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J Virol. Oct 2010; 84(19): 9695–9708.
Published online Jul 14, 2010. doi:  10.1128/JVI.00071-10
PMCID: PMC2937801

Characterization of a Putative Ancestor of Coxsackievirus B5 [down-pointing small open triangle]

Abstract

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) were discovered in the 1950s in a search for new poliovirus-like viruses (33, 61). Infections caused by CVBs are often asymptomatic but may occasionally result in severe diseases of the heart, pancreas, and central nervous system (99). CVBs are small icosahedral RNA viruses belonging to the Human enterovirus B (HEV-B) species within the family Picornaviridae (89). In the positive single-stranded RNA genome, the capsid proteins VP1 to VP4 are encoded within the P1 region, whereas the nonstructural proteins required for virus replication are encoded within the P2 and P3 regions (4). The 30-nm capsid has an icosahedral symmetry and consists of 60 copies of each of the four structural proteins. The VP1, VP2, and VP3 proteins are surface exposed, whereas the VP4 protein lines the interior of the virus capsid (82). The coxsackievirus and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, serves as the major cell surface attachment molecule for all six serotypes of CVB (5, 6, 39, 60, 98). Some strains of CVB1, CVB3 and CVB5 also interact with the decay-accelerating factor (DAF) (CD55), a member of the family of proteins that regulate the complement cascade. However, the attachment of CVBs to DAF alone does not permit the infection of cells (6, 7, 59, 85).

Picornaviruses exist as genetically highly diverse populations within their hosts, referred to as quasispecies (20, 57). This genetic plasticity enables these viruses to adapt rapidly to new environments, but at the same time, it may compromise the structural integrity and enzymatic functionality of the virus. The selective constraints imposed on the picornavirus genome are reflected in the different regions used for different types of evolutionary studies. The highly conserved RNA-dependent RNA polymerase (3Dpol) gene is used to establish phylogenetic relationships between more-distantly related viruses (e.g., viruses belonging to different genera) (38), whereas the variable genomic sequence encoding the VP1 protein is used for the classification of serotypes (13, 14, 69, 71, 72).

In 1963, Pauling and Zuckerkandl proposed that comparative analyses of contemporary protein sequences can be used to predict the sequences of their ancient predecessors (73). Experimental reconstruction of ancestral character states has been applied to evolutionary studies of several 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 the field of virology, reconstructed ancestral or consensus protein sequences have been used in attempts to develop vaccine candidates for human immunodeficiency virus type 1 (21, 51, 66, 81) but rarely to examine general phenotypic properties.

In this study, a CVB5 virus with a probable ancestral virion (CVB5-P1anc) was constructed and characterized. We first analyzed in detail the evolutionary relationships between structural genes of modern CVB5 isolates and inferred a time scale for their evolutionary history. An ancestral virion sequence was subsequently inferred by using a maximum likelihood (ML) method. This sequence was then synthesized de novo, cloned into a replicative backbone of an infectious CVB5 cDNA clone, and transfected into HeLa cells. The hypothetical CVB5-P1anc assembled into functional virus particles that displayed phenotypic properties similar to those of contemporary clinical isolates. This is the first report describing the reconstruction and characterization of a fully functional picornavirus with a putative ancestral capsid.

MATERIALS AND METHODS

Cell lines and viruses.

African green monkey kidney (GMK), human colon adenocarcinoma (HT29), Chinese hamster ovary (CHO), human lung carcinoma (A549), and human rhabdomyosarcoma (RD) cell lines were purchased from the American Type Culture Collection. HeLa Ohio cells were kindly provided by M. Roivainen (Helsinki, Finland). Recombinant CHO cells expressing CAR (CHO-CAR) or DAF (CHO-DAF) were constructed by H.-C. Selinka (77, 84). Cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% newborn calf serum (NCS). Recombinant CHO cells were grown in selective medium supplemented with 1 mg/ml G418 (Sigma) for cells expressing CAR and 0.75 mg/ml hygromycin B (Invitrogen) for cells expressing DAF.

