The major antigenic determinant of FMDV is 1D. Since the serotypic relatedness of different isolates is reflected in the phylogeny of the 1D gene region, it has traditionally been used in the phylogenetic analysis of FMDV sequences. However, radically incongruent tree topologies between the structural and nonstructural coding regions of FMDV isolates has suggested that the 1D phylogeny may not appropriately reflect the evolutionary histories of different FMDV isolates (7
). To address this issue, we analyzed the phylogenetic compatibility of successively generated sequence fragments across the complete genome sequences of 125 FMDV isolates. Phylogenetic-compatibility analysis indicated extensive incongruence between different regions of the genome (Fig. ). Whereas the phylogenies of the 1A, 1B, and 1C gene regions are consistent with that of 1D, the phylogeny of the remainder of the genome is largely irreconcilable with that of the P1 region. Phylogenetic-incompatibility values among different parts of the 3′ portion of the genome are only slightly higher than between different regions of P1, but the phylogeny of the 3′ half of the genome is also substantially incompatible with those of the 5′ nontranslated region and the L gene. The incongruence of different parts of the FMDV genome suggests that intertypic recombination may have played an important role in the evolution of FMDV.
FIG. 1. (a) Phylogenetic compatibility matrix of 125 FMDVs. To generate this plot, a 300-nucleotide window was moved across the alignment with 100-nucleotide steps, and at each position, a neighbor-joining tree was constructed. The compatibility of two trees (more ...)
To detect evidence of individual recombination events within the FMDV alignment, it was examined using a set of six recombination detection methods implemented in RDP3 (19
). RDP3 uses a mixture of statistical and phylogenetic methods to both identify evidence of probable recombination events within individual sequences and identify a minimal subset of unique events detectable within an entire alignment. Importantly, the program specifically avoids overestimating the amount of recombination in an alignment by identifying multiple descendants of individual recombination events. Eighty-six unique PREs among FMDV isolates were detected in this way (http://darwin.uvigo.es/rdp/heath2006.zip
). Included in these 86 PREs are all 8 that have been previously described (2
). Twenty-six of the 38 PREs for which two sequences closely related to the presumed recombinant's parental sequences were identified are between members of different serotypes. This implies that gene flow among serotypes is relatively common and that it does not necessarily incur severe fitness costs. Importantly, the results of this analysis are in close agreement with the phylogenetic-compatibility analysis in that the distribution of observed breakpoints appears to be nonrandom (Fig. ). Whereas there are locally significant breakpoint cold spots in the 1B, 1C, and 1D genes of the P1 region (Fig. ), the entire P1 region is bounded by two globally significant (P
< 0.01) breakpoint hot spots.
The results of these FMDV breakpoint distribution and phylogenetic-compatibility analyses mirror those recently reported for the enteroviruses, another picornavirus genus. Phylogenies of the structural and nonstructural genes of enteroviruses are apparently also largely incompatible with one another (14
). This suggested to us that a common mechanism may be influencing intertype recombination occurring among both aphthoviruses and enteroviruses and that evidence of the same mechanism might also be detectable in other picornavirus genera. To test this hypothesis, complete genome sequences of species A, B, and C enteroviruses (29
= 28, 61, and 51, respectively) and teschoviruses (27
= 29) were subjected to the same set of analyses as that used on the FMDV alignment. There are insufficient full genome sequences available for members of the other picornavirus genera for analysis of these to have yielded any meaningful results. We detected evidence of 32, 119, 58, and 46 independent PREs in the enterovirus species A, B, and C and teschovirus alignments, respectively (http://darwin.uvigo.es/rdp/heath2006.zip
Consistent with previous reports, significant recombination hot spots were detected at the boundaries of the P1 regions of all three enterovirus species (24
). These recombination hot spots are most clearly defined in the poliovirus and nonpoliovirus species C enteroviruses (Fig. ), for which two globally significant breakpoint hot spots are evident at or close to the boundaries of the P1 region. For the species A and B enteroviruses, globally significant hot spots are located close to the 5′ boundary of the P1 region (Fig. ). Athough there are breakpoint clusters close to the 3′ P1 boundary in both species A and B enterovirus genomes, the significance of these clusters is not statistically supported. A locally significant breakpoint hot spot is, however, apparent in both the enterovirus A and B genomes at the boundary between the 2A and 2B ORFs (Fig. ). It is perhaps also noteworthy that a nonsignificant breakpoint cluster between the 2A and 2B ORFs is also evident in the enterovirus C genomes (Fig. ). The globally significant (P
= 0.01) breakpoint hot spots at or close to the P1 boundaries in the teschoviruses closely resemble those observed in the FMDV and enterovirus C genomes (Fig. ). As with FMDV, the teschovirus 5′ untranslated region also contains a locally significant breakpoint cluster. The other locally significant teschovirus breakpoint cluster at the 2C-3AB ORF interface also seems to correspond with breakpoint clusters at or near the 2C-3AB ORF interface in the A, B, and C enteroviruses, with the clusters in enterovirus Bs being locally significant. Importantly, all of the recombination hot spots identified by the breakpoint-clustering analyses coincide very closely with the boundaries of genomic regions with maximum phylogenetic conflict indicated by the phylogenetic-compatibility analyses.
