Evidence for Positive Selection in the Capsid Protein-Coding Region of the Foot-and-Mouth Disease Virus (FMDV) Subjected to Experimental Passage Regimens

Mario Ali Fares*, Andrés Moya*, Cristina Escarmís{dagger}, Eric Baranowski{dagger}, Esteban Domingo{dagger} and Eladio BarrioGo,*

*Institut "Cavanilles" de Biodiversitat i Biología Evolutiva and Departament de Genètica, Universitat de València, Spain; and
{dagger}Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Spain


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We present sequence data from two genomic regions of foot-and-mouth disease virus (FMDV) subjected to several experimental passage regimens. Maximum-likelihood estimates of the nonsynonymous-to-synonymous rate ratio parameter (dN/dS) suggested the action of positive selection on some antigenic sites of the FMDV capsid during some experimental passages. These antigenic sites showed an accumulation of convergent amino acid replacements during massive serial cytolytic passages and also in persistent infections of FMDV in cell culture. This accumulation was most significant at the antigenic site A (the G-H loop of capsid VP1), which includes an Arg-Gly-Asp (RGD) cellular recognition motif. Our analyses also identified a subregion of VP3, part of the fivefold axis of FMDV particles, that also appeared to be subjected to positive selection of amino acid replacements. From these results, we can conclude that under the restrictive conditions imposed either by the presence of the monoclonal antibodies, by the persistent infections, or by the competition processes established between different variants of the viral population, amino acid replacement in some capsid-coding regions can be positively selected toward an increase of those mutants with a higher capability to infect the cell.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Because of the high rate with which RNA viral genes evolve compared with DNA-based organisms, RNA viruses provide an interesting type of replicon with which to study molecular evolution and to test the contribution of neutral drift versus positive selection to their rapid evolution (Kimura 1968, 1983Citation ). The lack of proofreading activity in viral RNA replicases, as evidenced in structural and biochemical studies (reviewed in Domingo and Holland 1997Citation ), provides one of the bases for high mutation rates and, as a consequence, the potential for frequent fixation of mutations and rapid evolutionary rates (Holland et al. 1982Citation ; Domingo and Holland 1994, 1997Citation ). On average, the evolutionary rate of the RNA virus is about three orders of magnitude higher than that of most functional genes from eukaryotic organisms (Moya et al. 2000Citation ).

Foot-and-mouth disease virus (FMDV) is an economically important animal pathogen that belongs to the genus Aphthovirus of the Picornaviridae family (Rueckert 1996Citation ). Its genome consists of a positive single-stranded RNA molecule of 8,500 nt that is translated to give a polyprotein, which is proteolytically processed to yield the mature structural and nonstructural viral proteins (Belsham 1993Citation ; Rueckert 1996Citation ). The coding region can be divided into three distinct regions: P1 (encoding capsid proteins VP4, VP2, VP3, and VP1, in that order), P2 (nonstructural proteins), and P3 (nonstructural proteins including the viral replicase).

In the present work, we studied the evolutionary dynamics of FMDV under different experimental regimes: cytolytic passages involving different viral population sizes, and persistent infections in which both host cells and FMDV survive and coevolve for many generations (de la Torre et al. 1985, 1988Citation ). Since in all cases the starting genome was the same FMDV clone, this experimental design allowed an evaluation of the evolutionary processes acting on this virus under different population dynamics by comparing the patterns of divergence between sequences from different FMDV isolates. This kind of study is of particular interest to unveil (1) the importance of convergent evolution in organisms with high mutation rates and huge population sizes, (2) the adaptive significance of convergent evolution, and (3) the effect of convergent evolution on phylogenetic reconstructions.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Cells, Viruses, and Infections
The experimental protocol described in the following paragraphs is summarized in figure 1 . The initial FMDV clone (C-S8c1) was a representative of the European serotype C, subtype C, which was biologically cloned (three times plaque-purified) in BHK-21 cells (Sobrino et al. 1983Citation ). Likewise, the BHK-21 host cells were derived from a single cell isolated by end-point dilution (de la Torre et al. 1985Citation ). The FMDV C-S8c1 clone was subjected to different experimental passages in BHK-21 cells. If not stated otherwise, cytolytic massive passages involved the infection of 5 x 106 cells at a multiplicity of infection (m.o.i.) of 1. The antibody-resistant clones HR and MARLS were derived from either C-S8c1 or C-S8c1p213 as escape mutants resistant to neutralization by monoclonal antibody (Martínez et al. 1991Citation ; Charpentier et al. 1996Citation ). Monoclonal antibody–resistant mutant HR was subjected to 100 serial cytolytic massive passages to obtain HRp100 (Holguín et al. 1997Citation ). Clones C-S8c1 and C-S8c1p113 (obtained after 113 serial cytolytic massive passages of C-S8c1) were subjected to plaque-to-plaque transfers, giving rise to series C and H, respectively (Escarmís et al. 1996Citation ). P1-P2 and Q1-Q2 were derived from C-S8c1 and C-S8c1p113, respectively, after 30 massive cytolytic passages.