The clinical CVB5 isolates used in this study were propagated in GMK cells according to standard procedures (63). CVB5 strain 1954UK85 (CVB5UK) (109) was provided by J. W. McCauley, Newbury, United Kingdom, and strain Dalldorf (CVB5D) was provided by R. L. Crowell, Philadelphia, PA (16, 79). As previously described (47), the genome sequence of the CVB5D virus is identical to that of prototype strain Faulkner (CVB5F) (55), except for one amino acid change in the VP1 protein. Viral titers of propagated viruses were determined by the 50% tissue culture infectious dose (TCID50) method according to standard procedures (41).

Flow cytometry analysis.

Flow cytometry analysis was performed as described previously (77). Briefly, CHO, CHO-CAR, CHO-DAF, and HeLa cells were stained with an anti-CAR (RmcB) antibody (43) (the hybridoma was kindly provided by L. Philipson and R. Pettersson, Karolinska Institute, Sweden [also available from the American Type Culture Collection {ATCC CRL-2379}]), an anti-DAF (BRIC110) antibody (Cymbus Biotechnologies), or a mouse IgG1 control antibody (X0931; Dako). After 1 h of incubation at 4°C, cells were washed and stained with a secondary R-phycoerythrin-labeled rabbit anti-mouse antibody (R0439; Dako). Data were acquired by using a FACSCalibur instrument (Becton Dickinson) and analyzed with CellQuest, version 3.3, software (Becton Dickinson).

Extraction, amplification, and sequencing.

Viral RNA was extracted from infected cell cultures (QIAamp viral RNA minikit; Qiagen), reverse transcribed (Superscript III; Invitrogen), and PCR amplified (PicoMaxx; Stratagene) by using virus-specific primers. PCR amplicons were visualized in agarose gels and purified (QIAquick gel extraction kit; Qiagen). The nucleotide sequences were determined with an ABI Prism 3130 automated sequencer (Applied Biosystems) by a primer-walking strategy on both strands using BigDye chemistry (ABI Prism BigDye Terminator cycle sequencing ready-reaction kit, version 1.1; Applied Biosystems). Sequences were analyzed by using the Sequencher, version 4.6, software package (Gene Codes Corporation).

Phylogenetic analysis.

Viral nucleotide sequences were aligned by using ClustalW (94), and phylogenetic signals were evaluated by using likelihood mapping (90). The presence of nucleotide substitution saturation was tested by using an approach described previously by Xia et al. (104). Phylogenetic relationships between the different CVB5 strains, based on the genomic VP1 and P1 regions, were inferred by the ML method as implemented in PhyML (35). Branch support values for inferred phylogeny were estimated by nonparametric bootstrapping consisting of 1,000 pseudoreplicates (30). The general time-reversible (GTR) substitution model (53) with a gamma-distributed rate heterogeneity was used for the VP1 analysis, while the same model including the proportion of invariable sites was used for the P1 sequence data. The phylogenetic relationship between viruses was also examined by the neighbor-joining method, as implemented in MEGA, version 4.0 (92). Previously determined CVB5 sequences (CVB5UK [GenBank accession number X67706] and CVB5D [47]) and swine vesicular disease virus (SVDV) sequences (Svdh3jap76 [accession number D00435], Svdj1jap73 [accession number D16364], Svd27uk72 [accession number X54521], Svd1spa93 [accession number AF039166], and Svd1net92 [accession number AF268065]) were included in the phylogenetic analysis of the VP1 gene. The sequences of CVB4 Tuscany (CVB4T) (accession number DQ480420) and CVB6 Schmitt (CVB6S) (accession number AF114384) were used as an outgroup. Phylogenetic trees were visualized with MEGA 4.0. Root-to-tip divergence as a function of sampling time was examined by using Path-O-Gen (available at http://tree.bio.ed.ac.uk/software).

Bayesian evolutionary analysis.

We inferred the time scale and tempo of CVB5 evolution using a Bayesian statistical approach implemented in BEAST (25). This approach employs a full probabilistic model of sequence evolution along rooted, time-measured phylogenies with a coalescent prior, using either a 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 nucleotide substitution (86) with gamma-distributed rate variation among sites, an uncorrelated log-normal relaxed-clock model, and a Bayesian skyline tree prior (26). Markov chain Monte Carlo analyses were run for 10 million generations and diagnosed by using Tracer (http://beast.bio.ed.ac.uk/Tracer). The evolutionary history was summarized in the form of a maximum clade credibility tree by using TreeAnnotator (http://beast.bio.ed.ac.uk/TreeAnnotator) and visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree). Bayesian credible intervals for continuous parameters are reported as the highest posterior density intervals, which are the smallest intervals that contain 95% of the posterior distribution.

