The evolution of foot-and-mouth disease virus: Impacts of recombination and selection

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Abstract

Foot-and-mouth disease virus is an economically important animal virus that exhibits extensive genetic and antigenic heterogeneity. To examine the evolutionary forces that have influenced the population dynamics of foot-and-mouth disease virus, individual genes and the coding genomes for the Eurasian (Asia1, A, C, and O) serotypes were examined for phylogenetic relationships, recombination, genetic diversity and selection. Our analyses demonstrate that paraphyletic relationships among serotypes are not as prevalent as previously proposed and suggest that convergent evolution might be obscuring phylogenetic relationships. We provide evidence that identification of recombinant sequences and recombination breakpoint patterns among and within serotypes are heavily dependent on the level of genetic diversity and convergent characters present in a particular data set as well as the methods used to detect recombination. Here, we also investigate the impact of adaptive positive selection on the capsid proteins and the non-structural genes 2B, 2C, 3A, and 3Cpro to identify genome regions involved in genetic diversity and antigenic variation. Two different categories of positive selection at the amino acid level were examined; conservative (stabilizing) selection that maintains particular phenotypic properties of an amino acid residue and radical (destabilizing), and selection that dramatically alters the phenotype and potentially the functional and/or structural features of the protein. Approximately, 29% of residues in the capsid proteins were under positive selection. Of those, 64% were under the influence of destabilizing selection, 80% were under the influence of stabilizing selection, and 44% had phenotypic properties influenced by both selection types. The majority of residues under selection (74%) were located outside of known antigenic sites; suggestive of additional uncharacterized epitopes and genomic regions involved in antigenic drift.

Introduction

Foot-and-mouth disease virus (FMDV) is one of the two species in the genus Aphthovirus which belongs to the viral family Picornaviridae. FMDV is the etiological agent responsible for one of the most economically important and contagious animal diseases that afflicts wild and domesticated cloven-hoofed animals including cattle, sheep, pigs, goats, and water buffalo. The virus is enzootic to all continents except North America and Australia (Grubman and Baxt, 2004). FMDV exhibits extensive antigenic variability and exists as seven immunologically distinct serotypes; four Euroasian serotypes: Asia1, A, C, and O distributed throughout South America, Middle East, Asia and some parts of Africa, and three South African Territories (SAT) serotypes: SAT1, SAT2, and SAT3.

FMDV has a positive-sense, single-stranded RNA genome of ∼8.4 kb. The virus is infective upon entry into the host's cytoplasm where the genome is translated as a single polypeptide which is co- and post-translationally cleaved by viral encoded proteases to produce four structural proteins that form the viral capsid and eight non-structural proteins that control the viral life cycle while within host cells. In addition to FMDV's high infectivity and transmissivity, it has been characterized by high mutation rates 10−3 to 10−5 per replication cycle (Batschelet et al., 1976, Drake and Holland, 1999). As a consequence, populations of FMDV show extensive genetic and antigenic heterogeneity which is reflected in the characterization of greater than 65 strains among the seven serotypes (Domingo et al., 2004, Jenkins et al., 2001, Mateu et al., 1994). Nucleotide substitutions resulting in altered antigenic properties generally occur on the external surface of the viral capsid in regions directly involved in binding antibodies.

FMDV genomes are believed to evolve primarily through genetic drift driven by error-prone replication, recombination, large population sizes, and relatively high numbers of replication cycles per unit time (Boerlijst et al., 1996, Domingo et al., 2004, Jenkins et al., 2001, Miralles et al., 1999). FMDV is known to undergo recombination in vitro and in natural populations (Krebs and Marquardt, 1992, McCahon et al., 1985, Wilson et al., 1988). Detectable recombinants in tissue culture predominantly involve single cross-over events occurring within the non-structural genes (McCahon et al., 1985). Recent bioinformatic studies using whole genomes have depicted recombination hotspots occurring primarily at the boundary between the capsid and non-structural genes (Heath et al., 2006, Jackson et al., 2007, McCahon et al., 1985, Simmonds, 2006). These authors propose that the capsid genes function as a single evolutionary unit that can be exchanged among strains and serotypes during recombination events of co-infected hosts.

Some of the most promising approaches to controlling and eradicating FMDV involve the development of genetically engineered vaccines that lack the capacity to escape into the environment, provide immunity against multiple serotypes, and induce an immune response distinguishable from that produced by exposure to the live virus (Bareling, 2004, Pacheco et al., 2005, Song et al., 2005). Achievement of this goal requires a solid understanding of the evolutionary history of the virus and a detailed characterization of the genetic features (such as the location of epitopes and genetic regions involved in viral attenuation) and processes (recombination and selection) that contribute to the generation and maintenance of genetic diversity. Sequence data are becoming the standard for establishing viral classifications and species concepts (Oberste et al., 2005, Samuel and Knowles, 2001a). However, few phylogenetic hypotheses have been proposed for the Euroasian serotypes (Asia1, A, C, and O) using both individual gene and the coding genome sequences. Merging disciplines into a coherent framework has enabled researchers to develop more realistic experimental models and interpretations of results; which ultimately assists in designing public health policies (Galvani, 2003, Grenfell et al., 2004, Tibayrenc, 2005). To contribute to this integrative experimental approach we explored the molecular epidemiology of FMDV by (1) testing the hypothesis that FMDV serotypes are monophyletic; (2) rigorously testing for recombination using multiple detection programs and assessing the effects of genetic diversity on recombination breakpoint patterns within and among serotypes; and (3) characterizing the population genetic structure and population dynamics of FMDV through estimates of genetic diversity and selection at the protein level.

Section snippets

Data collection and sequence alignment

Polyprotein sequences (excluding non-structural genes 3B1, 3B2, and 3B3) for serotypes O (n = 74), A (n = 47), C (n = 7), Asia1 (n = 10), SAT1 (n = 10), SAT2 (n = 6), SAT3 (n = 5) and Equine Rhinitis A virus (n = 7), the other species in the genus Aphthovirus, were obtained from GenBank and the Picornavirus Home Page (http://www.picornaviridae.com) (Table S1 in supplementary data). To conserve the correct reading frame during the alignment process, each gene sequence was converted into amino acids using the

Phylogenetic analyses

For all gene partitions and data sets, both BI and ML produced similar tree topologies with only minor differences in the placement of terminal taxa outside of well-supported clades. However, the level of phylogenetic clarity differed among gene partitions and data sets. Most of the deeper relationships were unresolved due to lack of character support. However, the majority of clades depicted by the capsid genes VP1–VP3 that had significant support (pP > 0.95; BP > 70) were recovered by the

Phylogenetics

The four data sets examined in this study incorporated varying levels of genetic diversity as the number of taxa increased from 47 individuals in the serotype A data set to 148 in the Aphthovirus data set. There were two interesting findings from contrasting the phylogenetic results among these data sets. The first was incongruence in paraphyletic relationships recovered from different gene partitions and data sets. None of the paraphyletic relationships depicted in the smaller data sets was

Acknowledgements

We thank Duke S. Rogers and two anonymous reviewers who provided helpful comments toward improving this manuscript. Our research was supported by the USDA grant CSREES 2007-01737 to K.A.C. and a Brigham Young University, Graduate Research Fellowship to N.L.R. Data analyses were performed using the College of Life Sciences computational Debian Linux Cluster, Brigham Young University.

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