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Fig. 1.—Scheme of the passage history of the foot-and-mouth disease virus isolates and clones analyzed in the present study. Plaque isolations are indicated with filled squares, large population passages with empty circles, and passages of persistently infected cells with diamonds. Each viral genome analyzed evolved from the same C-S8c1 clone (Sobrino et al. 1983Citation ). Throughout the scheme, "p" means passage number. C-S8c1 was used to derive a mutant clone resistant to MAb SD6, termed HR, which was subjected to 100 serial cytolytic passages (Holguín et al. 1997Citation ). C-S8c1 was passaged 213 times and used to derive MARLS, a mutant clone resistant to MAb SD6 (Charpentier et al. 1996Citation ; Baranowski et al. 1998Citation ). C-S8c1 was also used to establish persistently infected BHK-21 cells (de la Torre et al. 1985, 1988Citation ; Díez et al. 1990Citation ). At passage 95, two sublineages of carrier cells were maintained, one up to passage 146, and the other up to passage 100 (Toja, Escarmís, and Domingo 1999Citation ). A cytolytic amplification of the latter was the starting population for cytolytic passage series S and L (Sevilla and Domingo 1996Citation ). In these series, thin and thick arrows indicate that the infection involved 2 x 105 PFUs infecting 5 x 106 cells, and 2 x 108 PFUs infecting 2 x 108 cells per passage, respectively. From populations C-S8c1p2 and C-S8c1p113, two series of plaque-to-plaque transfers (series C and H, respectively) as well as two large population passages (P1, P2, and Q1, Q2, respectively) were carried out as described (Escarmís et al. 1996Citation ). Series C and H clones are denoted with a subscript indicating the transfer number and a superscript corresponding to the clone series. The origin of C-S8c1 and the numbers of cells and virus involved in each lineage are given in Materials and Methods. Further information, as well as the description of the genomic nucleotide sequences for the different clones and populations, is given in the references cited in this legend.

 
A cell line persistently infected with FMDV C-S8c1 was established by growth of BHK-21 cells that survived standard cytolytic infections with this clone (de la Torre et al. 1985, 1988Citation ). Viruses produced endogenously and shed by the persistently infected BHK-21 cells were isolated at different cell passages. These isolates are denoted by "R" followed by the cell passage number (R99, R100, and R146; Toja, Escarmís, and Domingo 1999Citation ). VR100 was derived from R100 by cytolytic amplification from about 103 PFUs to about 108 PFUs (Díez et al. 1990Citation ). Isolates S-1, S-2, S-3, L-1, L-2, and L-3 were derived by subjecting VR100 to 100 cytolytic passages. The first 60 passages were carried out at an m.o.i. of 0.1 (5 x 106 cells infected per passage). Then, each lineage was split into two lineages. In one, serial passages were continued up to passage 100 using the same infection conditions (isolates S1, S2, and S3). In the second, 2 x 108 cells were infected at each passage with an m.o.i. of 1 (isolates L1, L2, and L3)(Sevilla and Domingo 1996Citation ).

cDNA Synthesis, PCR Amplifications, and Nucleotide Sequencing
Viral RNA extraction, reverse transcription for the synthesis of cDNA, PCR amplification, and the subsequent direct sequencing of the amplified product to obtain consensus sequences of the viral isolates or clones were performed as previously described (Escarmís et al. 1996Citation ; Escarmís, Dávila, and Domingo 1999Citation ). The accession numbers of the complete viral genome sequences available are as follows: C-S8c1, AJ133357; Rp99, AJ133358; Rp146, AJ133359; MARLS, AF274010.

Phylogenetic Analysis
Nucleotide sequences were aligned using CLUSTAL X, a Windows version of the CLUSTAL W program (Thompson, Higgins, and Gibson 1994Citation ). Alignments are available from the authors on request.

For phylogenetic inference, we used three different methods: (1) the neighbor-joining (NJ) distance-based method (Saitou and Nei 1987Citation ), (2) maximum-parsimony (MP) analysis (Fitch 1971Citation ), and (3) maximum-likelihood (ML) procedures (Felsenstein 1981Citation ). The MEGA program (Kumar, Tamura, and Nei 1993Citation ) was used to calculate distances and to construct NJ trees with bootstrap support values based on 1,000 replications. MP and ML trees were constructed by using the computer programs DNAPARS and DNAML, respectively, from the PHYLIP package, version 3.5 for Windows (Felsenstein 1993Citation ).

Alternative topologies were viewed with the TreeView program, version 1.5.2 (Page 1992), and compared using the test of MP proposed by Templeton (1983)Citation and developed by Felsenstein (1985)Citation . This test was also performed with the program DNAPARS from the PHYLIP package.

