Characterization of a Putative Ancestor of Coxsackievirus B5ᰔ Maria Gullberg,1 Conny Tolf,1 Nina Jonsson,1 Mick N

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Characterization of a Putative Ancestor of Coxsackievirus B5ᰔ Maria Gullberg,1 Conny Tolf,1 Nina Jonsson,1 Mick N. Mulders,2 Carita Savolainen-Kopra,3 Tapani Hovi,3 Marc Van Ranst,4 Philippe Lemey,4 Susan Hafenstein,5 and A. Michael Lindberg1* School of Natural Sciences, Linnaeus University, SE-391 82 Kalmar, Sweden1; Centre for Infectious Disease Control Netherlands, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands2; Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), Helsinki, Finland3; Department of Microbiology and Immunology, Rega Institute for Medical Research, Katholieke Universiteit, Leuven, Belgium4; and Department of Microbiology and Immunology, the Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 170335

Received 13 January 2010/Accepted 6 July 2010

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the ﬁrst report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.

The group B coxsackieviruses (CVBs) (serotypes 1 to 6) ever, the attachment of CVBs to DAF alone does not permit were discovered in the 1950s in a search for new poliovirus-like the infection of cells (6, 7, 59, 85). viruses (33, 61). Infections caused by CVBs are often asymp- Picornaviruses exist as genetically highly diverse populations tomatic but may occasionally result in severe diseases of the within their hosts, referred to as quasispecies (20, 57). This heart, pancreas, and central nervous system (99). CVBs are genetic plasticity enables these viruses to adapt rapidly to new small icosahedral RNA viruses belonging to the Human en- environments, but at the same time, it may compromise the terovirus B (HEV-B) species within the family Picornaviridae structural integrity and enzymatic functionality of the virus. (89). In the positive single-stranded RNA genome, the capsid The selective constraints imposed on the picornavirus genome proteins VP1 to VP4 are encoded within the P1 region, are reflected in the different regions used for different types of whereas the nonstructural proteins required for virus replica- evolutionary studies. The highly conserved RNA-dependent tion are encoded within the P2 and P3 regions (4). The 30-nm RNA polymerase (3Dpol) gene is used to establish phyloge- capsid has an icosahedral symmetry and consists of 60 copies of netic relationships between more-distantly related viruses (e.g., each of the four structural proteins. The VP1, VP2, and VP3 viruses belonging to different genera) (38), whereas the vari- proteins are surface exposed, whereas the VP4 protein lines able genomic sequence encoding the VP1 protein is used for the interior of the virus capsid (82). The coxsackievirus and the classification of serotypes (13, 14, 69, 71, 72). adenovirus receptor (CAR), a cell adhesion molecule of the In 1963, Pauling and Zuckerkandl proposed that compar- immunoglobulin superfamily, serves as the major cell surface ative analyses of contemporary protein sequences can be attachment molecule for all six serotypes of CVB (5, 6, 39, 60, used to predict the sequences of their ancient predecessors 98). Some strains of CVB1, CVB3 and CVB5 also interact with (73). Experimental reconstruction of ancestral character the decay-accelerating factor (DAF) (CD55), a member of the states has been applied to evolutionary studies of several family of proteins that regulate the complement cascade. How- different proteins, e.g., galectins (49), G protein-coupled receptors (52), alcohol dehydrogenases (95), rhodopsins (15), ribonucleases (46, 88, 110), elongation factors (32), steroid receptors (10, 96, 97), and transposons (1, 45, 87). In * Corresponding author. Mailing address: School of Natural Sci- the field of virology, reconstructed ancestral or consensus ences, Linnaeus University, SE-391 82 Kalmar, Sweden. Phone: 46 480 446240. Fax: 46 480 446262. E-mail: [email protected]. protein sequences have been used in attempts to develop ᰔ Published ahead of print on 14 July 2010. vaccine candidates for human immunodeficiency virus type 1