Ancestral sequence reconstruction.

The ancestral sequences were reconstructed from the CVB5 ingroup taxa of the phylogenetic trees based on the genetic VP1 and P1 regions but without the reference strains (CVB5D and CVB5UK). These reference strains were not included in the reconstruction because their passage history is unknown. ML ancestral sequences were inferred by using a standard codon substitution model (M0, which assumes a homogenous nonsynonymous/synonymous substitution rate among sites and among lineages), as implemented in codeml of the PAML package (34, 105). After ML optimization under the codon model, this approach considers the assignment of a set of characters to all interior nodes at a site as a reconstruction and selects the reconstruction that has the highest posterior probability, i.e., so-called joint reconstruction (106). This procedure was efficiently performed by using an algorithm described previously by Pupko and colleagues (78). A corresponding reconstruction was also performed by using the best-fitting empirical amino acid model. The inference of the ancestor sequence was facilitated by the absence of gaps or evidence of recombination within the genomic P1 region, as confirmed by using the phi test (12).

Construction of CVB5-P1anc.

The complete CVB5D genome was amplified and cloned into the pCR-Script Direct SK(+) vector (Stratagene) by using the AscI and NotI restriction enzyme cleavage sites, as previously described (Fig. (Fig.1)1) (56). In this infectious full-length cDNA clone of CVB5D (pCVB5Dwt), a ClaI site was introduced at nucleotide position 3340 to generate a cassette vector (pCVB5D-cas). This modification resulted in one amino acid change in the 2A protein (valine to leucine at amino acid position 17). This substitution was accepted, as leucine 17 is present in the 2A protein of other enteroviruses, including echovirus 30 and echovirus 21. In addition, a synonymous mutation was introduced into the P1 ancestral sequence to remove a SalI site. In order to construct a CVB5 clone with the inferred ancestral capsid sequence (pCVB5-P1anc), a pUC57 plasmid containing the ancestral P1 sequence flanked by SalI and ClaI sites was purchased (GenScript). Subsequently, the P1 genomic region of this pUC57 plasmid was amplified and then cloned into pCVB5D-cas. The constructs were propagated in Escherichia coli DH5α cells and purified (Midiprep kit; Promega). The nucleotide sequences of all constructs were verified by sequencing as described above.

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 ...

HeLa cell monolayers were transfected with 2.5 μg of the CVB5 cDNA clones by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Recombinant viruses were collected on day 5 posttransfection and subsequently sequenced to confirm their identity. The titers of these viruses were determined by a TCID50 assay with HeLa cells. In order to ensure the safety of laboratory workers, the environment, and the public, the generation of CVB5-P1anc was performed under conditions of biosafety level 2 containment.

Virus infection.

Cell monolayers were infected with virus according to standard procedures (63). Briefly, subconfluent monolayers of cells grown in 25-cm2 flasks were inoculated with viruses at a multiplicity of infection (MOI) of 10 TCID50/cell. Following virus adsorption at room temperature for 1 h, the cells were washed three times before the addition of DMEM supplemented with 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Incubation of the virus infection at 37°C in a 7.5% CO2 atmosphere was continued for 5 days or until a cytopathic effect (CPE) was observed.

Viral replication was quantified by analyses of samples taken immediately after infection and 5 days postinfection (p.i.) or when a complete CPE was observed. After three cycles of freezing and thawing, the viral titers were determined by a TCID50 assay with HeLa cells as described above.

The molecular evolutions of CVB5-P1anc, 151rom70, 4378fin88, and wild-type CVB5 (CVB5Dwt) were compared after 10 serial rounds of infection in GMK, HeLa, and RD cells. After the 10th passage, the P1 regions of these viruses were sequenced as described above.

Immunofluorescence.

Infected HeLa cells cultured on Lab-Tek II chamber glass slides (Nalge Nunc International) were fixed in 4% formaldehyde for 30 min at 4°C and stained for 1 h at room temperature with an enterovirus-specific polyclonal rabbit antiserum (KTL-482) (42). The primary antibody was visualized with a secondary goat anti-rabbit antibody labeled with Alexa Fluor 488 (A11034; Molecular Probes Inc.). Finally, slides were mounted with Vectashield (Immunkemi) containing 4′,6-diamidino-2-phenylindole (DAPI), and images were captured with an epifluorescence microscope.