For distance estimation and ML analysis, different models of evolution were compared using conventional likelihood ratio tests (Huelsenbeck and Crandall 1997Citation ). The models tested for a given phylogenetic tree of FMDV sequences were those described by Jukes and Cantor (1969)Citation , Kimura (1980)Citation , Felsenstein (1981, 1984Citation [described in Felsenstein 1993Citation ]), Hasegawa, Kishino, and Yano (1985)Citation , Tamura and Nei (1993)Citation , and Yang (1994)Citation . In addition, models assuming equal substitution rates among sites (Poisson distribution) or heterogeneous substitution rates (the {Gamma}-distributed rates model) were also compared using likelihood ratio tests. The nucleotide changes experienced by the capsid-coding region were better explained by Tamura and Nei's (1993)Citation model with a gamma distribution of substitution rates across sites ({alpha} = 0.02). the model that best accounted for the evolution of the L protein region was Kimura's (1980)Citation two-parameter model with a Poisson distribution. Log likelihood values were obtained using the BASEML program of the PAML, version 3.0 package (Yang 1997Citation ).

Detecting Positive Selection
Different codon-based models for the evolution of protein-coding sequences that allow for variable selection intensity among sites (Nielsen and Yang 1998Citation ; Yang et al. 2000Citation ) were also tested with the use of conventional likelihood ratio tests. In these models, the nonsynonymous/synonymous substitution rate ratio ({omega} = dN/dS), which reflects the selection intensity at the amino acid level, was allowed to vary among amino acid sites.

The 13 different models tested were those described in Nielsen and Yang (1998)Citation and Yang et al. (2000)Citation . Several models assumed discrete distributions to model heterogeneous {omega} among sites. These models were the "neutral" model (M1 according to Yang et al. 2000Citation ), with two categories of {omega} ratio sites ({omega} = 0 and {omega} = 1); the "selection" model (M2), including a third class of codons with the underlying {omega} ratio estimated from the data; the "discrete" model (M3), with an unconstrained discrete distribution of K {omega} ratios; and the "freqs" model (M4), with five classes of codons with prespecified values of {omega}.

Other models assumed continuous distributions for heterogeneous {omega} ratios among sites, although they used a discrete distribution of 10 categories with the same probabilities as an approximation in the likelihood calculation. These models were the "gamma" model (M5), assuming a simple gamma distribution; the "double gamma" model, which used a mixture of two gamma distributions; and the "beta" model (M7), which did not allow for positive selected sites ({omega} > 1). The following models (M8–M11) accounted for positively selected sites: the "beta & {omega}" model (M8); the "beta & gamma" model (M9), the "beta & gamma + 1" model (M10), with a gamma distribution shifted to the right by one unit, accounting for positively selected sites ({omega} > 1); the "beta & normal > 1" model (M11), with the distributions left-truncated by 1; the "0 & 2 normal > 0" model (M12), which assumed a proportion of sites with {omega} = 0 and a proportion from a mixture of two normal distributions truncated at {omega} = 0; and the "3 normal > 0" model (M13), which used a mixture of three normal distributions truncated at {omega} = 0. A more detailed description of the models is given in the original references.

These models, implemented in the CODEML program of the PAML, version 3.0, package (Yang 1997Citation ), are very useful for testing adaptive molecular evolution and also for identifying amino acid sites under positive selection by an empirical Bayes approach (Nielsen and Yang 1998Citation ).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Phylogenetic Reconstructions and Convergent Evolution
Two regions of the FMDV genome were analyzed in the present study. The first one encompassed a 2,142-nt segment coding for the four capsid proteins: VP4 (partial sequence lacking the first 51 nt, positions 52–255 according to the nucleotide numbering of Díez et al. [1990Citation ]), VP2 (positions 256–909), VP3 (positions 910–1515), and VP1 (positions 1516–2193). The second region corresponded to 603 nt coding for the L protein. The nucleotide sequences analyzed for each viral lineage amounted to one third of the entire FMDV genome.

Sequences of the capsid region were obtained by consensus sequencing (i.e., direct sequencing of PCR fragments amplified by RT-PCR from a heterogeneous population of viruses) for each of the 38 FMDV isolates indicated in bold in the scheme presented in figure 1 . A total of 31 of the 38 sequences analyzed were different and yielded 2,142 nucleotide positions in the alignment because there were no indels. In the alignment, 78 sites were variable (3.6%). Of the inferred amino acid positions, 35 were variable (out of 714). There was a very slight predominance of variable sites exhibiting nonsynonymous changes (40 nonsynonymous vs. 38 synonymous).

With respect to the L protein region, sequences also obtained by consensus sequencing were available for the same isolates except for HR and HRp100. Of the 36 L-protein sequences, 22 were different and yielded a 603-nt alignment with no gaps. This set of aligned sequences contained 34 variable sites (5.6%). Of the inferred amino acid positions, 33 were variable (out of 201). In this case, there was a clear predominance of variable nucleotide sites exhibiting nonsynonymous changes (29) over synonymous changes (5).