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(21, 51, 66, 81) but rarely to examine general phenotypic for the VP1 analysis, while the same model including the proportion of invariable properties. sites was used for the P1 sequence data. The phylogenetic relationship between In this study, a CVB5 virus with a probable ancestral virion viruses was also examined by the neighbor-joining method, as implemented in MEGA, version 4.0 (92). Previously determined CVB5 sequences (CVB5UK (CVB5-P1anc) was constructed and characterized. We first [GenBank accession number X67706] and CVB5D [47]) and swine vesicular analyzed in detail the evolutionary relationships between struc- disease virus (SVDV) sequences (Svdh3jap76 [accession number D00435], tural genes of modern CVB5 isolates and inferred a time scale Svdj1jap73 [accession number D16364], Svd27uk72 [accession number X54521], for their evolutionary history. An ancestral virion sequence was Svd1spa93 [accession number AF039166], and Svd1net92 [accession number subsequently inferred by using a maximum likelihood (ML) AF268065]) were included in the phylogenetic analysis of the VP1 gene. The sequences of CVB4 Tuscany (CVB4T) (accession number DQ480420) and method. This sequence was then synthesized de novo, cloned CVB6 Schmitt (CVB6S) (accession number AF114384) were used as an out- into a replicative backbone of an infectious CVB5 cDNA group. Phylogenetic trees were visualized with MEGA 4.0. Root-to-tip diver- clone, and transfected into HeLa cells. The hypothetical gence as a function of sampling time was examined by using Path-O-Gen (avail- CVB5-P1anc assembled into functional virus particles that dis- able at http://tree.bio.ed.ac.uk/software). Bayesian evolutionary analysis. played phenotypic properties similar to those of contemporary We inferred the time scale and tempo of CVB5 evolution using a Bayesian statistical approach implemented in BEAST clinical isolates. This is the first report describing the recon- (25). This approach employs a full probabilistic model of sequence evolution struction and characterization of a fully functional picornavirus along rooted, time-measured phylogenies with a coalescent prior, using either a with a putative ancestral capsid. fixed or relaxed molecular clock model (23, 24). For rapidly evolving viruses, the molecular clock is calibrated based on the divergence accumulation between sequences sampled at different points in time. We used the SRD06 model of MATERIALS AND METHODS nucleotide substitution (86) with gamma-distributed rate variation among sites, an uncorrelated log-normal relaxed-clock model, and a Bayesian skyline tree Cell lines and viruses. African green monkey kidney (GMK), human colon prior (26). Markov chain Monte Carlo analyses were run for 10 million gener- adenocarcinoma (HT29), Chinese hamster ovary (CHO), human lung carcinoma ations and diagnosed by using Tracer (http://beast.bio.ed.ac.uk/Tracer). The evo- (A549), and human rhabdomyosarcoma (RD) cell lines were purchased from the lutionary history was summarized in the form of a maximum clade credibility tree American Type Culture Collection. HeLa Ohio cells were kindly provided by M. by using TreeAnnotator (http://beast.bio.ed.ac.uk/TreeAnnotator) and visual- Roivainen (Helsinki, Finland). Recombinant CHO cells expressing CAR (CHO- ized with FigTree (http://tree.bio.ed.ac.uk/software/figtree). Bayesian credible CAR) or DAF (CHO-DAF) were constructed by H.-C. Selinka (77, 84). Cells intervals for continuous parameters are reported as the highest posterior density were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supple- intervals, which are the smallest intervals that contain 95% of the posterior mented with 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and distribution. 10% newborn calf serum (NCS). Recombinant CHO cells were grown in selec- Ancestral sequence reconstruction. The ancestral sequences were recon- tive medium supplemented with 1 mg/ml G418 (Sigma) for cells expressing CAR structed from the CVB5 ingroup taxa of the phylogenetic trees based on the and 0.75 mg/ml hygromycin B (Invitrogen) for cells expressing DAF. genetic VP1 and P1 regions but without the reference strains (CVB5D and The clinical CVB5 isolates used in this study were propagated in GMK cells CVB5UK). These reference strains were not included in the reconstruction according to standard procedures (63). CVB5 strain 1954UK85 (CVB5UK) because their passage history is unknown. ML ancestral sequences were inferred (109) was provided by J. W. McCauley, Newbury, United Kingdom, and strain by using a standard codon substitution model (M0, which assumes a homogenous Dalldorf (CVB5D) was provided by R. L. Crowell, Philadelphia, PA (16, 79). As nonsynonymous/synonymous substitution rate among sites and among lineages), previously described (47), the genome sequence of the CVB5D virus is identical as implemented in codeml of the PAML package (34, 105). After ML optimi- to that of prototype strain Faulkner (CVB5F) (55), except for one amino acid zation under the codon model, this approach considers the assignment of a set of change in the VP1 protein. Viral titers of propagated viruses were determined by characters to all interior nodes at a site as a reconstruction and selects the the 50% tissue culture infectious dose (TCID ) method according to standard 50 reconstruction that has the highest posterior probability, i.e., so-called joint procedures (41). reconstruction (106). This procedure was efficiently performed by using an al- Flow cytometry analysis. Flow cytometry analysis was performed as described gorithm described previously by Pupko and colleagues (78). A corresponding previously (77). Briefly, CHO, CHO-CAR, CHO-DAF, and HeLa cells were reconstruction was also performed by using the best-fitting empirical amino acid stained with an anti-CAR (RmcB) antibody (43) (the hybridoma was kindly model. The inference of the ancestor sequence was facilitated by the absence of provided by L. Philipson and R. Pettersson, Karolinska Institute, Sweden [also gaps or evidence of recombination within the genomic P1 region, as confirmed by available from the American Type Culture Collection {ATCC CRL-2379}]), an using the phi test (12). anti-DAF (BRIC110) antibody (Cymbus Biotechnologies), or a mouse IgG1 Construction of CVB5-P1anc. control antibody (X0931; Dako). After1hofincubation at 4°C, cells were The complete CVB5D genome was amplified ϩ washed and stained with a secondary R-phycoerythrin-labeled rabbit anti-mouse and cloned into the pCR-Script Direct SK( ) vector (Stratagene) by using the antibody (R0439; Dako). Data were acquired by using a FACSCalibur instru- AscI and NotI restriction enzyme cleavage sites, as previously described (Fig. 1) ment (Becton Dickinson) and analyzed with CellQuest, version 3.3, software (56). In this infectious full-length cDNA clone of CVB5D (pCVB5Dwt), a ClaI (Becton Dickinson). site was introduced at nucleotide position 3340 to generate a cassette vector Extraction, amplification, and sequencing. Viral RNA was extracted from (pCVB5D-cas). This modification resulted in one amino acid change in the 2A infected cell cultures (QIAamp viral RNA minikit; Qiagen), reverse transcribed protein (valine to leucine at amino acid position 17). This substitution was (Superscript III; Invitrogen), and PCR amplified (PicoMaxx; Stratagene) by accepted, as leucine 17 is present in the 2A protein of other enteroviruses, using virus-specific primers. PCR amplicons were visualized in agarose gels and including echovirus 30 and echovirus 21. In addition, a synonymous mutation was purified (QIAquick gel extraction kit; Qiagen). The nucleotide sequences were introduced into the P1 ancestral sequence to remove a SalI site. In order to determined with an ABI Prism 3130 automated sequencer (Applied Biosystems) construct a CVB5 clone with the inferred ancestral capsid sequence (pCVB5- by a primer-walking strategy on both strands using BigDye chemistry (ABI Prism P1anc), a pUC57 plasmid containing the ancestral P1 sequence flanked by SalI BigDye Terminator cycle sequencing ready-reaction kit, version 1.1; Applied and ClaI sites was purchased (GenScript). Subsequently, the P1 genomic region Biosystems). Sequences were analyzed by using the Sequencher, version 4.6, of this pUC57 plasmid was amplified and then cloned into pCVB5D-cas. The software package (Gene Codes Corporation). constructs were propagated in Escherichia coli DH5␣ cells and purified (Mi- Phylogenetic analysis. Viral nucleotide sequences were aligned by using Clust- diprep kit; Promega). The nucleotide sequences of all constructs were verified by alW (94), and phylogenetic signals were evaluated by using likelihood mapping sequencing as described above. (90). The presence of nucleotide substitution saturation was tested by using an HeLa cell monolayers were transfected with 2.5 ␮g of the CVB5 cDNA clones approach described previously by Xia et al. (104). Phylogenetic relationships by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s pro- between the different CVB5 strains, based on the genomic VP1 and P1 regions, tocol. Recombinant viruses were collected on day 5 posttransfection and subse- were inferred by the ML method as implemented in PhyML (35). Branch support quently sequenced to confirm their identity. The titers of these viruses were values for inferred phylogeny were estimated by nonparametric bootstrapping determined by a TCID50 assay with HeLa cells. In order to ensure the safety of consisting of 1,000 pseudoreplicates (30). The general time-reversible (GTR) laboratory workers, the environment, and the public, the generation of CVB5- substitution model (53) with a gamma-distributed rate heterogeneity was used P1anc was performed under conditions of biosafety level 2 containment. VOL. 84, 2010 CVB5 PHYLOGENETICS AND ANCESTRAL RECONSTRUCTION 9697

