outcome. The pathogenicity of the virus therefore remains unknown, and the rare association in the critically ill of HSV-1 isolation with mortality may represent reactivation of the virus in immunodepressed patients with multiple organ failure and poor outcome [1, 2, 11, 14, 15] , rather than a symptomatic primary infection or superinfection contributing to death.

Assessing pulmonary capillary protein permeability noninvasively at the bedside to yield the pulmonary leak index (PLI) could help in determining the extent of tissue injury, as was previously described [18] [19] [20] . This radionuclide technique involves gallium-67-labelled transferrin ( 67 Gatransferrin) and technetium-99m-labelled red blood cells ( 99m Tc-RBCs). In bacterial pneumonia, for instance, the PLI is elevated and the increase above normal directly relates to the severity of pneumonia, expressed as the lung injury score (LIS) [19] . In patients with acute lung injury (ALI) or ARDS during the course of bacterial pneumonia, the PLI is uniformly and greatly elevated above normal (up to 14.1 × 10 -3 /min) when LIS is greater than 2.5; the PLI is also elevated in 80% of patients with mild injury and a LIS between 1.5 and 2.5 [19] . Hence, the technique is a direct measure of permeability and an indirect measure of capillary injury in the lungs. The PLI is also elevated in interstitial lung disease [21] .

In order to help differentiate between symptomatic and asymptomatic viral shedding and spread, which could inform the decision regarding whether to institute antiviral therapy and help in determining the pathogenicity of the virus, we measured the PLI in four consecutive critically ill patients with persistent pulmonary infiltrates of unknown origin on ventilatory support, in whom a HSV-1 had been isolated.

We studied a small series of consecutive patients in whom respiratory secretions, sent for viral culture because of persistent pulmonary infiltrates of unknown origin, were found to be positive for HSV-1 (Table 1) . Tracheal aspirates or bronchoalveolar lavage fluid were transported directly to the microbiology laboratory or placed in viral transport medium (Copan Diagnostics Inc., Corona, CA, USA). For isolation of HSV-1, specimens were inoculated using standard procedures in triplicate flat bottom tubes on human embryonal lung fibroblasts and incubated at 37°C. Cultures were studied three times weekly for 10 days to identify the presence of a cytopathic effect. If a cytopathic effect, indicating the presence of HSV-1, was apparent or otherwise at days 2 and 7, the cells were fixed in methanol:acetone (1:1) and typed by immunofluorescence with labelled specific HSV-1 and HSV-2 antibodies (Syva Mikrotac HSV-1/HSV-2 typing kit, Palo Alto, CA, USA). In the four patients studied, the results were available within 3 days after samples had been inoculated in culture medium.

On the day of specimen collection for viral culture, demographic, chest radiographic and respiratory data were recorded, as were clinical features. In three out of four patients on mechanical ventilation after intubation, the total respiratory compliance was calculated from ventilator settings as follows (ml/cmH 2 O): tidal volume/(plateau -end-expiratory pressure). From the radiographic score (ranging from 0 to 4 depending on the number of quadrants with radiographic opacities), the ratio of arterial oxygen tension to fractional inspired oxygen, the level of positive end-expiratory pressure and the compliance, the LIS was calculated [22] . (LIS ranges between 0 and 4, with values up to 2.5 denoting ALI and those above 2.5 ARDS.) None of the patients had visible oropharyngeal vesicles.

To characterize further the persistent pulmonary infiltrates, the PLI was measured using a modification to a method described previously [19, 20] . Because this is a routine procedure, informed consent was waived. Autologous RBCs were labeled with 99m Tc (11 MBq, physical half-life 6 hours; Mallincrodt Diagnostica, Petten, The Netherlands), using a modified in vitro method. Ten minutes after injection of the labelled RBCs, transferrin was labelled in vivo, following intravenous injection of 67 Ga-citrate (6 MBq, physical half-life 78 hours; Mallincrodt Diagnostica). Patients were in the supine position, and two scintillation detection probes were positioned over the right and left lung apices. The probe system (manufactured by Eurorad C.T.T., Strasbourg, France) consists of two small cesium iodide scintillators (15 × 15 × 15 mm 3 ), each in a 2-mm tungsten and 1-mm aluminium housing cover (35 mm in diameter and 40 mm in height). The front end of each probe has an aluminium flange attached (3 mm in thickness and 70 mm in diameter) to facilitate easy fixation to the patient's chest with tape. Each probe weighs approximately 255 g. The probe signals are led into a dual amplifier, from which the output is fed into a multichannel analyzer system connected to a personal computer. Because the probes have separate channels, there is no electronic crossover.