Phylogenetic analyses of the capsid sequences with three methods (MP, NJ, and ML) gave similar topologies, but relationships differed from those of the actual experimental evolutionary history of the FMDV isolates. The MP method gave eight equally parsimonious trees (but only four alternative topologies) requiring 86 changes. These trees differed in the alternative positions of three pairs of ambiguous changes due to convergent mutations (we use the term "convergence" as including both parallel and reverse changes according to Doolittle [1994Citation ]) that could be interpreted either as two parallel substitutions or as a forward substitution and its subsequent reversion or back mutation. Table 1 describes convergent changes, as well as adjacent (taking place in neighbor positions of the same codon) and multiple-hit changes, which occurred during the evolution of FMDV capsid region sequences according to the MP phylogenetic reconstructions. Of these changes, 14 correspond to nonsynonymous changes, and only 1 corresponds to a synonymous substitution.


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Table 1 Convergent, Multiple-Hit, and Adjacent (occurring in neighbor positions of the same codon) Substitutions as Deduced from the Maximum-parsimony Phylogenetic Reconstructions of the Capsid and Protein L Regions of the FMDV Genome (figs. 2 and 3, respectively)

 
Figure 2 shows the MP trees with the three convergences (sites 1703, 2003, and 2147) treated either as parallel changes (fig. 2A ) or as reversions (fig. 2B ); different combinations of parallel changes and reversions gave the other six MP trees, which correspond to a total of four different topologies.



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Fig. 2.—Two of the eight equally parsimonious trees obtained for the foot-and-mouth disease virus capsid region sequences, which correspond to four alternative topologies. These eight different MP trees were produced by the presence of three ambiguous convergences (sites 1703, 2003, and 2147; described in table 1 ) that can be treated either as parallel changes (A) or as reversions (B). The possible combinations of parallel changes and reversions gave the other six MP trees. The numbers of nonsynonymous (followed by "N") or synonymous (followed by "S") substitutions required to connect the different sequences are indicated on the branches. Lettering identifies those branches where convergent, multiple-hit, and adjacent (taking place in neighbor positions of the same codon) nonsynonymous substitutions occurred (described in table 1 )

 
The topology of the ML tree (not shown) was identical to that of one of the MP trees, and that of the NJ tree was similar to those obtained by the other two methods, but the relationships among isolates R99, R146, and VR100 were less congruent with the experimental history of these isolates (results not shown).

The topologies obtained with the three methods of phylogenetic inference (MP, ML, and NJ) corresponded to a similar phylogenetic reconstruction. In fact, the parsimony-based method of Templeton (1983)Citation (modified by Felsenstein 1985Citation ), used to test if alternative topologies were significantly better or worse, indicated that the five topologies were not significantly different (results not shown). However, the same test suggested that the tree corresponding to the experimental history of the isolates required significantly more nucleotide changes to explain the evolution of the capsid sequences than the trees obtained with the different methods of phylogenetic reconstruction.

The application of the parsimony method to the L-coding region gave a unique MP tree of length 36 (fig. 3 ). Convergent, adjacent, and multiple-hit changes, accumulated during the evolution of FMDV L-protein sequences according to the MP phylogenetic reconstructions, are also given in table 1 , and all correspond to nonsynonymous changes. As in the case of the capsid-coding region, the ML tree (not shown) had the same topology as the MP tree, and the NJ method gave a slightly different tree (results not shown). The Templeton (1983)Citation test indicated that the topologies of the ML and MP trees were better than that of the distance-based tree, but the difference was not significant. Again, the trees obtained with the different methods of phylogenetic reconstruction were significantly better for explaining the evolution of the sequences of the L-protein region than was the tree corresponding to the experimental history of the isolates.



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Fig. 3.—Maximum-parsimony tree of the foot-and-mouth disease virus protein L sequences. The numbers of nonsynonymous (followed by "N") or synonymous (followed by "S") nucleotide substitutions required to connect the different sequences are indicated on the branches. Lettering identifies those branches where convergent, multiple-hit, and adjacent (taking place in neighbor positions of the same codon) nonsynonymous substitutions occurred. These nonsynonymous changes are described in table 1

 
When the nonsynonymous nucleotide substitutions were located at the genome positions where they occurred, a particular distribution was observed (not shown). Nonsynonymous nucleotide substitutions were more or less randomly distributed along the four capsid proteins in series C and H, with no more than two to three mutations located in an interval of less than 10 codon positions. However, in the persistence lineage, nonsynonymous substitutions mainly concentrated within two narrow regions, with nine (out of 24) nonsynonymous substitutions occurring within an interval of eight codons located at the amino terminal end of VP3, and the eight other substitutions occurring in a nine-codon interval located within antigenic site A of capsid protein VP1.