FIG. 1. Genomic structures of CVB5D and CVB5-P1anc. An illustration of the genome organization of CVB5 is shown at the top, including positions of relevant restriction enzyme sites used to construct infectious viral cDNA clones. The ClaI site (*) was introduced to construct a cassette vector. Infectious CVB5D cDNA clone variants, as they were inserted into the pCR-Script Direct SK(ϩ) vector, are depicted at the bottom. The ancestral P1 sequence is indicated in gray. Names of constructed cDNA clones and viruses generated from these clones (given within parentheses) are used throughout the text. UTR, untranslated region.

Virus infection. Cell monolayers were infected with virus according to stan- measured according to a method described previously (2, 62). Briefly, adherent dard procedures (63). Briefly, subconfluent monolayers of cells grown in 25-cm2 CHO, CHO-CAR, CHO-DAF, and HeLa cells were detached by EDTA treat- flasks were inoculated with viruses at a multiplicity of infection (MOI) of 10 ment. Approximately 2.5 ϫ 105 cells were incubated with radiolabeled virus ϳ TCID50/cell. Following virus adsorption at room temperature for 1 h, the cells ( 30,000 dpm) in DMEM with 2% NCS, 2 mM L-glutamine, 100 U/ml penicillin, were washed three times before the addition of DMEM supplemented with 2 and 0.1 mg/ml streptomycin. Following virus adsorption to cells for2hatroom mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Incubation of temperature with gentle agitation, unbound virus was removed by three washes, the virus infection at 37°C in a 7.5% CO2 atmosphere was continued for 5 days and cell-bound radioactivity was quantified by liquid scintillation counting. The or until a cytopathic effect (CPE) was observed. virus binding was analyzed in triplicate, and data are presented as means Ϯ Viral replication was quantified by analyses of samples taken immediately after standard errors of the means (SEM). infection and 5 days postinfection (p.i.) or when a complete CPE was observed. Mapping of structural differences between CVB5-P1anc and the clinical CVB5 After three cycles of freezing and thawing, the viral titers were determined by a isolates. Based on a sequence alignment of CVB3 strain M, the clinical CVB5

TCID50 assay with HeLa cells as described above. isolates, and CVB5-P1anc (ClustalW) (94), the sequence-equivalent residues in The molecular evolutions of CVB5-P1anc, 151rom70, 4378fin88, and wild-type the ancestor were mapped to the X-ray crystallographic structure of CVB3 CVB5 (CVB5Dwt) were compared after 10 serial rounds of infection in GMK, (Protein Data Bank [PDB] accession number 1COV) (64). The footprints of HeLa, and RD cells. After the 10th passage, the P1 regions of these viruses were CAR (39) and DAF (36) on the CVB3 virion surface were used to locate the sequenced as described above. equivalent CVB5-P1anc residues within the receptor binding footprints. The Immunofluorescence. Infected HeLa cells cultured on Lab-Tek II chamber X-ray crystal structure of the CVB3 capsid was modeled and visualized by using glass slides (Nalge Nunc International) were fixed in 4% formaldehyde for 30 Chimera (75, 83). min at 4°C and stained for1hatroom temperature with an enterovirus-specific Nucleotide sequence accession numbers. The VP1 and P1 nucleotide se- polyclonal rabbit antiserum (KTL-482) (42). The primary antibody was visualized quences determined in this study were submitted to the GenBank sequence with a secondary goat anti-rabbit antibody labeled with Alexa Fluor 488 (A11034; database under accession numbers GU300033 to GU300065 and GU323569 to Molecular Probes Inc.). Finally, slides were mounted with Vectashield (Im- GU323576, respectively. Inferred ancestral P1 and VP1 nucleotide sequences munkemi) containing 4Ј,6-diamidino-2-phenylindole (DAPI), and images were were deposited under accession numbers GU323568 and GU323577, respec- captured with an epifluorescence microscope. tively. Plaque formation assay. A semisolid gum tragacanth medium was used as previously described (18) to assess the plaque morphology of viruses. Briefly, confluent monolayers of HeLa cells in six-well plates were incubated with 1 ml of RESULTS virus in 10-fold dilutions for1hat37°C. Following adsorption, the virus inocu- lum was aspired, and cells were overlaid with DMEM supplemented with 1% Phylogenetic relationships of CVB5 viruses. The gene en- NCS, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.8% coding the VP1 capsid protein is considered to be the most (wt/vol) gum tragacanth (Sigma). The plaques were visualized by staining cells with a crystal violet-ethanol solution after 48 h of incubation at 37°C. informative genomic region for studying molecular epidemiol- Viral growth kinetics. To assess the growth kinetics of viruses, HeLa cells in ogy and evolutionary relationships among enteroviruses (13,