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 virus in 10-fold dilutions for 1 h at 37°C. Following adsorption, the virus inoculum was aspired, and cells were overlaid with DMEM supplemented with 1% NCS, 2 mM l-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.8% (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.

Viral growth kinetics.

To assess the growth kinetics of viruses, HeLa cells in 24-well plates were infected with virus at an MOI of 10 TCID50/cell as described above. Cells and medium were harvested and frozen at various time points postinfection. Virus titers in collected samples were determined by a TCID50 assay.

Neutralization assay.

Serial 2-fold dilutions of antiserum against CVB1, CVB2, CVB3, or CVB4 (kindly provided by H. Norder and L. Magnius, Swedish Institute for Infectious Disease Control, Sweden); CVB5F (V032-501-560, 1965; NIH research reagent); or CVB6 (V033-501-560, 1965; NIH research reagent) 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 at 37°C for 1 h. The virus-antibody mixtures were applied onto HeLa cells in 96-well plates (quadruplicate). After 5 days of incubation at 37°C, cells were examined microscopically for evidence of virus-induced CPE. The highest serum dilution that completely inhibited CPE was determined to be the endpoint titer.

Virus binding assay.

Viruses were metabolically labeled with [35S]methionine-cysteine (Perkin-Elmer) and purified by sucrose gradient centrifugation as described previously for echoviruses 1 and 8 (8). Virus attachment to cells was measured according to a method described previously (2, 62). Briefly, adherent CHO, CHO-CAR, CHO-DAF, and HeLa cells were detached by EDTA treatment. Approximately 2.5 × 105 cells were incubated with radiolabeled virus (~30,000 dpm) in DMEM with 2% NCS, 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Following virus adsorption to cells for 2 h at room temperature with gentle agitation, unbound virus was removed by three washes, and cell-bound radioactivity was quantified by liquid scintillation counting. The virus binding was analyzed in triplicate, and data are presented as means ± standard errors of the means (SEM).

Mapping of structural differences between CVB5-P1anc and the clinical CVB5 isolates.

Based on a sequence alignment of CVB3 strain M, the clinical CVB5 isolates, and CVB5-P1anc (ClustalW) (94), the sequence-equivalent residues in the ancestor were mapped to the X-ray crystallographic structure of CVB3 (Protein Data Bank [PDB] accession number 1COV) (64). The footprints of CAR (39) and DAF (36) on the CVB3 virion surface were used to locate the equivalent CVB5-P1anc residues within the receptor binding footprints. The X-ray crystal structure of the CVB3 capsid was modeled and visualized by using Chimera (75, 83).

RESULTS

Phylogenetic relationships of CVB5 viruses.

The gene encoding the VP1 capsid protein is considered to be the most informative genomic region for studying molecular epidemiology and evolutionary relationships among enteroviruses (13, 14, 69, 71, 72). Hence, in order to evaluate the phylogenetic relationships among 41 clinical CVB5 strains, the VP1-encoding gene was sequenced. These viruses were isolated in Europe, Asia, North America, and South America between 1970 and 1996. The ML analysis of aligned VP1 nucleotide sequences of these clinical isolates together with two CVB5 reference strains (CVB5D and CVB5UK) and five SVDV isolates revealed a dichotomous phylogenetic relationship, which distinguishes the existence of two coevolving clusters of genetic lineages (indicated as clusters I and II) (Fig. (Fig.22 A). Some CVB5 strains isolated in Finland clustered together in cluster I, whereas other Finnish strains were more closely related to viruses isolated in other parts of Europe and North America, indicating a geographic mixture of cluster I and II viruses. The phylogenetic analysis also showed that SVDV was more closely related to CVB5 viruses in cluster II. In addition to the VP1 sequences, the complete sequence of the structural genes (the entire P1 region) of four isolates from each of the two clusters, separated both in time of isolation and by geographic location, was determined. The result from the subsequent phylogenetic analysis based on the P1 sequences showed a relationship between viruses corresponding to the phylogeny of the VP1 gene (Fig. (Fig.2B).2B). Tree topologies corresponding to those resulting from the ML analyses were also observed for trees inferred with the neighbor-joining method (data not shown).