Starting at the time of the intravenous injection of 67 Ga, radioactivity was measured each minute for 1 hour. For each measurement interval, the entire spectrum of photon energies was stored on disk. During processing, the 99m Tc and 67 Ga -and plotted against time. The PLI was calculated, using linear regression analysis, from the slope of increase of the radioactivity ratio divided by the intercept, in order to correct for physical factors in radioactivity detection.

By taking pulmonary blood volume and thus presumably surface area into account, the radioactivity ratio represents the ratio of extravascular to intravascular 67 Ga radioactivity. The PLI represents the transport rate of 67 Ga-transferrin from the intravascular to the extravascular spaces in the lungs, and it is therefore a measure of pulmonary capillary permeability to transferrin [19, 20] . The mean PLI from the two lungs was taken. The upper limit of normal PLI is 14.1 × 10 -3 /min. Where appropriate, numbers are summarized as median (range).

Patient data are presented in Table 1 . The patients had stayed for some time in the hospital or intensive care unit before HSV-1 was isolated, and they had been admitted primarily because of respiratory insufficiency during the course of pneumonia. Patient 4 was admitted into the coronary care unit a few days before intensive care unit admission for cardiogenic pulmonary oedema. All patients had been dependent on mechanical ventilatory support for some time before sampling. They had received adequate antibiotic therapy for pneumonia and had ALI at the time of sampling, which was of otherwise unknown origin. Table 1 shows that patients had radiographic abnormalities but without an increased PLI. Central venous pressure was not elevated, which suggests that the persistent pulmonary infiltrates were not caused by overhydration. In patients 1 and 3 a high-resolution computed tomography scan of the lungs with contrast was obtained; the findings were nonspecific, however, with alveolar consolidations and pleural fluid, even in the presence of interstitial abnormalities with a ground glass appearance in patient 3. In patient 1 a bronchoscopy was performed and there were no mucosal lesions. There was a normal distribution of lymphocyte subtypes in the lavage fluid. A transbronchial biopsy revealed interstitial inflammation with many macrophage deposits, and immunohistochemical staining for HSV-1 was negative. No multinucleated cells or cell inclusions were observed, either in bronchoalveolar lavage fluid from patient 1 or in tracheal aspirates from the other patients. In patients 1-3 concomitant isolation of bacteria by culture was regarded as bacterial colonization. Antibody testing was not done in patients 2-4 but was found to be positive for anti-HSV-1 IgG in patient 1, which is indicative of prior HSV-1 infection.

The antiviral agent aciclovir (10 mg/kg three times daily) was started when cultures became positive in two patients, at the discretion of the treating physician. Aciclovir was withheld in the other two patients because it was presumed that the pulmonary infiltrates were not caused by HSV-1, on the basis of a normal PLI among other findings. In patient 1, who had a normal PLI, a course of steroids was initiated on the day after the PLI was measured, and was continued despite positivity for HSV-1, reported 5 days later. All patients survived until discharge from the intensive care unit.

The 67 Ga-transferrin PLI is a sensitive and specific measure of pulmonary capillary permeability, which is utilized for noninvasive assessment of severity of a broad range of pulmonary conditions [19] [20] [21] . The PLI roughly parallels clinical severity (i.e. the LIS) [19, 20] . Although it involves the use of relatively routine equipment, the diagnostic method has not gained broad application, partly because of its laborious nature [20] . It has the advantage that bedside measurements are possible in mechanically ventilated critically ill patients, who cannot easily be transported. Pulmonary inflammation, of whatever cause, increases the PLI up to four times normal values in the most severe forms of lung injury, including ARDS. In less severe injury, such as impending ARDS and interstitial lung disease, the PLI is also elevated, albeit to a lesser extent, as reported by us and other groups [20, 21] .