24-well plates were infected with virus at an MOI of 10 TCID50/cell as described 14, 69, 71, 72). Hence, in order to evaluate the phylogenetic above. Cells and medium were harvested and frozen at various time points relationships among 41 clinical CVB5 strains, the VP1-encod- postinfection. Virus titers in collected samples were determined by a TCID50 ing gene was sequenced. These viruses were isolated in Eu- assay. Neutralization assay. Serial 2-fold dilutions of antiserum against CVB1, rope, Asia, North America, and South America between 1970 CVB2, CVB3, or CVB4 (kindly provided by H. Norder and L. Magnius, Swedish and 1996. The ML analysis of aligned VP1 nucleotide se- Institute for Infectious Disease Control, Sweden); CVB5F (V032-501-560, 1965; quences of these clinical isolates together with two CVB5 ref- NIH research reagent); or CVB6 (V033-501-560, 1965; NIH research reagent) erence strains (CVB5D and CVB5UK) and ﬁve SVDV isolates was mixed with an equal volume of virus (100 TCID50) in DMEM with 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and then incubated revealed a dichotomous phylogenetic relationship, which dis- at 37°C for 1 h. The virus-antibody mixtures were applied onto HeLa cells in tinguishes the existence of two coevolving clusters of genetic 96-well plates (quadruplicate). After 5 days of incubation at 37°C, cells were lineages (indicated as clusters I and II) (Fig. 2A). Some CVB5 examined microscopically for evidence of virus-induced CPE. The highest serum strains isolated in Finland clustered together in cluster I, dilution that completely inhibited CPE was determined to be the endpoint titer. whereas other Finnish strains were more closely related to Virus binding assay. Viruses were metabolically labeled with [35S]methionine- cysteine (Perkin-Elmer) and puriﬁed by sucrose gradient centrifugation as de- viruses isolated in other parts of Europe and North America, scribed previously for echoviruses 1 and 8 (8). Virus attachment to cells was indicating a geographic mixture of cluster I and II viruses. The 9698 GULLBERG ET AL. J. VIROL.

FIG. 2. Phylogenetic relationships among CVB5 isolates. (A) Phylogram based on the nucleotide sequences of the VP1 gene. F, CVB5 isolates selected for sequencing of the entire P1 region. (B) Phylogeny of selected CVB5 isolates based on the nucleotide sequences of the P1 region. The scale bars represent the genetic distance (nucleotide substitutions per site). Both ML trees were evaluated by nonparametric bootstrap analysis and 1,000 pseudoreplicates. Only bootstrap values Ն70% are denoted. Clusters are indicated by roman numerals (clusters I and II). The trees were rooted with CVB4T and CVB6S. Abbreviations in isolate names are as follows: den, Denmark; dor, Dominican Republic; ecu, Ecuador; est, Estonia; ﬁn, Finland; fra, France; hon, Honduras; jap, Japan; kyr, Kyrgyzstan; net, Netherlands; pak, Pakistan; rom, Romania; rus, Russia; spa, Spain; uk, United Kingdom; usa, United States of America. The last two numbers of isolates depict the year of isolation, except for isolates from France, where the year of isolation is shown by the two ﬁrst numbers after fra.

phylogenetic analysis also showed that SVDV was more closely Timed evolutionary history of CVB5 viruses. By plotting related to CVB5 viruses in cluster II. In addition to the VP1 root-to-tip divergence from the ML tree as a function of sam- sequences, the complete sequence of the structural genes (the pling time, we observed a clear accumulation of nucleotide entire P1 region) of four isolates from each of the two clusters, substitutions over the sampling time interval (Fig. 3). To es- separated both in time of isolation and by geographic location, tablish a time scale for the CVB5 evolutionary history and was determined. The result from the subsequent phylogenetic estimate the viral rate of evolution, we performed Bayesian analysis based on the P1 sequences showed a relationship be- evolutionary analysis of the VP1 sequences sampled over time tween viruses corresponding to the phylogeny of the VP1 gene using BEAST (25). In this approach, we used Markov chain (Fig. 2B). Tree topologies corresponding to those resulting Monte Carlo analyses to average over tree space, with each from the ML analyses were also observed for trees inferred tree having branch lengths in units of time. Figure 4 shows the with the neighbor-joining method (data not shown). maximum clade credibility tree that summarizes the evolution- VOL. 84, 2010 CVB5 PHYLOGENETICS AND ANCESTRAL RECONSTRUCTION 9699

(1913 versus 1933) albeit with overlapping credible intervals (1887 to 1935 versus 1916 to 1947). The evolutionary rate estimate resulted in 0.0042 (0.0033 to 0.0052) nucleotide substitutions per site per year. Despite the rate of evolution and the relatively old MRCA, no signal for substitution saturation was detected by using the saturation index reported previously by Xia et al. (104). The coefﬁcient of variation (0.24 [0.09 to 0.42]) indicated that the rate of evolution varies among branches within about 25% of the mean rate. The highest rate (Fig. 3) was observed for the branch leading to the most recent SVDV lineages, which probably reﬂects the ongoing process of adaptation to a new host species after interspecies transmission to pigs. These SVDV lineages were also noted as outliers in the root-to-tip divergence plot (Fig. 3). Ancestral reconstruction. In order to reconstruct a putative FIG. 3. Root-to-tip divergence plot. Shown is a linear regression ancestral character state for the CVB5 virion, we applied a plot for root-to-tip divergence versus sampling year. The SVDV iso- codon-based ML approach to the inferred CVB5 phylogeny. In lates included in the analysis are encircled. the sequence alignment of the reconstructed CVB5 capsid residue state (P1anc) and eight clinical isolates, 51 variable ary history estimated by using a relaxed molecular clock (23). amino acid residue positions were observed (Table 1). The The most recent common ancestor (MRCA) of all CVB5 lin- robustness of the P1anc reconstruction was assessed by com- eages (clusters I and II) dated back to 1854 (1807 to 1898). paring the ancestral reconstruction of the P1 sequence with an Cluster II had a somewhat older MRCA than did cluster I ancestor sequence based on the VP1 gene alone (VP1anc).

FIG. 4. Maximum clade credibility tree representing the CVB5 evolutionary history inferred by using Bayesian evolutionary analysis. The tree has branch lengths in time units and is depicted on a time scale. The uncertainty (95% highest posterior density intervals) for the node times is indicated with blue bars. Branches with an asterisk are supported with posterior probabilities higher than 0.85. Rate variation among branches is indicated by using a blue-black-red (slow-average-high) color scheme. Clusters are indicated by roman numerals (clusters I and II). 9700 GULLBERG ET AL. J. VIROL.