FIG. 2.
Phylogenetic relationships among CVB5 isolates. (A) Phylogram based on the nucleotide sequences of the VP1 gene. •, CVB5 isolates selected for sequencing of the entire P1 region. (B) Phylogeny of selected CVB5 isolates based on the nucleotide ...

Timed evolutionary history of CVB5 viruses.

By plotting root-to-tip divergence from the ML tree as a function of sampling time, we observed a clear accumulation of nucleotide substitutions over the sampling time interval (Fig. (Fig.3).3). To establish a time scale for the CVB5 evolutionary history and estimate the viral rate of evolution, we performed Bayesian evolutionary analysis of the VP1 sequences sampled over time using BEAST (25). In this approach, we used Markov chain Monte Carlo analyses to average over tree space, with each tree having branch lengths in units of time. Figure Figure44 shows the maximum clade credibility tree that summarizes the evolutionary history estimated by using a relaxed molecular clock (23). The most recent common ancestor (MRCA) of all CVB5 lineages (clusters I and II) dated back to 1854 (1807 to 1898). Cluster II had a somewhat older MRCA than did cluster I (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. (Fig.3)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. (Fig.33).

FIG. 3.
Root-to-tip divergence plot. Shown is a linear regression plot for root-to-tip divergence versus sampling year. The SVDV isolates included in the analysis are encircled.
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 ...

Ancestral reconstruction.

In order to reconstruct a putative ancestral character state for the CVB5 virion, we applied a codon-based ML approach to the inferred CVB5 phylogeny. In the sequence alignment of the reconstructed CVB5 capsid residue state (P1anc) and eight clinical isolates, 51 variable amino acid residue positions were observed (Table (Table1).1). The robustness of the P1anc reconstruction was assessed by comparing the ancestral reconstruction of the P1 sequence with an ancestor sequence based on the VP1 gene alone (VP1anc). The reconstruction of VP1 was based on the sequences of 41 clinical isolates. When comparing the ancestral VP1 sequence derived from P1anc with the corresponding VP1 ancestor based on the clinical strains, the two sequences matched completely except for one amino acid residue (aspartic acid in P1anc to asparagine in VP1anc at position 85). The high level of similarity between the two ancestors indicates that the prediction of P1anc is consistent among genome regions of different sizes and robust to sampling variations. Reconstructions using an empirical amino acid model, however, resulted in three different amino acid residues in the reconstructed P1 ancestor, i.e., in the VP2 protein (threonine to serine at amino acid position 160), the VP3 protein (aspartic acid to glutamic acid at position 35), and the VP1 protein (aspartic acid to asparagine at position 85).

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

To predict the localization of amino acid differences between 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 CVB3, and the equivalent residues were mapped onto the crystal structure of CVB3 (PDB accession number 1COV) (64). The structural analysis showed that amino acid differences were clustered into five defined regions, i.e., around the VP3 β-B knob, in a linear cluster on the internal surface, in the hydrophobic pocket, as well as around regions corresponding to the CAR (39) and DAF (36) binding sites on CVB3 (Fig. (Fig.55 and Table Table1).1). Furthermore, in a CVB5-SVDV comparison, 7 of the 51 variable residue positions mapped to regions corresponding to antigenic sites of SVDV (Table (Table11).

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 ...

Construction and characterization of CVB5-P1anc.

In the picornavirus genome, the P2-P3 region is generally highly conserved among members of a given HEV species, as it codes for proteins essential for viral replication (44). The P1 region, on the other hand, seems less crucial for RNA replication. This was clearly illustrated when a recombinant poliovirus clone lacking large parts of the P1 region was able to replicate its RNA in transfected cells (74). In this study, a replication-competent backbone (pCVB5D-cas) based on a full-length infectious CVB5D cDNA clone (pCVB5Dwt) was constructed (Fig. (Fig.1).1). To generate a CVB5 construct with an ancestral capsid (pCVB5-P1anc), the P1 region of the pCVB5D-cas vector was replaced with a synthesized ancestral P1 sequence. Viruses were derived from the generated constructs, pCVB5Dwt, pCVB5D-cas, and pCVB5-P1anc, by transfection into HeLa cells. All the recombinant CVB5 viruses caused complete cytolysis at 96 h posttransfection and replicated to high titers (109 TCID50/ml) in HeLa cells (Fig. (Fig.6).6). The identity of progeny viruses was confirmed by sequencing. Continued functional characterization of HeLa cells infected with CVB5-P1anc (MOI of 10) showed that the virus induced a complete destruction of the cell monolayer within 12 h p.i. (Fig. (Fig.77 A). Furthermore, infection with CVB5-P1anc was verified by the detection of viral antigens using an enterovirus-specific antiserum (Fig. (Fig.7B7B).

