Pervasive within-host recombination and epistasis as major determinants of the molecular evolution of the foot-and-mouth disease virus capsid.

Author summary There are 7 serotypes of Foot-and-Mouth Disease Virus and multiple strains of each serotype. The emergence of new strains can result in widespread outbreaks of disease and requires new vaccines to be developed. The major mechanisms driving variation are thought to be substitutions in the viral genome. Recombination in the capsid-coding region of the virus genome has been described at phylogenetic scales but not thought to play a major role in generating variants. In the current experiment, a co-infection of African buffaloes with closely related sub-populations of viruses allowed us to detect recombination events. For structural protein-coding sequences, the genetic composition of the population is driven by extensive within-host recombination. During the acute infection phase the intra-host recombination rates of 0.1 per base per year are comparable to the typical mutation rates of the virus. The recombination map reveals two strongly linked regions within the VP1 protein-coding sequence. Epistatic interactions between co-evolved mutations in VP1 are caused by intra-host selection at the RNA and protein level and are present both within and between the two regions. Our findings in this experimental setting support a major role for recombination and epistasis in the intra-host evolution of FMDV. Although recombination is known to occur in foot-and-mouth disease virus (FMDV), it is considered only a minor determinant of virus sequence diversity. Analysis at phylogenetic scales shows inter-serotypic recombination events are rare, whereby recombination occurs almost exclusively in non-structural proteins. In this study we have estimated recombination rates within a natural host in an experimental setting. African buffaloes were inoculated with a SAT-1 FMDV strain containing two major viral sub-populations differing in their capsid sequence. This population structure enabled the detection of extensive within-host recombination in the genomic region coding for structural proteins and allowed recombination rates between the two sub-populations to be estimated. Quite surprisingly, the effective recombination rate in VP1 during the acute infection phase turns out to be about 0.1 per base per year, i.e. comparable to the mutation/substitution rate. Using a high-resolution map of effective within-host recombination in the capsid-coding region, we identified a linkage disequilibrium pattern in VP1 that is consistent with a mosaic structure with two main genetic blocks. Positive epistatic interactions between co-evolved variants appear to be present both within and between blocks. These interactions are due to intra-host selection both at the RNA and protein level. Overall our findings show that during FMDV co-infections by closely related strains, capsid-coding genes recombine within the host at a much higher rate than expected, despite the presence of strong constraints dictated by the capsid structure. Although these intra-host results are not immediately translatable to a phylogenetic setting, recombination and epistasis must play a major and so far underappreciated role in the molecular evolution of the virus at all scales.