TABLE 1. Primary structural differences between the capsid proteins of CVB5-P1anc and those of eight clinical CVB5 isolatesa

Amino acid Residue Locationb CVB5-P1anc Cluster I Cluster II SVDV CVB3 VP1 7 V V V/I M/T I Disordered, N terminus 19 G G/S G G G Interior, N terminus 52 H H/R H H H Interior 54 K K/R K K K Interior 75 Y Y Y/F F Y Exterior 85 D D D/N N - Exterior, BC loop, antigenic site 87 D D/N D D K Exterior, BC loop, antigenic site 95 S S/N S/N N T Buried, hydrophobic pocket, antigenic site 99 V V V/A V A Buried 125 T T T/S T T Exterior 156 V V/I V V V Exterior 180 M M/I M M M Buried, hydrophobic pocket 246 T T/A T T A Buried 272 E E E/K E T Exterior, antigenic site 273 S S S/G G T Exterior, C terminus 275 T T T/A T Q Exterior, C terminus, antigenic site 276 D E D D S Exterior, C terminus 279 T A T T T Exterior, C terminus 281 Q Q Q/R K T Exterior, C terminus

VP2 37 V T/I V/I V V Interior, N terminus 45 D E D/E E S Interior 76 P P/S P P P Exterior, BC loop 104 A A A/S A T Buried 108 I V I I I Buried 137 L L/I/V L L L Exterior, EF loop, putative DAF binding site 153 S N S/N S/N K Exterior, EF loop 154 M T M M/T E Exterior, EF loop, antigenic site 156 E E/D E E A Exterior, EF loop 160 T T S S S Exterior, EF loop, putative DAF binding site 161 T T T/S T G Exterior, EF loop, putative DAF binding site 185 F F F/Y F F Buried, EF loop 205 I I I/V I T Interior 223 I I I/V V V Buried 233 T T T/A T/A P Exterior, HI loop, antigenic site

VP3 35 D A/E D/E/N D R Interior, N terminus 39 Q Q Q/R Q E Interior 58 T T/I T/A T V Exterior, ␤-B knob 63 L L/S L/M M N Exterior, ␤-B knob, putative DAF binding site 67 A A A/S A A Buried, ␤-B knob 80 S S S/T S T Exterior, BC loop 88 T T/I T T Q Exterior 168 I I/V I I I Buried 181 M M/V M/T M S Exterior, putative CAR binding site 183 E E/D E E E Exterior, putative CAR binding site 232 K K K/Q K S Exterior, putative DAF binding site 235 N N/S N/S N N Exterior, C terminus

VP4 16 G G G/S S G Disordered 18 S S S/N S/N N Disordered 20 S S S/A A S Disordered 45 D D/E D D D Interior 47 T T/A T T T Interior

a Amino acid differences identiﬁed in aligned sequences of CVB5-P1anc, the eight clinical CVB5 isolates, SVDV (strain SPA/2Ј/93), SVDV (strain UKG/27/72), and CVB3 (strain M). The crystallographic information from CVB3 (PDB accession number 1COV) (64) and SVDV (PDB accession numbers 1OOP and 1MQT) (31, 101) as well as the footprints of CAR (39) and DAF (36) on the surface of CVB3 were used. The numbering of amino acids is according to the residue positions in the CVB5 sequence. Identiﬁed antigenic sites of SVDV are indicated (9, 48, 68). b Disordered, the corresponding residue in the X-ray crystallographic structure of CVB3 was not ordered; exterior, exposed on the outside of the capsid; interior, exposed on the inside of the capsid; buried, completely contained within the capsid protein shell. VOL. 84, 2010 CVB5 PHYLOGENETICS AND ANCESTRAL RECONSTRUCTION 9701

FIG. 5. Structural differences between CVB5-P1anc and clinical CVB5 isolates. (A, left) A CVB3 (PDB accession number 1COV) (64) protomer in a ribbon diagram with VP1, VP2, VP3, and VP4 (light blue, light green, pink, and light yellow, respectively) with a symmetry-related copy of VP3 included to complete the canyon. Based on a ClustalW alignment of CVB5-P1anc and the CVB5 isolates with CVB3, the sequence-equivalent residues that differ between CVB5-P1anc and the CVB5 isolates are depicted as spheres, and VP1, VP2, VP3, and VP4 are shown in dark blue, green, red, and yellow, respectively. The asymmetric unit is indicated by a black triangle. (Right) A single pentamer of the virus capsid is surface rendered to show the location of the amino acid differences exposed to the viral surface. (B) Surface-rendered close-up of the pocket with VP1 residues 93 and 178 (i.e., residues 95 and 180 of CVB5) that line the pocket. The pocket factor is shown in orange (64). On the right is the pentamer showing the amino acid differences exposed to the interior surface of the capsid. (C) Residues predicted to interact with CAR and DAF. (Left) The protomer is shown in a ribbon diagram, with residues within the CAR and DAF footprints shown as magenta and cyan spheres (36, 39). A symmetry-related copy of VP3 is shown to provide the entire CAR footprint on CVB3 in one asymmetric unit. (Right) In the surface-rendered pentamer, the CAR footprint on CVB3 is in magenta, and the DAF binding footprint is in cyan.

The reconstruction of VP1 was based on the sequences of 41 pletely except for one amino acid residue (aspartic acid in clinical isolates. When comparing the ancestral VP1 sequence P1anc to asparagine in VP1anc at position 85). The high level derived from P1anc with the corresponding VP1 ancestor of similarity between the two ancestors indicates that the pre- based on the clinical strains, the two sequences matched com- diction of P1anc is consistent among genome regions of dif- 9702 GULLBERG ET AL. J. VIROL.