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 ± SEM (n = 3).
FIG. 7.
CVB5-P1anc infection in HeLa cells. (A) Light microscopic image of CVB5-P1anc-infected (MOI of 10) HeLa cells (12 h p.i.). Bar, 100 μm. (B) Production of viral antigen in HeLa cells infected with CVB5-P1anc (MOI of 10) and analyzed at 5 h after ...

Comparative analyses of CVB5-P1anc and modern clinical isolates.

To further characterize CVB5-P1anc, properties including molecular evolution, receptor preferences, plaque morphology, and cell tropism were analyzed and compared with the features of CVB5Dwt and two clinical isolates, one from each phylogenetic cluster (4378fin88 from cluster I and 151rom70 from cluster II).

Like other RNA viruses, CVB5 has a high mutation rate, which facilitates a rapid adaptation to new environmental conditions. In this study, the accumulation of mutations during replication in the P1 regions of CVB5-P1anc, 4378fin88, 151rom70, and CVB5Dwt, after 10 consecutive passages in three different cell lines (i.e., GMK, HeLa, and RD cells), was analyzed. Sequence analyses of the CVB5-P1anc progeny virus collected after passages in GMK cells (two independent experiments) revealed that the original ancestral P1 sequence was completely retained (Table (Table2).2). After passages in HeLa cells, a single-amino-acid substitution in the VP2 protein (in the first experiment) or the VP1 protein (in the second experiment) was fixed in the final progeny population. In a corresponding experiment with CAR-deficient RD cells (77), CVB5-P1anc replicated without signs of CPE, and serial infections resulted in an introduction of three nonsynonymous mutations, located in the VP1 and the VP3 genes. Interestingly, one substitution (lysine to methionine at position 259 in the VP1 protein) was introduced into both viral progeny populations after passages in RD cells. Comparative studies of the accumulation of mutations of CVB5-P1anc and 4378fin88, 151rom70, and CVB5Dwt after 10 passages in GMK, HeLa, and RD cells showed that the number of mutations introduced into the ancestral sequence was not higher than that in sequences of contemporary viruses. In conclusion, these results showed that the ancestral P1 region of CVB5-P1anc was tolerated during serial passages in cell culture.

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

Some viruses use multiple cell surface receptors for initial host cell attachment. Previously, it was reported that CVB5 binds to CAR as a primary receptor (6, 60) but that it also uses DAF as a coreceptor (6, 85). The capacity of radiolabeled CVB5 viruses to bind either CAR or DAF alone or the two receptors in combination was assessed by utilizing CHO cell lines transfected with either CAR or DAF and HeLa cells expressing both CAR and DAF. The expression of cell surface receptors was verified by flow cytometry (Fig. (Fig.8).8). At the level of virus binding, no significant interaction between radiolabeled CVB5 variants and CHO cells was detected (Fig. (Fig.99 A). The viruses bound with equal efficiencies to HeLa and CHO-DAF cells, whereas a lower level of binding to CHO-CAR cells was measured. Hence, the expression of CAR on CHO cells resulted in a 10-fold increase in the binding of the CVB5 strains, whereas a significantly higher level of binding was observed in the presence of DAF (450-fold). Overall, these results indicated that CVB5-P1anc, CVB5Dwt, and two clinical CVB5 isolates use both CAR and DAF as cellular attachment molecules.

FIG. 8.
Flow cytometric analysis of CAR and DAF expression on CHO, CHO-CAR, CHO-DAF, and HeLa cells. For the detection of CAR and DAF, monoclonal anti-CAR (RmcB) and anti-DAF (BRIC110) antibodies were used (black histograms). A mouse IgG1 antibody was used as ...
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. ...