The patients had in common a prior infectious episode, followed by a relatively prolonged period of respiratory insufficiency. They had persistent and nonspecific pulmonary infiltrates of unknown origin, after treatment of their primary disease, which prompted viral culture. The normal PLI observed suggests the involvement of a relatively harmless reactivation of HSV-1, rather than the presence of a primary and damaging infection. Indeed, critically ill patients with sepsis may have late immunodepression, with lymphocytic apoptosis, lymphocytopenia and T-cell anergy, promoting viral reactivation [23, 24] . Apparently, the virus must have been latent in the nerve endings of the mucous membranes of the upper respiratory tract in these patients [2, 15] . Herpesviruses (HSV-1) have frequently been isolated in vivo from respiratory secretions of patients with ARDS [3, 4] and detected in surveillance cultures from the respiratory tract of patients following burns, trauma, transplantation, major surgery and others. However, these viruses are detected in only 3% of lung biopsies from patients with prolonged and unresolving ARDS [3, 7, [9] [10] [11] [12] [13] 15] . The literature is thus widely divergent on the precise role of the virus in pulmonary disease in the critically ill and its contribution to patient morbidity and mortality [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] .

We believe that the tracheal aspirates were representative of lower respiratory tract secretions, in the absence of herpes orolabialis and oral epithelial cells in smears for Gram stain of the secretions. Concurrent colonization with other pathogens has previously been described [5, 13] . Because there was no overlap in the duration of stay of the patients, transmission of the virus from one patient to another can be excluded. This further suggests that respiratory HSV-1 infections in the critically ill may result from relatively harmless endogenous reactivation. Although the normal PLI argues against pulmonary parenchymal pathogenicity, tracheobronchitis caused by the virus [18, 25] cannot be ruled out, even in the absence of orolabial lesions, because bronchoscopy was not performed in three of the four patients, even though it was unremarkable in patient 1. The persistent pulmonary infiltrates in our patients may thus relate to slow radiographic resolution of prior bacterial or aspiration pneumonia, rather then superimposed infection. Moreover, computed tomographic images of the lung may be largely nonspecific [26] , and so the precise diagnostic criteria for HSV-1 pneumonia remain unclear. When properly standardized, for instance with respect to cell numbers in bronchoalveolar fluid or tracheal aspirates, quantitative cultures, viral RNA and DNA by polymerase chain reaction, could be helpful together with the PLI in further studies to quantitate viral load and the ratio of replication to shedding, and therefore the pathogenicity of the virus in the lower respiratory tract.

In conclusion, the anecdotal data presented here suggest that isolation of HSV-1 from respiratory secretions in the critically ill patient with a persistent pulmonary infiltrate may warrant evaluation of tissue injury potentially caused by the virus to judge its pathogenicity. This could be done using a radionuclide PLI measurement, and would help to inform decisions regarding antiviral therapy, which may have adverse effects. In some patients a normal PLI may argue against viral pathogenicity, and withholding of aciclovir in such patients may be safe. Logistics of community smallpox control through contact tracing and ring vaccination: a stochastic network model BACKGROUND: Previous smallpox ring vaccination models based on contact tracing over a network suggest that ring vaccination would be effective, but have not explicitly included response logistics and limited numbers of vaccinators. METHODS: We developed a continuous-time stochastic simulation of smallpox transmission, including network structure, post-exposure vaccination, vaccination of contacts of contacts, limited response capacity, heterogeneity in symptoms and infectiousness, vaccination prior to the discontinuation of routine vaccination, more rapid diagnosis due to public awareness, surveillance of asymptomatic contacts, and isolation of cases. RESULTS: We found that even in cases of very rapidly spreading smallpox, ring vaccination (when coupled with surveillance) is sufficient in most cases to eliminate smallpox quickly, assuming that 95% of household contacts are traced, 80% of workplace or social contacts are traced, and no casual contacts are traced, and that in most cases the ability to trace 1–5 individuals per day per index case is sufficient. If smallpox is assumed to be transmitted very quickly to contacts, it may at times escape containment by ring vaccination, but could be controlled in these circumstances by mass vaccination. CONCLUSIONS: Small introductions of smallpox are likely to be easily contained by ring vaccination, provided contact tracing is feasible. Uncertainties in the nature of bioterrorist smallpox (infectiousness, vaccine efficacy) support continued planning for ring vaccination as well as mass vaccination. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity) and should be accompanied by increased public awareness and surveillance. Concerns about intentional releases of smallpox have prompted extensive preparations to improve our ability to detect and respond to an outbreak of smallpox [1, 3, 4, 2] . Many factors contribute to the public health challenge of understanding and preparing for smallpox, including the age and quality of epidemiological data on native smallpox and the smallpox vaccine, the difficulty of extrapolating that data to our current populations, the possible terrorist use of altered smallpox, our ignorance of terrorist methods of release, and the relatively high risk of adverse events caused by the smallpox vaccine.