FIG. 6. Viral titers of cDNA clone-derived viruses. The titers were determined at 5 days after transfection of cDNA clones into HeLa cells by endpoint titration. Values shown are means Ϯ SEM (n ϭ 3).

ferent sizes and robust to sampling variations. Reconstructions using an empirical amino acid model, however, resulted in FIG. 7. CVB5-P1anc infection in HeLa cells. (A) Light micro- three different amino acid residues in the reconstructed P1 scopic image of CVB5-P1anc-infected (MOI of 10) HeLa cells (12 ␮ ancestor, i.e., in the VP2 protein (threonine to serine at amino h p.i.). Bar, 100 m. (B) Production of viral antigen in HeLa cells infected with CVB5-P1anc (MOI of 10) and analyzed at 5 h after acid position 160), the VP3 protein (aspartic acid to glutamic infection. Antigen was detected with an enterovirus-specific poly- acid at position 35), and the VP1 protein (aspartic acid to clonal rabbit antibody (KTL-482) and a secondary antibody labeled asparagine at position 85). with Alexa Fluor 488 (green). The cellular nuclei were visualized ␮ To predict the localization of amino acid differences be- with DAPI (blue). Bar, 50 m. tween the phylogenetically reconstructed ancestral virion and the clinical isolates on which this ancestor sequence was based, the CVB5 P1 sequences were aligned with that of Furthermore, infection with CVB5-P1anc was verified by the CVB3, and the equivalent residues were mapped onto the detection of viral antigens using an enterovirus-specific an- crystal structure of CVB3 (PDB accession number 1COV) tiserum (Fig. 7B). (64). The structural analysis showed that amino acid differ- Comparative analyses of CVB5-P1anc and modern clinical ences were clustered into five defined regions, i.e., around the isolates. To further characterize CVB5-P1anc, properties in- VP3 ␤-B knob, in a linear cluster on the internal surface, in the cluding molecular evolution, receptor preferences, plaque hydrophobic pocket, as well as around regions corresponding morphology, and cell tropism were analyzed and compared to the CAR (39) and DAF (36) binding sites on CVB3 (Fig. 5 with the features of CVB5Dwt and two clinical isolates, one and Table 1). Furthermore, in a CVB5-SVDV comparison, 7 from each phylogenetic cluster (4378fin88 from cluster I and of the 51 variable residue positions mapped to regions corre- 151rom70 from cluster II). sponding to antigenic sites of SVDV (Table 1). Like other RNA viruses, CVB5 has a high mutation rate, Construction and characterization of CVB5-P1anc. In the which facilitates a rapid adaptation to new environmental con- picornavirus genome, the P2-P3 region is generally highly conditions. In this study, the accumulation of mutations during served among members of a given HEV species, as it codes for replication in the P1 regions of CVB5-P1anc, 4378fin88, proteins essential for viral replication (44). The P1 region, on 151rom70, and CVB5Dwt, after 10 consecutive passages in the other hand, seems less crucial for RNA replication. This three different cell lines (i.e., GMK, HeLa, and RD cells), was was clearly illustrated when a recombinant poliovirus clone analyzed. Sequence analyses of the CVB5-P1anc progeny virus lacking large parts of the P1 region was able to replicate its collected after passages in GMK cells (two independent exper- RNA in transfected cells (74). In this study, a replication- iments) revealed that the original ancestral P1 sequence was competent backbone (pCVB5D-cas) based on a full-length completely retained (Table 2). After passages in HeLa cells, a infectious CVB5D cDNA clone (pCVB5Dwt) was constructed single-amino-acid substitution in the VP2 protein (in the first (Fig. 1). To generate a CVB5 construct with an ancestral cap- experiment) or the VP1 protein (in the second experiment) sid (pCVB5-P1anc), the P1 region of the pCVB5D-cas vector was fixed in the final progeny population. In a corresponding was replaced with a synthesized ancestral P1 sequence. Viruses experiment with CAR-deficient RD cells (77), CVB5-P1anc were derived from the generated constructs, pCVB5Dwt, replicated without signs of CPE, and serial infections resulted pCVB5D-cas, and pCVB5-P1anc, by transfection into HeLa in an introduction of three nonsynonymous mutations, located cells. All the recombinant CVB5 viruses caused complete cy- in the VP1 and the VP3 genes. Interestingly, one substitution tolysis at 96 h posttransfection and replicated to high titers (109 (lysine to methionine at position 259 in the VP1 protein) was

TCID50/ml) in HeLa cells (Fig. 6). The identity of progeny introduced into both viral progeny populations after passages viruses was conﬁrmed by sequencing. Continued functional in RD cells. Comparative studies of the accumulation of mu- characterization of HeLa cells infected with CVB5-P1anc tations of CVB5-P1anc and 4378ﬁn88, 151rom70, and (MOI of 10) showed that the virus induced a complete CVB5Dwt after 10 passages in GMK, HeLa, and RD cells destruction of the cell monolayer within 12 h p.i. (Fig. 7A). showed that the number of mutations introduced into the VOL. 84, 2010 CVB5 PHYLOGENETICS AND ANCESTRAL RECONSTRUCTION 9703

TABLE 2. Mutations and amino acid substitutions in the genomic P1 region of CVB5-P1anc, 151rom70, 4378ﬁn88, or CVB5Dwt after 10 passages in different cell lines

Sample 1 Sample 2 Virus and cell line Genomic region Mutation Amino acid substitution Genomic region Mutation Amino acid substitution CVB5-P1anc GMKa HeLa VP2 1661T3A 238Y3N VP1 3219A3G 258Q3R RDb VP3 1908A3G 59E3G VP1 3222A3T 259K3M VP3 2164A3A/G VP1 2524G3G/T 26E3D VP1 3222A3T 259K3M

151rom70 GMK VP1 2099A3A/G 132Q3Q/R VP1 2099A3A/G 132Q3Q/R HeLaa RDb VP3 1163C3T 58T3I VP3 1163C3T 58T3I VP1 2099A3G 132Q3R VP3 1335G3G/A VP1 2099A3G 132Q3R VP1 2480A3T 259K3M

4378ﬁn88 GMK VP1 2090C3T 129S3F VP1 2090C3T 129S3F HeLa VP1 1883C3C/T 60S3S/F VP1 1883C3C/T 60S3S/F VP1 2090C3T 129S3F VP1 2090C3T 129S3F RDb VP1 1986C3T VP1 2098C3A 132Q3K VP1 2098C3A 132Q3K VP1 2218A3A/G 172T3T/A VP1 2228G3A 175S3N VP1 2377G3A 225D3N VP1 2377G3A 225D3N

CVB5Dwt GMK VP1 2148T3C VP1 2087A3A/G 128Q3Q/R VP1 2148T3C HeLa VP1 1998A3A/G VP1 1998A3A/G RDb VP3 1057T3C 23S3P VP2 723C3T VP1 2087A3G 128Q3R VP1 2087A3G 128Q3R VP1 2109C3C/T VP1 2148T3C VP1 2148T3C VP1 2299G3A 199A3T VP1 2248A3T 182I3L

a No mutations were detected. b Noncytolytic infection.