We further complemented our comparative analyses of CVB5-P1anc by investigating the plaque morphology and growth properties of the virus in HeLa cells. The plaque phenotype of CVB5-P1anc in HeLa cells was similar to those of plaques formed by CVB5Dwt and the two clinical isolates at 48 h p.i. (Fig. (Fig.9B).9B). The one-step growth curve analysis of the CVB5-P1anc infection in HeLa cells showed that virus production started as early as 4 h p.i. (Fig. (Fig.9C).9C). This initial sign of viral replication was followed by a steep rise in virus titers and a plateau that was reached 8 h after infection. Signs of cytopathogenicity were already observed at 6 h after viral exposure. Furthermore, CVB5-P1anc replicated as efficiently as CVB5Dwt and the two clinical isolates.

Human enteroviruses infect cells mainly of human or other primate origin. Consequently, studies of infectivity in a variety of cell lines were undertaken as an additional approach to analyze the host cell specificity of the CVB5-P1anc phenotype. As shown in Fig. Fig.9D,9D, CVB5-P1anc bound to and replicated efficiently in HeLa, A549, HT29, and GMK cells. This virus replication caused a CPE typical of enteroviruses. However, although CVB5-P1anc caused a productive infection in RD cells, no signs of CPE were detected. In contrast, no active replication or induction of CPE was observed for CHO cells in spite of the detected virus binding to the cell surface. In addition, CVB5Dwt and the two clinical isolates showed a cell tropism corresponding to that of CVB5-P1anc (Table (Table3).3). Conclusively, progeny CVB5 virus production was clearly detectable in all cells assessed except CHO cells.

TABLE 3.
Comparisons of cell tropisms and CVB5 antiserum cross-reactivities between CVB5-P1anc and modern CVB5 isolates

In order to analyze if specific antiserum generated against CVB5F (which is the CVB5 prototype strain and identical to CVB5Dwt except for one amino acid substitution in the VP1 protein) could neutralize CVB5-P1anc, a neutralization assay 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 equally efficient in protecting HeLa cells from infection by CVB5-P1anc (Table (Table3).3). These results suggest that the CVB5-P1anc virion shares neutralizing epitopes with the CVB5F laboratory strain as well as with recently isolated CVB5 viruses. Further studies of CVB5-P1anc serology showed that neither antiserum against CVB1 to CVB4 nor antiserum against CVB6 protected HeLa cells against infection.

Taken together, these results showed that the CVB5-P1anc virus constructed using ML phylogenetic methods displayed characteristics corresponding to those of present-day circulating CVB5 viruses, including properties such as receptor binding, 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 America, 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 separation 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 multifurcating 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.

The SVDV isolates included in the phylogenetic analyses constituted a monophyletic group that was most closely related to CVB5 viruses of cluster II, in agreement with data from a 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 virus and is therefore economically important (27, 54, 67).

The high rate of RNA virus evolution allows a rapid adaptation to new environments (20, 57, 76, 102). Among picornaviruses, it has been shown for poliovirus that every progeny RNA molecule on average contains one mutation (22). By using a relaxed-clock model, the accumulation of mutations in the heterochronous CVB5 sequence data was transformed into an estimated evolutionary rate of approximately 0.004 nucleotide substitutions per site per year, which in turn corresponds to an MRCA dating back to the mid-19th century. This evolutionary rate is within the range estimated for other picornaviruses (11, 29, 108). For example, an evolutionary rate of 0.0038 nucleotide substitutions per site per year was estimated for enterovirus 70 based on VP1 sequence analyses (91). The use of a relaxed-clock model allowed a more confident estimate of divergence times in the face of rate variation among lineages, and it also enabled us to investigate sources of rate variation. For example, a rapid rate of evolution was observed for the branch leading to the most recent SVDV isolates, strongly suggesting adaptation after interspecies transmission.

In studies of picornaviruses, the evolution of virus sequences has been assayed by serial transfers of large viral populations pertubated by bottleneck events (3, 19, 28), while molecular structure-function relationships of viral proteins have been evaluated by site-directed mutagenesis (37, 40, 58, 100, 107). Recent advances in phylogenetics and DNA synthesis techniques enable ancestral sequence reconstruction, providing an important new approach to investigate evolutionary events and the conservation of structural features (96). Although it is impossible to claim that reconstructed ancestral sequences are correct in all details, since computational models simplify biological processes, the resurrection of molecular ancestors with a detectable biological activity, such as enzymes and viruses, offers a possibility to verify their structural integrity. The proper folding of viral capsid proteins, plus intra- and intermolecular interactions, plays a crucial role for the capsid to maintain its multifaceted properties, including structural stability to protect the genome and, at the same time, flexibility to enable uncoating after interactions with host cell receptors (103). Despite the advantage of using virus infectivity to verify the functionality of hypothetical ancestral sequences, few studies of resurrected viral sequences have been presented (21, 51, 66, 81).