With respect to L protein, convergent changes occurred only twice in the persistence lineage and corresponded to nonsynonymous substitutions; the other nonsynonymous changes were single events per codon, and there was no concentration of nonsynonymous changes in adjacent codons.

Tests of Positive Selection and Identification of Positively Selected Sites
To determine the possible action of positive selection on subregions of the FMDV capsid and L-protein regions, different codon-based models for the evolution of protein-coding sequences that allowed for variable selection intensity among sites (Nielsen and Yang 1998Citation ; Yang et al. 2000Citation ) were compared with the use of likelihood ratio tests.

The log likelihood values obtained from the phylogenetic analysis of the two FMDV protein-coding regions under different models (Nielsen and Yang 1998Citation ; Yang et al. 2000Citation ) are listed in tables 2 (capsid region) and 3 (L protein). When two models are nested, they can be compared using the likelihood ratio test; twice the log likelihood difference follows a {chi}2 distribution, with the number of degrees of freedom being the difference in the numbers of free parameters between the two models. However, nonnested models can also be compared using the Akaike Information Criterion (AIC; Akaike 1974Citation ): AIC = -2 (estimated log likelihood of the model) + 2 (number of free parameters of the model), which is also given in table 2 . A model that minimizes the AIC is considered the most appropriate model.


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Table 2 Parameter Estimates and Log Likelihood Values Under Different Models of Evolution for the FMDV Capsid Genome Region According to the Maximum-Likelihood (ML) Phylogenetic Reconstruction

 
For the capsid region (table 2 ), the average {omega} ranged from 0.49 to 0.54 among all models except for M1 (neutral) and M7 (beta), which did not allow for positively selected sites, fitted the data badly, and gave smaller estimates of {omega} (0.31 and 0.25, respectively). The average {omega} was <1, indicating that, on average, purifying selection dominates the evolution of this genome region. The neutral (M1) model (Nielsen and Yang 1998Citation ) and the Goldman and Yang (1994)Citation model (M0) were not nested and therefore could not be tested using the likelihood ratio test. Nevertheless, as both models had the same number of parameters, their log likelihood values were comparable. For the capsid region, the log likelihood value under the neutral model was higher than that under the Goldman and Yang model by 11.1 units, indicating that the neutral model is a much more realistic representation of the evolution of the FMDV capsid region than the Goldman and Yang model. The one-ratio model (M0) had the highest AIC values and was easily rejected when compared with all other models, which allowed the {omega} ratio to vary among sites.

Models that allowed for positively selected sites (M2, M3, M5, M6, and M8–M13), all suggested the presence of positively selected sites. For example, the selection model (M2) suggested ~1.3% of sites under strong positive selection, with {omega}2 = 10. Model M3 (discrete) suggested an important fraction of sites (4.2%) under strong diversifying selection, with {omega}2 = 10.45. Both models had significantly higher likelihood values than models M0 and M1, providing evidence for adaptive evolution. The likelihood ratio test statistics for the comparison of M2 (selection) to M0 (one-ratio) and M1 (neutral) were 48.0 (P = 3.8 x 10-11, df = 2) and 25.8 (P = 2.5 x 10-6, df = 2), respectively. The likelihood ratio test statistics for the comparison of M3 (discrete) to M0 (one-ratio) and M1 (neutral) were 48.6 (P = 6.9 x 10-10, df = 4) and 26.5 (P = 2.6 x 10-5, df = 4), respectively. Similarly, M8 (beta & {omega}) suggested ~1.4% of sites under very strong positive selection, with {omega}2 = 17.2. The likelihood ratio test statistic for the comparison of M7 (beta) and M8 (beta & {omega}) was 24.7 (P = 4.3 x 10-6, df = 2). No other continuous models involving a beta distribution (M9–M11) fitted the data significantly better than M7; however, these models (with the exception of M10), together with models M5 (gamma) and M6 (double gamma), did not allow for positively selected sites.

Because positive-selection models (M2, M3, and M8) provided a better explanation of the evolution of the capsid sequences than neutral models (M1 and M7), an identification of individual positively selected amino acid positions was performed by the empirical Bayesian approach (Nielsen and Yang 1998Citation ). According to this approach, the category (with the associated dN/dS ratio) that maximizes the posterior probability is the most likely category for the amino acid site. The posterior probabilities provide a measure of the accuracy of that inference.