ancestral sequence was not higher than that in sequences of isolates use both CAR and DAF as cellular attachment mole- contemporary viruses. In conclusion, these results showed that cules. the ancestral P1 region of CVB5-P1anc was tolerated during We further complemented our comparative analyses of serial passages in cell culture. CVB5-P1anc by investigating the plaque morphology and Some viruses use multiple cell surface receptors for initial growth properties of the virus in HeLa cells. The plaque phe- host cell attachment. Previously, it was reported that CVB5 notype of CVB5-P1anc in HeLa cells was similar to those of binds to CAR as a primary receptor (6, 60) but that it also uses plaques formed by CVB5Dwt and the two clinical isolates at DAF as a coreceptor (6, 85). The capacity of radiolabeled 48 h p.i. (Fig. 9B). The one-step growth curve analysis of the CVB5 viruses to bind either CAR or DAF alone or the two CVB5-P1anc infection in HeLa cells showed that virus produc- receptors in combination was assessed by utilizing CHO cell tion started as early as 4 h p.i. (Fig. 9C). This initial sign of viral lines transfected with either CAR or DAF and HeLa cells replication was followed by a steep rise in virus titers and a expressing both CAR and DAF. The expression of cell surface plateau that was reached 8 h after infection. Signs of cyto- receptors was verified by flow cytometry (Fig. 8). At the level of pathogenicity were already observed at 6 h after viral exposure. virus binding, no significant interaction between radiolabeled Furthermore, CVB5-P1anc replicated as efficiently as CVB5 variants and CHO cells was detected (Fig. 9A). The CVB5Dwt and the two clinical isolates. viruses bound with equal efficiencies to HeLa and CHO-DAF Human enteroviruses infect cells mainly of human or other cells, whereas a lower level of binding to CHO-CAR cells was primate origin. Consequently, studies of infectivity in a variety measured. Hence, the expression of CAR on CHO cells re- of cell lines were undertaken as an additional approach to sulted in a 10-fold increase in the binding of the CVB5 strains, analyze the host cell specificity of the CVB5-P1anc phenotype. whereas a significantly higher level of binding was observed in As shown in Fig. 9D, CVB5-P1anc bound to and replicated the presence of DAF (450-fold). Overall, these results indi- efficiently in HeLa, A549, HT29, and GMK cells. This virus cated that CVB5-P1anc, CVB5Dwt, and two clinical CVB5 replication caused a CPE typical of enteroviruses. However, 9704 GULLBERG ET AL. J. VIROL.

ing, plaque morphology, growth kinetics, cell tropism, and overall structure.

DISCUSSION Previous studies have provided some insight into the genetic diversity within the CVB5 serotype (50, 80, 93). In the present study, an ML approach was applied to extend previous studies and to analyze the global genetic diversity among contemporary CVB5 isolates collected over a 25-year period. Consistent with a study of local CVB5 isolates in Belgium (93), our phylogenetic analysis of isolates from Europe, Asia, North Amer- ica, and South America showed that CVB5 viruses have evolved from their MRCA into two major evolutionary lineages. The two major cocirculating clades were observed by analysis of the VP1 gene but also in a corresponding analysis of the entire P1 region. There are several possible explanations to this bimodal CVB5 evolution, including the geographic sepa- ration of ancestral viruses or adaptation processes during early host switch events. Possibly, in the future, viruses in these two clusters will evolve into two distinct serotypes. Interestingly, the evolution of other serotypes within the HEV-B species, based on a phylogenetic analysis of the VP1 gene, exhibits a different tree topology. For example, CVB4 shows a multifur- cating topology (65), whereas echovirus 30 displays a tree- trunk-like pattern (70). However, the evolutionary events leading to these differences in tree topology are not known. FIG. 8. Flow cytometric analysis of CAR and DAF expression on The SVDV isolates included in the phylogenetic analyses CHO, CHO-CAR, CHO-DAF, and HeLa cells. For the detection of constituted a monophyletic group that was most closely related CAR and DAF, monoclonal anti-CAR (RmcB) and anti-DAF (BRIC110) antibodies were used (black histograms). A mouse IgG1 to CVB5 viruses of cluster II, in agreement with data from a antibody was used as a negative control (gray histograms). previous report (108). The adaptation of CVB5 from human to pig has been estimated to have occurred between 1945 and 1965 (108). This highly contagious porcine CVB5 variant causes symptoms similar to those of foot-and-mouth disease although CVB5-P1anc caused a productive infection in RD virus and is therefore economically important (27, 54, 67). cells, no signs of CPE were detected. In contrast, no active The high rate of RNA virus evolution allows a rapid adap- replication or induction of CPE was observed for CHO cells in tation to new environments (20, 57, 76, 102). Among picorna- spite of the detected virus binding to the cell surface. In addi- viruses, it has been shown for poliovirus that every progeny tion, CVB5Dwt and the two clinical isolates showed a cell RNA molecule on average contains one mutation (22). By tropism corresponding to that of CVB5-P1anc (Table 3). Con- using a relaxed-clock model, the accumulation of mutations in clusively, progeny CVB5 virus production was clearly detect- the heterochronous CVB5 sequence data was transformed into able in all cells assessed except CHO cells. an estimated evolutionary rate of approximately 0.004 nucleo- In order to analyze if specific antiserum generated against tide substitutions per site per year, which in turn corresponds CVB5F (which is the CVB5 prototype strain and identical to to an MRCA dating back to the mid-19th century. This evo- CVB5Dwt except for one amino acid substitution in the VP1 lutionary rate is within the range estimated for other picorna- protein) could neutralize CVB5-P1anc, a neutralization assay viruses (11, 29, 108). For example, an evolutionary rate of was performed. Infections by CVB5Dwt and two field strains included in this comparative study were neutralized by the antiserum. Interestingly, this anti-CVB5F antiserum was TABLE 3. Comparisons of cell tropisms and CVB5 antiserum equally efficient in protecting HeLa cells from infection by cross-reactivities between CVB5-P1anc and CVB5-P1anc (Table 3). These results suggest that the CVB5- modern CVB5 isolates a P1anc virion shares neutralizing epitopes with the CVB5F lab- Cell tropism Neutralization Virus c oratory strain as well as with recently isolated CVB5 viruses. CHO GMK HeLa RDb A549 HT29 dilution Further studies of CVB5-P1anc serology showed that neither Ϫϩϩϩϩϩ antiserum against CVB1 to CVB4 nor antiserum against CVB6 CVB5-P1anc 1:4,096 CVB5Dwt Ϫϩϩϩϩϩ 1:8,192 protected HeLa cells against infection. 151rom70 Ϫϩϩϩϩϩ 1:4,096 Taken together, these results showed that the CVB5-P1anc 4378fin88 Ϫϩϩϩϩϩ 1:4,096 virus constructed using ML phylogenetic methods displayed a Plus sign, cytolytic infection; minus sign, no infection. characteristics corresponding to those of present-day circulat- b Noncytolytic infection. ing CVB5 viruses, including properties such as receptor bind- c Highest dilution of serum that neutralizes virus infection. VOL. 84, 2010 CVB5 PHYLOGENETICS AND ANCESTRAL RECONSTRUCTION 9705