In the present study, an ancestral state, i.e., the most likely ancestral CVB5 capsid sequence, was inferred from sequences of contemporary CVB5 isolates by ML ancestral reconstruction (78) and de novo gene synthesis. Although CVB5 most likely exists as a swarm of closely related variants within hosts, similar to poliovirus and foot-and-mouth disease virus (20), this has little impact on our evolutionary reconstruction because it essentially involves an interhost evolutionary process. All variants sampled from a patient at a particular point in time will generally coalesce to a common ancestor prior to the time of infection. Therefore, whatever variants are sampled from the patients, we expect the same common ancestor for viruses obtained from different patients. Inference techniques and evolutionary models may have a more important impact on ancestral reconstructions. We have used likelihood-based codon models, which can accommodate a detailed evolutionary process. For a few highly variable positions, however, this was not always consistent with amino acid reconstructions. Further research that can take into account the uncertainty of reconstructed ancestral sequences is therefore needed.

Characterization of the inferred ancestral P1 sequence showed that capsid proteins expressed from the CVB5-P1anc clone assembled into functional infectious virions. In addition, the recombinant CVB5-P1anc virus shared several features with present-day clinical isolates, including immunogenic epitopes, preferences for the CAR and DAF receptors, cell tropism, plaque morphology, and growth characteristics. So far, no evidence of functional impairments has been observed, suggesting that the proposed ancestral capsid proteins fold into native conformations, which facilitate the assembly of functional virions. Sequence analysis of the P1 region after 10 passages of CVB5-P1anc and two clinical CVB5 isolates in three different cell lines demonstrated that a number of substitutions had been introduced into the VP3 β-B knob region as well as the VP1 C terminus. This is consistent with previous reports describing these regions as neutralizing immunogenic epitopes (9, 48, 68). This analysis also showed that no substitutions were introduced at the “ancestral” amino acid positions in the CVB5-P1anc sequence and, hence, no indication of an evolution toward the sequence of present-day isolates. However, it is important that these 10 passages in cell culture represent a simple model system that is lacking a selective pressure imposed by an immune response, including antibodies. Possibly, the evolution that is observed for CVB5-P1anc has more to do with adaptation to the different cell types used in this experiment. Taken together, the data presented shed light on the properties of current CVB5 isolates but also showed that the likelihood-based phylogenetic method enabled an ancestral reconstruction of the four different structural proteins of CVB5. In future studies of CVB5-P1anc, additional phenotypic properties, including virion stability and pathogenicity in animal models, will be investigated.

The ancestral capsid sequence described is a reflection of eight related CVB5 sequences on which the reconstruction was based. As more CVB5 sequences become available, it will be of interest to compare the ancestral states of these sequences with CVB5-P1anc. Encouraged by the initial characterizations of the CVB5-P1anc virus and with consideration of modern ancestral reconstruction techniques, including methods for inferences of insertion-deletion scenarios (17), the resurrection of the ancestors of more distantly related viruses, such as the ancestor of all CVBs, offers new possibilities in future evolutionary studies and possibly also for the development of new antiviral treatments.

Overall, the present study shows that phylogenetic tools can be used to construct functional ancestral virions as well as provide information on conserved structural features important for the function of the virus. In the future, this type of ancestral reconstruction may also contribute to an increased understanding of the molecular evolution of enteroviruses.

Acknowledgments

We thank J. M. Bergelson for his contribution to the binding studies; K. Edman for valuable discussions; and M. Roivainen, H. Norder, L. Magnius, J. W. McCauley, R. L. Crowell, L. Philipson, and R. Pettersson for providing cells, viruses, and antibodies.

Funding was provided by grants from the Swedish Knowledge Foundation, the Graninge Foundation, the Helge Ax:son Johnsons Foundation, the Sparbanken Kronan Foundation, and the University of Kalmar. S.H. was supported by NIH grant K22 AI-07927. Figure Figure55 was produced by using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH grant P41 RR-01081).

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 July 2010.

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