Posterior probability distributions for sites along the FMDV capsid region were plotted for the three best fitting models, M2 (selection), M3 (discrete), and M8 (beta & {omega}), as shown in figure 4 . At the 99% level, model M2 identified three positively selected codons: 277, 310, and 568. At the same level, model M3 identified codons 310, 312, and 568. At the 95% level, both models also identified codons 666 and 670. Amino acid 277 was placed within the VP2 protein, sites 310 and 312 were located in the N-terminal region of the VP3 protein, amino acid 568 corresponded to the central region of the VP1 protein, and sites 666 and 670 were located within antigenic site A of the VP1 protein, the first one involved in the generation of the Mab escape mutant MARLS. Codons with a posterior probability smaller than 95% but larger than 90% according to model M3 were 668 (P = 0.949), also located within antigenic site A of the VP1 protein and involved in the generation of the Mab escape mutant HR, and 716 (P = 0.936), the single codon located between antigenic sites D1 and C. Model M8 (beta & {omega}) identified fewer sites because the parameter estimates under this model suggested a smaller proportion of very strongly selected sites ({omega} = 17.2). Other models including components specifically designed to allow for positively selected sites identified all codons exhibiting nonsynonymous substitutions as being under positive selection ({omega} = 1.5 for models M5, M9, and M11; {omega} = 3 for model M4; and {omega} = 4.0–4.2 for models M12 and M13, respectively) with a posterior probability of >0.99, except M6, which identified the same eight codons as model M3 as being under positive selection ({omega} = 3.8) with a posterior probability of >99%.



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Fig. 4.—Locations of positively selected codons within the capsid protein region of foot-and-mouth disease virus according to models M2, M3, and M8. Codons included with a higher probability in the positive-selection category according to an empirical Bayesian approach are plotted against their corresponding posterior probabilities. The discontinuous line shows the 95% level of significance

 
For the L-protein region (table 3 ), the best model according to the AIC was the one-ratio Goldman and Yang model (M0). This model also was the best-fitting model when compared with any of the more general discrete models that allowed for variable ratios among sites according to the likelihood ratio test. In contrast, continuous {omega} distribution models suggested the presence of positively selected sites in the L protein. The gamma (M5) and double gamma (M6) models had categories with {omega} > 1 (6.6 in both cases) when discretized. Similarly, M8 (beta & {omega}) also suggested that a small fraction of sites (0.7%) were under very strong positive selection, with {omega} = 18.4. The likelihood ratio statistic for the comparison of M7 (beta) and M8 (beta & {omega}) was 10.7 (P = 4.8 x 10-3, df = 2). Other continuous models, such as M9 (beta & gamma + 1) and M11 (beta & normal > 1) were also significantly better than M7 ({chi}2 = 3.86, P = 0.15, df = 2). In summary, several continuous models provided for the existence of a small fraction of positively selected sites in this FMDV L-protein region, which was not detected according to the discrete models.


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Table 3 Parameter Estimates and Log Likelihood Values Under Different Models of Evolution for the FMDV L-Protein Region According to the Maximum-Likelihood (ML) Phylogenetic reconstruction

 
The identification of positively selected amino acid positions according to the different models was obtained by the empirical Bayesian approach proposed by Nielsen and Yang (1998)Citation . Models M5, M6, M9, and M11–M13 identified five codons (10, 18, 23, 162, and 190) as being under positive selection ({omega} = 6.6) with a posterior probability of >0.98. Model M8 identified only codon 23 as being under very strong positive selection ({omega} = 18.4) with a posterior probability of 0.993.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The Effect of Convergent Evolution on Phylogenetic Reconstruction
A phylogenetic analysis of FMDV sequences with a known evolutionary history was performed, and the phylogenetic reconstructions obtained appeared to be incongruent with the experimental history of the clones and populations analyzed. In some cases, discrepancies can be ascribed to the presence of polymorphism in the viral isolates. For example, each series H clone directly derives from the isolate C-S8c1p113 according to the experimental procedure, but some of them share common ancestors different from the C-S8c1p113 sequence, according to the phylogenetic reconstructions based on either the capsid region or the L-protein. As these sequences were obtained by "consensus" sequencing of the viral population present in the different isolates, these phylogenetic discrepancies can be explained by the coexistence of several variants in C-S8c1p113 that became fixed by chance in the different plaque-to-plaque transfers.

In other cases, discrepancies can only be attributed to convergent evolution. Thus, for both gene regions, series L sequences are closely related and series S sequences are identical according to the reconstructed trees, but, according to the experimental procedure, S-1 and L-1 share a common experimental history, and the same goes for S-2 and L-2 and for S-3 and L-3. These discrepancies can be explained either by convergent nucleotide changes or by the presence of several variants in the parental VR100 population, with one variant becoming more frequent under one of the infection conditions (series S sequence) and the other becoming more frequent under the new infection conditions (the ancestor of sequences L-1, L-2, and L-3). Two of the most relevant differences between series L and series S sequences are the two nonsynonymous substitutions located in the antigenic A region of VP1 involving two adjacent amino acid replacements (codons 666 and 667) from Leu-Ala to Val-Pro in series L sequences. A sequence analysis of this region from isolates at intermediate passages was performed by Sevilla and Domingo (1996)Citation . In their sequencing gels, bands diagnostic of the two changes were not visible at passages 60–80 and increased from passage 82 to passage 100. An analysis of individual biological clones derived from L-1 at different intermediate passages showed the presence of the intermediate form Leu-Pro and confirmed the progressive increase of the frequency of the variant Val-Pro (0%; 11%, 68%, and 100% at passages 80, 85, 90, and 100, respectively). The results are compatible with both alternative hypotheses, but if several variants were present in the parental VR100 isolate, the derived variants had to be present at very low frequencies and increase in a parallel fashion in the three series L.

