The RNA-dependant RNA polymerase is characterized by a high mutation rate, estimated around 10-4 mutations per nucleotide. This represents almost one mutation per HRV genome per replication cycle [56]. During an infection, the viral population is thus not genetically homogeneous, but each genome differs by one or several point mutation(s). Such a viral population harbouring a “cloud” of related but slightly different genomes is called a quasispecies (Figure 8). The advantage of such a heterogeneous genetic repertoire is that upon environmental changes, one or several genomes may already be suited to the new environmental conditions. This or these will be selected and further mutate to recreate a diverse population.
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Figure 8. Generation of a viral quasispecies. This scheme illustrates in a simplified manner how a quasispecies arises starting from a single viral genome. In these trees, each branch links together viral genomes differing by one point mutation, and each circle represents a replication cycle (Adapted from [57]).
Recombination
Viral recombination involves the exchange of genomic fragments between two different viruses. These two viruses need to be sufficiently similar to be compatible and to generate functional recombinant progeny. This classically occurs between two viruses belonging to the same species.
Intraspecies recombination events have been extensively described regarding HEVs and are considered as an evolutive driving force for this virus group [58, 59]. The HEV recombination breakpoints mostly map around the 5‟ (VP4) and 3‟ ends (VP1-2AB junction) of the P1 region, while they are almost absent in the VP2-VP3-VP1 capsid region (Figure 9A)[58, 60].
A frequently observed HEV intraspecies recombination phenomenon concerns circulating vaccine-derived poliovirus (cVDPV) strains. These cVDPVs, whose pathogenicity can be
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similar to that of wildtype PV, result from recombination between attenuated oral polioviruses and co-circulating non-poliovirus HEV-C members [61-63].
Some recent natural interspecies recombination events have also been described among circulating HEVs [64-66]. The recombination sites in these instances have been mapped to the 5‟UTR and the 3D region (Figure 9B).
Recombination events seem to occur less frequently among circulating HRVs. The recombination breakpoints identified such circulating HRV recombinants are situated at the 3‟
end of the 5‟UTR and at the 5‟ end of the 3C gene (Figure 9C) [67].
Whether the difference in recombination frequency between the two virus groups is related to the type and site of infection, the frequency of co-infection or genomic features remains an open question.
Phylogenetic studies indicate that interspecies HRV recombination occurred in the past. For instance, recombination between the 5‟UTR of HRV-A and the polyprotein of HRV-C was proposed as the mechanism at the origin of the HRV-Ca subgroup that harbours HRV-A-like 5‟UTR sequences [68, 69]. The remainder HRV-C strains, called HRV-Cc, exhibit 5‟UTR sequences divergent from those of HRV-A, HRV-B and HRV-Ca members. Three putative interspecies recombination breakpoints in the 5‟UTR have been mapped for HRV-Ca strains around position 481, 565, in the polypyrimidine tract, and 523, within stem-loop 5 of the IRES (Figure 9D)[69]. In the majority of the sequences analyzed, recombination presumably occurred in either one of the last two recombination hotspots, which are located in highly conserved sequence stretches. These two particular locations may therefore represent preferred sites for other interspecies 5‟UTR recombination within the Enterovirus genus.
Furthermore, some HRV-C strains harbour short HRV-A sequences in their 2A region (Figure 9D) [68, 69]. Analysis of the full-length sequences of all known HRV types in 2009 suggests that some HRV types resulted from ancient intraspecies recombination [70]. The majority of
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the recombination sites revealed by this analysis are situated in the 5‟UTR and the adjacent P1 region (Figure 9E).
Figure 9. Major genomic regions involved in HEV and HRV recombination (in red). HEV intraspecies recombination among circulating strains (A). HEV interspecies recombination sites among circulating strains (B). HRV intraspecies recombination sites among circulating strains (C). Ancient HRV interspecies recombination between HRV-A and HRV-C species (D). Ancient HRV intraspecies recombination resulting in new HRV types (E).
Finally, based on full genome phylogenetic analysis, it was proposed that ancient recombination events between HRV-A and HEV members gave rise to the HRV-B species (Figure 10) [71].
A.
B.
C.
D.
E.
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Figure 10. The HRV-B species might have arisen from recombination between HRV-A and HEV members.
The whole-polyprotein, maximum likelihood phylogenetic tree shows a closer relation between HRV-B and HEV than between HRV-A and HRV-B. The percentage of bootstraps (out of 1000) supporting corresponding clades is indicated. The sequence of simian picornavirus 1 (SV-2) was used as an outgroup. The branch lengths are measured in substitutions per site (adapted from [71]).
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However, based on sequence homology, all the above proposed natural interspecies recombination events likely occurred between ancestors of the current HRV circulating strains.
Two RNA virus recombination models have been proposed. The first is referred to as the
“copy-choice” or “template switch model” (Figure 11A). The viral RNA polymerase synthesizes an RNA fragment complementary to the positive sense RNA template originating from one of the two viruses co-infecting the same cell, before being released from that template and resuming RNA synthesis on a neighbour genome originating from the other infecting virus [72]. Gmyl et al unraveled an alternative poliovirus recombination mechanism in vitro, called “non replicative RNA” recombination (Figure 11B), in which two different viral genomes are cleaved and joined together, resulting in a chimeric genome [73]. In their study, deleted poliovirus genomes were designed to promote non replicative recombination in the spacer region of the poliovirus RNA, situated between the IRES and the coding sequence and known to tolerate sequence variations without altering viability. Poliovirus genomes consisting of intact CL and IRES elements but lacking the polyprotein and 3‟UTR sequences were co-transfected with genomes harbouring intact polyprotein and 3‟UTR sequences but lacking an essential cis-acting element of the 5‟UTR. Rescued polioviruses were sequenced, allowing recombination sites mapping. The vast majority of them were found in the spacer region. The exact molecular reactions underlying this type of recombination are not understood.
While both mechanisms may be implicated in recombination events, it is currently not possible to determine which one is predominant, although it is generally believed that the copy-choice model is responsible for natural recombination.
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Figure 11. RNA viruses recombination models. Template switch model (A, adapted from [74]), and the non replicative RNA recombination model (B, adapted from [75].