The Centers for Disease Control and Prevention (CDC) established ring vaccination (selective epidemiological control [5] ), a strategy in which contacts of cases are identified and vaccinated, as the preferred control measure in the event of a smallpox outbreak (interim plan). The successful use of ring vaccination during the smallpox eradication campaign and its logical emphasis of case-contacts for immediate vaccination support its use (though the attribution of the success of the eradication program to ring vaccination has been challenged [6] ). Health Officers should initiate ring vaccination upon identification of the first cases of smallpox. However, there are legitimate concerns regarding the ability of public health practitioners to mount a quick, comprehensive and successful ring vaccination program, particularly in the face of a moderatesized or large smallpox outbreak. To guide preparation efforts and inform incident decision-making, we attempt to identify outbreak characteristics and response capacities that significantly impact the ability of ring vaccination to control a smallpox outbreak and to determine whether ring vaccination is useful in the presence of a mass vaccination campaign. Our analysis uses a newly developed mathematical model: a continuous-time, event-driven network simulation model of smallpox ring vaccination.

Mathematical models can advance our understanding of how a smallpox outbreak might progress. Several mathematical and computer models address the question of smallpox transmission [7] [8] [9] [10] [11] [12] [13] . The first model to appear [8] concluded that ring vaccination would be effective, but did not treat response logistics in detail; the model was linear and did not treat the depletion of susceptibles as the epidemic progressed (appropriate, however, for assessing control early in an epidemic, when the number infected is small compared to the number of susceptibles, e.g. [14] ). The innovative model by Kaplan et al. [9] emphasized the importance of resource limitation and the logistics of smallpox response, but assumed that full infectiousness began before the onset of symptoms (and the subsequent identification and removal), and did not separately monitor close epidemiological contacts of patients (which are at greatest risk, but also easiest to find and vac-cinate); the conclusions were highly critical of ring vaccination. The model by Halloran et al. [11] , a stochastic, discrete-time network model omitted the explicit inclusion of response logistics while otherwise used parameter values similar to those in Kaplan et al. [9] ; the inclusion of residual immunity from individuals vaccinated prior to the discontinuation of routine vaccination, however, led to a more favorable view of ring vaccination. The model by Bozzette et al. [12] assumed that ring vaccination would reduce the number of transmissions and focused on health care workers (but did not explicitly include the network structure of the population nor the response logistics of ring vaccination). The model by Eichner [15] did not explicitly include the network structure of the population nor the logistics of ring vaccination, but did use parameters based on data from an outbreak in Nigeria, and did distinguish close and casual contacts, case isolation, and surveillance of contacts; it concluded that case isolation and contact tracing could prevent the spread of smallpox. Finally, the individual-based model by Epstein et al. [16] presented scenarios illustrating certain alternatives to pure mass vaccination and ring vaccination of contacts of cases in preventing smallpox transmission in small populations of 800 individuals; this model includes no homogeneity assumptions, but did not analyze tracing of contacts of contacts.