Finally, in other examples, the wrong interpretation of convergent nucleotide changes as homologies in the phylogenetic reconstructions is the only explanation. Thus, isolate HR100 was derived from the antibody-resistant clone HR but appears in the MP trees clustered with the persistence lineage. This HR100 sequence shares only one single nucleotide substitution with HR, causing the replacement of His with Arg in the antigenic A region of the VP1 capsid protein selected by the antibody SD6. However, HR100 shares four nonsynonymous substitutions with the sequences of the isolates from the persistence lineage, which explains why they are clustered together in the phylogenetic reconstructions. In this case, ancestral polymorphism and differential sorting of variants cannot be proposed as an alternative hypothesis to the convergent nucleotide changes to explain the incongruence between the experimental tree and the phylogenetic reconstructions, since HR is not a heterogeneous isolate, but a clone.

Eleven other convergences (parallel or reverse changes) are also required to explain the evolution of the persistence lineage, and these were correctly interpreted as homoplasic changes in the phylogenetic reconstructions.

Adaptive Significance of Convergence
Evidence demonstrating that convergent changes play an important evolutionary role has been obtained from both natural populations (Weisblum 1995Citation ; Crandall et al. 1999Citation ) and experimental systems (Bull et al. 1997Citation ; Cunningham et al. 1997Citation ; Wichman et al. 1999Citation ; Crill, Wichman, and Bull 2000Citation ). Also, phylogenetic reconstructions based on molecular data always reveal homoplasy, indicating that a certain level of convergence is common, if not universal (Sanderson and Donoghue 1996Citation ; Wells 1996Citation ). Although a certain amount of convergence is expected even under neutral random substitutions, the rate of convergence is higher when strong mass selection is operating on very large populations, e.g., drug resistance in bacteria (Weisblum 1995Citation ) or viruses (Crandall et al. 1999Citation ) or viral adaptation to different hosts (Bull et al. 1997Citation ; Cunningham et al. 1997Citation ; Wichman et al. 1999Citation ; Crill, Wichman, and Bull 2000Citation ).

In the case of the FMDV capsid region, convergent changes (all but one nonsynonymous) occurred in short sequence stretches coding for antigenic sites of the capsid proteins and are involved in the generation of MAb escape mutants during the evolution of the persistence lineage or after the subsequent recovery of the cytolytic capability. Under the restrictive conditions imposed either by the presence of the SD6 monoclonal antibody, by the persistent infections, or by the competition processes established between different variants of the viral population, amino acid replacement in the capsid-coding region can therefore be positively selected and thus increase fitness. These processes imply viral adaptation to changing conditions that results in the generation of C-S8c1 derivates with increased virulence (de la Torre et al. 1988Citation ; Sevilla and Domingo 1996Citation ; Baranowski et al. 1998Citation ).

Adaptive Evolution in FMDV
Rapid divergence driven by positive selection has rarely been demonstrated at the molecular level, since it can be confounded with variation at RNA and protein sites that tolerate genetic drift. However, the convergent evolution of the FMDV lineages may imply adaptive change. In this study, adaptive molecular evolution in FMDV has been demonstrated by the application of ML methods that allow for variable intensity of selection pressure among codons. Codons of the FMDV genome undergoing positive selection correspond to those where convergent and adjacent nonsynonymous substitutions accumulate (table 1 and fig. 4 ), which supports the adaptive value of such convergences.

The application of the likelihood ratio tests to determine the evolutionary dynamics of the capsid-coding region strongly suggests that positive selection is operating on the antigenic A region located in the VP1 capsid protein. Several other codons under positive selection (666, 668, and 670) are located within the region coding for this antigenic site at the G-H loop of the VP1 capsid protein. Previous studies have shown that large population passages may produce FMDV variants with unique amino acid substitutions in this antigenic A region of the VP1 protein and drastic alterations in antigenic specificity even in the absence of immune selection (Díez et al. 1990Citation ; Sevilla, Verdaguer, and Domingo 1996Citation ; Holguín et al. 1997Citation ). In fact, evidence of selection acting on this antigenic A region can be deduced from the study by Holguín et al. (1997)Citation , where the nonsynonymous/synonymous mutant frequencies ratios in the absence of immune selection are ~1 and 1 for antigenic sites C and D, respectively, but 16.9 for site A.