FIG. 2. Phylogenetic-compatibility matrices and breakpoint density plots of enteroviruses and teschoviruses constructed as outlined for Fig. . (a) Enterovirus species A. (b) Enterovirus species B. (c) Enterovirus species C. (d) Teschoviruses. The (more ...)
Our study supports recent proposals that the constant generation of variants possessing different combinations of structural and nonstructural proteins is an important feature of enterovirus evolution (13
). Moreover, our work suggests that this reassortment of structural- and nonstructural-protein coding regions has played a similarly significant role in the evolution of other picornavirus genera. Recombination in picornaviruses may be analogous to component swapping or pseudorecombination that occurs among viruses with multipartite genomes. Such “pseudopseudorecombination” may be strongly facilitated by the partitioning of structural and nonstructural genes that is common in picornavirus genomes. Clustering of structural-protein genes, for example, will massively increase the probability that single recombination events will transfer them as an intact genome module.
Although it is likely that there is a common mechanism influencing the recombination patterns we have observed in FMDV and other picornaviruses, it is not immediately obvious what that mechanism might be. The recombination hot spots described here could be facilitated by conserved biochemical and/or folded nucleotide structural features at, for example, the interfaces between the structural- and nonstructural-protein coding sequences. There does not, however, appear to be any correlation between the positions of enterovirus genomic-RNA secondary structures and favored recombination breakpoint positions detected in these viruses (29
It is also possible that recombination occurs throughout these genomes at roughly the same rate but that recombinants containing breakpoints outside certain well-defined genome regions (such as at the interfaces between the structural and nonstructural genes) are generally less fit than parental viruses. Evaluation of the replicative abilities of recombinant FMDV and poliovirus genomes provides some experimental support for the notion that many newly formed recombinant picornaviruses, if not the vast majority, are substantially less viable than either of their parents (23
). We and others have previously demonstrated that selection seems to favor the survival of recombinants in which the regions of sequence inherited from different sources either work well together or do not encode proteins that interact extensively with one another (10
). The viability of a newly formed recombinant genome is apparently inversely correlated with both the complexity of interactions between genome regions inherited from its different parents and the relatedness of those parents to one another (10
). It is conceivable that picornavirus recombinants with complexly interacting ORFs inherited from divergent parents will be removed by purifying selection. If this is true, what should remain is evidence of a set of recombination events that accurately anticipate protein-protein, protein-RNA, and RNA-RNA interactions between viral components. The apparent paucity of recombination breakpoints within P1 observed in all five picornavirus data sets that we examined may, for example, indicate that breakpoints occurring in this region have a high probability of disrupting complex interactions between the various structural proteins that play crucial roles in the stability and maturation of picornavirus virions (9
The results presented here should be of considerable use in future analyses of picornavirus evolution. Partitioning picornavirus genomes at the recombination hot spots we have identified should both increase the power of and decrease the rate of false-positive inferences in population-genetic and selection studies. Our RDP project files (http://darwin.uvigo.es/rdp/heath2006.zip
) can also be directly used with the program RDP3 to obtain mostly recombination-free picornavirus data sets that should be of great use in such studies. Besides this, the files, when loaded into RDP3, are essentially a highly interactive database of probable aphthovirus, enterovirus, and teschovirus recombination. These project files will enable detailed analyses of how, when, and where any of the many individual PREs detected in this study may have occurred.