FIG. 9. Receptor preferences, plaque phenotypes, and virus growth kinetics of CVB5-P1anc, CVB5Dwt, 151rom70, and 4378fin88 as well as CVB5-P1anc infectivity in different cell lines. (A) Binding of radiolabeled CVB5 viruses to CHO, CHO-CAR, CHO-DAF, or HeLa cells. Cells were incubated with [35S]methionine-cysteine-labeled CVB5-P1anc, CVB5Dwt, 151rom70, or 4378fin88 at room temperature for 2 h. Following the removal of unbound virions, the cell-associated radioactivity was determined by scintillation counting. Results are presented as means Ϯ SEM (n ϭ 3). (B) Plaques were visualized at 48 h p.i. by crystal violet staining of HeLa cells. Virus-infected cell lysates were diluted 10Ϫ7 times in order to distinguish individual plaques. (C) HeLa cells were infected with the indicated viruses at an MOI of 10 TCID50/cell. At various times postinfection, samples were frozen, and the total yield of infectious virus was quantified by the TCID50 method. Results shown are representative of three independent experiments. (D) CVB5-P1anc titer determined at time point zero and after complete CPE or 5 days p.i. by endpoint titration in HeLa cells. Results are presented as means Ϯ SEM (n ϭ 3).

0.0038 nucleotide substitutions per site per year was estimated proper folding of viral capsid proteins, plus intra- and inter- for enterovirus 70 based on VP1 sequence analyses (91). The molecular interactions, plays a crucial role for the capsid to use of a relaxed-clock model allowed a more confident esti- maintain its multifaceted properties, including structural sta- mate of divergence times in the face of rate variation among bility to protect the genome and, at the same time, flexibility to lineages, and it also enabled us to investigate sources of rate enable uncoating after interactions with host cell receptors variation. For example, a rapid rate of evolution was observed (103). Despite the advantage of using virus infectivity to verify for the branch leading to the most recent SVDV isolates, the functionality of hypothetical ancestral sequences, few stud- strongly suggesting adaptation after interspecies transmission. ies of resurrected viral sequences have been presented (21, 51, In studies of picornaviruses, the evolution of virus sequences 66, 81). has been assayed by serial transfers of large viral populations In the present study, an ancestral state, i.e., the most likely pertubated by bottleneck events (3, 19, 28), while molecular ancestral CVB5 capsid sequence, was inferred from sequences structure-function relationships of viral proteins have been of contemporary CVB5 isolates by ML ancestral reconstruc- evaluated by site-directed mutagenesis (37, 40, 58, 100, 107). tion (78) and de novo gene synthesis. Although CVB5 most Recent advances in phylogenetics and DNA synthesis tech- likely exists as a swarm of closely related variants within hosts, niques enable ancestral sequence reconstruction, providing an similar to poliovirus and foot-and-mouth disease virus (20), important new approach to investigate evolutionary events and this has little impact on our evolutionary reconstruction be- the conservation of structural features (96). Although it is cause it essentially involves an interhost evolutionary process. impossible to claim that reconstructed ancestral sequences are All variants sampled from a patient at a particular point in time correct in all details, since computational models simplify bi- will generally coalesce to a common ancestor prior to the time ological processes, the resurrection of molecular ancestors of infection. Therefore, whatever variants are sampled from with a detectable biological activity, such as enzymes and vi- the patients, we expect the same common ancestor for viruses ruses, offers a possibility to verify their structural integrity. The obtained from different patients. Inference techniques and 9706 GULLBERG ET AL. J. VIROL. evolutionary models may have a more important impact on ACKNOWLEDGMENTS ancestral reconstructions. We have used likelihood-based We thank J. M. Bergelson for his contribution to the binding studies; codon models, which can accommodate a detailed evolutionary K. Edman for valuable discussions; and M. Roivainen, H. Norder, L. process. For a few highly variable positions, however, this was Magnius, J. W. McCauley, R. L. Crowell, L. Philipson, and R. Petters- not always consistent with amino acid reconstructions. Further son for providing cells, viruses, and antibodies. Funding was provided by grants from the Swedish Knowledge Foun- research that can take into account the uncertainty of recon- dation, the Graninge Foundation, the Helge Ax:son Johnsons Foun- structed ancestral sequences is therefore needed. dation, the Sparbanken Kronan Foundation, and the University of Characterization of the inferred ancestral P1 sequence Kalmar. S.H. was supported by NIH grant K22 AI-07927. Figure 5 was produced by using the UCSF Chimera package from the Computer showed that capsid proteins expressed from the CVB5-P1anc Graphics Laboratory, University of California, San Francisco (sup- clone assembled into functional infectious virions. In addition, ported by NIH grant P41 RR-01081). the recombinant CVB5-P1anc virus shared several features REFERENCES with present-day clinical isolates, including immunogenic 1. Adey, N. B., T. O. Tollefsbol, A. B. Sparks, M. H. Edgell, and C. A. epitopes, preferences for the CAR and DAF receptors, cell Hutchison III. 1994. 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