Moreover, the same Thr-to-Lys-or-Ala replacements in amino acid position 670 (148 in VP1) have been found in an independent number of passage series of FMDV C-S8c1 or derivatives (Díez et al. 1990Citation ; Borrego et al. 1993Citation ; Holguín et al. 1997Citation ), including its fixation in the process of fitness gain of a weakened FMDV clone (Escarmís, Dávila, and Domingo 1999Citation ). Something similar happens with position 666 (144 in VP1): Leu is replaced by Ser in MAR mutants derived both from C-S8c1p100 (Martinez et al. 1997Citation ) and from C-S8c1p213 (virus MARLS), or by a Val in MAR mutants derived from C-S8c1p100 (Martinez et al. 1997Citation ), and in the L series isolates.

These results are not unexpected, because capsid proteins establish contacts with the host cell surface as the first step in the intracellular replication of FMDV, especially the highly conserved Arg-Gly-Asp triplet of VP1 (positions 663–665), which mediates cell adhesion by being the major determinant in the interaction of protein ligands with cell surface receptors of the integrin superfamily (Mateu et al. 1996Citation ) not only in FMDV, but also in other viral pathogens. In FMDV, it has further been demonstrated that the contiguous residues 666–668, on which selection is acting according to the present study, are also involved in cell recognition (Mateu et al. 1996Citation ).

In addition, it has recently been shown that FMDV harbors the potential for adaptation to multiple cellular receptors, including heparan sulfate and several integrin molecules (Jackson et al. 1996, 2000a, 2000bCitation ). In the case of FMDV C-S8c1, changes in receptor specificity have been associated with various capsid surface alterations (Baranowski et al. 1998, 2000Citation ), such as that at position 666 (144 in VP1), where positive selection has been demonstrated.

In this study, we also observed that positive selection was operating on codons located in other capsid regions, such as sites 277 (located in VP2), 310, 312 (located at the 5' end of the VP3 region), and 568 (located in VP1). The antigenic importance of these regions has not been demonstrated. However, site 568 is located in a region, called antigenic site 3, that has been demonstrated to be antigenic in FMDV type O. In addition, Escarmís et al. (1998)Citation selected variants of FMDV C3Arg/85 (a type C clone isolated in Argentina) in a single infection of partially resistant BHK-21 cells (obtained after persistent infection with C-S8c1 and subsequent elimination of virus from the cells with ribavirin) and found that the selected variants (C3-Rb) showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect other cells (Chinese hamster ovary cells). The comparison of the genomic sequences of the parental (C3Arg/85) and the modified (C3-Rb) clones revealed only two amino acid differences, the fixed Asp-9 to Ala in VP3 (codon 312 in the numbering used in the present study), and either His-108 to Arg or Gly-110 to Arg in VP1 (codons 613 and 615). The same phenotypic and genotypic modifications occurred in a number of independent infections with C3Arg/85 or with clones derived from it. Of the two amino acid differences observed, the constant Asp to Ala in VP3 involved the same nonsynonymous substitution occurring in the persistence lineage analyzed in the present study (table 1 , codon 312). This convergence proves that adaptation to the same cells promotes the selection of virus variants showing the same alterations in cell tropism.

Finally, evidence of positive selection acting on the FMDV genome was obtained from the statistical analysis of the ratio between nonsynonymous and synonymous substitution rates in coding regions. However, an excess of nonsynonymous substitutions is an indicator of positive selection acting at the amino acid level, but genomic RNA is involved in many RNA-RNA and RNA-protein interactions that affect viral replication. This is obvious for noncoding, regulatory regions, but there is increasing evidence that capsid-coding regions in picornaviruses may also have an effect on viral replication (McKnight and Lemon 1998Citation ). Therefore, the RNA itself (apart from its protein-coding capacity) may contribute to the viral phenotype, and fitness may also be affected by synonymous replacements not detected according to this kind of analysis.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
This work was supported by grants PM97-0060-C02-01 to E.D. and PM97-0060-C02-02 to A.M. from the Spanish DGESIC-MEC. M.A.F. acknowledges a fellowship from Conselleria de Cultura, Educació i Ciència, Generalitat Valenciana. Computer-based analyses were carried out using the Servei de Bioinformàtica, University of Valencia, Spain.


    Footnotes
 
Edward Holmes, Reviewing Editor

1 Abbreviations: FMDV, foot-and-mouth disease virus; MAb, monoclonal antibody; m.o.i., multiplicity of infection; nt, nucleotide(s); PCR, polymerase chain reaction; PFU, plaque-forming unit. Back

2 Keywords: foot-and-mouth disease virus experimental phylogeny convergent evolution parallel evolution positive selection Back

3 Address for correspondence and reprints: Eladio Barrio, Institut "Cavanilles" de Biodiversitat i Biologia Evolutiva, Universitat de València, Edifici d'Instituts del Campus de Paterna. P.O. Box 2085, E-46071 València, Spain. E-mail: eladio.barrio{at}uv.es Back


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Accepted for publication September 5, 2000.