Because none of the available models includes both network structure (with explicit contact tracing) and response logistics limited by the number of available disease control investigators [9] , we included these features in a continuous-time event-driven network simulation model of smallpox ring vaccination. Specifically, the model we developed includes the following features: exposed individuals and vaccinate them in time, resulting in a "race to trace" [9] .

Mild, ambulatory cases of smallpox may spread disease because such cases may be harder to recognize.

Vaccination of individuals prior to the discontinuation of routine vaccination may provide some, possibly considerable, protection against infection [11, 23, 24] , although it may also result in more mild cases which may be harder to detect.

Public awareness may lead to more rapid detection of cases.

We use this model to determine what factors promote or hinder the success of ring vaccination during a smallpox outbreak, and whether ring vaccination is useful in the presence of a mass vaccination campaign. In particular, the goal of this paper is to examine the control of smallpox by contact tracing and ring vaccination using a network model which includes response logistics [9] .

Natural history of smallpox We briefly review relevant features of the natural history and epidemiology of smallpox [17, [25] [26] [27] 8, 28] . Following infection by the variola virus, individuals exhibited an incubation period of approximately 7-19 days with 10-14 being most typical. Sudden onset of fever and malaise, often with accompanying headache and backache, began the initial (or pre-eruptive) phase of smallpox. After 2-3 (or perhaps 4) days, individuals with the most common form, ordinary type smallpox, developed the characteristic focal rash, preceded in many cases by oropharyngeal lesions. In fatal cases of ordinary smallpox, death often occurred between the tenth and sixteenth day of symptoms; among survivors, most scabs had separated by day 22-27 of illness [26] .

The course of smallpox varied widely between individuals, and several different clinical classifications were developed [29] [30] [31] 17, 26] . Consideration of the clinical features and severity of smallpox is important from the standpoint of mathematical transmission modeling because (1) the clinical features affect the ease of diagnosis (and thus of case identification), (2) more severe forms of smallpox may result in more transmission, (3) vaccinated individuals may develop less severe disease. We utilize a modified or simplified version of the classification system developed by Rao [32, 31, 26] ; for the mathematical model, we will classify smallpox into five categories: early hemorrhagic, flat and late hemorrhagic, ordinary, modified, and mild. However, the clinical features and severity of smallpox in different populations may have been affected by underlying host factors, differences in viral strains, or differences in the infectious dose owing to different prevailing modes of transmission, and thus robust and precise quantitative estimates of the effects of (pre-or post-exposure) vaccination on the resulting smallpox severity, or of the infectivity differences between individuals exhibiting different forms of smallpox, are not available. The significance of such differences will be revealed through sensitivity analysis. Further details are given in Appendix 1 [see Additional file: 1].

Vaccination with vaccinia virus provided substantial protection against infection. Dixon assessed the risk of infection for an individual successfully vaccinated 3 years prior to exposure to be 0.1% the infection risk of an unvaccinated individual [17] . However, smallpox vaccination did not always take when applied, and moreover, in many instances, individuals who experienced a repeated vaccination failure developed severe smallpox upon exposure. The probability of a successful take depended on the vaccination method used; we assume that the take rate is between 95% and 100% [22, 28] . In addition to protection against infection, vaccination could in many cases modify the course of infection and reduce the severity. Vaccine protection waned over time, but individuals vaccinated 20 years prior to exposure were believed to still have half the infection probability that an unvaccinated person had [17] , and to have some protection against the most severe manifestations of smallpox. Dixon [17] believed that vaccine protection had at least three components, which decayed at different rates; for the purpose of this paper, we will assume that the severity of smallpox in previously any (recently or otherwise) vaccinated individuals follows the same distribution as for the vaccinated subjects seen in the case series observed by Rao in Madras [26] , except that anywhere from 0 to 5% of vaccinated subjects develop smallpox too mild to diagnose without special surveillance or awareness. Observe that the vaccinated cases studied by Rao were vaccinated (at some point in their lives) before exposure, rather than after exposure to smallpox.