Rice Yellow Mottle Virus, an RNA Plant Virus, Evolves as Rapidly as Most RNA Animal Viruses

The rate of evolution of an RNA plant virus has never been estimated using temporally spaced sequence data, by contrast to the information available on an increasing range of animal viruses. Accordingly, the evolution rate of Rice yellow mottle virus (RYMV) was calculated from sequences of the coat protein gene of isolates collected from rice over a 40-year period in different parts of Africa. The evolution rate of RYMV was estimated by pairwise distance linear regression on five phylogeographically defined groups comprising a total of 135 isolates. It was further assessed from 253 isolates collected all over Africa by Bayesian coalescent methods under strict and relaxed molecular clock models and under constant size and skyline population genetic models. Consistent estimates of the evolution rate between 4 × 10?4 and 8 × 10?4 nucleotides (nt)/site/year were obtained whatever method and model were applied. The synonymous evolution rate was between 8 × 10?4 and 11 × 10?4 nt/site/year. The overall and synonymous evolution rates of RYMV were within the range of the rates of 50 RNA animal viruses, below the average but above the distribution median. Experimentally, in host change studies, substitutions accumulated at an even higher rate. The results show that an RNA plant virus such as RYMV evolves as rapidly as most RNA animal viruses. Knowledge of the molecular clock of plant viruses provides methods for testing a wide range of biological hypotheses. The mutation rates of RNA viruses (i.e., the number of nucleotide misincorporations per site and per round of replication) are 104 to 105 times higher than those of their DNA hosts (8, 9). Such a high mutation rate is attributed to the lack of repair function of the RNA polymerase of these viruses, the short replication times, and the large populations in infected hosts (7). A high mutation rate often results in rapid evolution of RNA animal viruses. This allowed the measure of the evolution rates of an extensive range of animal viruses through the analysis of heterochronous sequences, i.e., sequences of viral genes isolated at different times (11, 36). A large variation in the evolution rates of RNA animal viruses was subsequently found and was attributed mostly to differences in replication rates (24, 26). Estimates of evolution rates are used increasingly to date the emergence and to reconstruct the population dynamics of major viral epidemics (6). Interestingly, some RNA viruses change little or not at all over time. The best-documented example is an RNA plant virus, Tobacco mild green mosaic virus, which showed no increase in genetic diversity over the 90 years considered, in the longest series of isolates with known isolation times for any virus (20). Indeed, many studies have shown the remarkable genetic stability of RNA plant virus populations from different geographical regions, hosts, and collection times (21). It was claimed that most tobamovirus populations are very stable and do not evolve at a measurable rate (22). It was even observed that populations of Turnip yellow mosaic virus from Europe and Australia that probably separated more than 12,000 years ago differed by less than 1% (4). Actually, the lack of estimates of evolution rates of RNA plant viruses over time may merely reflect the absence of a large enough number of isolates collected over a sufficiently long period. Heterochronous data for plant virus isolates are particularly difficult to gather compared to data for animal viruses, for which isolates are readily recovered from blood samples that have been collected over many decades and stored for medical purposes. Even then, the temporal component of variation can be blurred or biased by other sources of diversity, such as long range dispersal, recombinant events, and subpopulation division, which are common features of plant viruses. Since the 1920s, experimental evidence has established that RNA plant viruses can evolve rapidly, especially under selection pressures such as a change of host (21, 22). The evolution rate of Wheat streak mosaic virus was extrapolated from serial passage experiments (38). This method assumed that the mean rate of change measured in the laboratory reflected that of the natural viral populations, although constraints on evolution in nature and in experiments may differ. Recently, the evolution rate of Barley yellow dwarf virus was calculated by comparing an isolate preserved in old herbarium specimens to present-day specimens (29). This method postulates that the genetic diversity of the population at the time of sampling is negligible, so that sequences isolated at different times differ only by substitutions accumulated during the time interval. If this condition is not met, this method has an upward bias and overestimates the evolution rate (11). In addition, both attempts assumed that the molecular clock remained constant during the evolution of the viruses. The estimates of the evolution rates of these two RNA plant viruses therefore rely on critical but nontestable assumptions. Estimates of the evolution rates of plant viruses based on historical evidence such as outbreak records can be tentative only. Altogether, the evolution rate of an RNA plant virus has never been estimated by applying statistical methods developed to analyze temporally spaced sequences. This contrasts with the recent advances made for RNA animal viruses by using this statistical approach (11). Rice yellow mottle virus (RYMV), of the Sobemovirus genus, was used to estimate the evolution rate of an RNA plant virus. RYMV has a high natural molecular diversity (18) and reaches a high concentration in rice (19). Experimentally, RYMV adapts rapidly to alternative hosts through accumulation of point mutations (25, 34). These features make RYMV an appropriate model with which to estimate the evolution rate of an RNA plant virus species. RYMV is an emergent virus, indigenous to Africa. It was first noticed in Kenya in East Africa in 1966 (3) and since then in almost all African countries where rice is grown, including Madagascar (39). RYMV is a major threat to rice cultivation (28). It has a narrow host range, restricted to wild and cultivated rice species and a few related grasses (2). RYMV is transmitted primarily by coleopterous beetles of the family Chrysomelidae and is disseminated by contact during cultural practices (28). Its genome contains four open reading frames (ORFs) (18). ORF1, located at the 5? end of the genome, encodes a protein involved in virus movement and gene silencing suppression. ORF2, which encodes the central polyprotein, comprises two overlapping ORFs. ORF2a encodes a serine protease and a viral-genome-linked protein (VPg). ORF2b, which is translated through a ?1 ribosomal frameshift mechanism as a fusion protein, encodes the RNA-dependent RNA polymerase. The coat protein (CP) gene (ORF4) is expressed by a subgenomic RNA at the 3? end of the genome. RYMV isolates were collected in 16 African countries between 1966 and 2006. The CP genes of 253 isolates were sequenced in this or in earlier studies (1, 18, 33, 39). Such a large collection of sequences over a 40-year period is unique for a plant virus and is used here to estimate the rate of change of RYMV. RYMV diversity is geographically structured, with different strains in East, West, and Central Africa (1, 33, 39). Substitution rates were first estimated by pairwise distance linear regressions from five phylogeographically based groups of isolates. Each group comprised isolates collected over the longest possible period of epidemiological record while other factors influencing diversity, which might adversely affect the analysis of temporally spaced viral sequences, were minimized (11). The five groups comprised 135 isolates in total. Rates were further assessed by Bayesian coalescent methods using 253 isolates originating from all regions of Africa. The rates were calculated under strict and relaxed molecular clock hypotheses (10) and under constant-size and skyline population models (14). The synonymous evolution rate was estimated from the evolution rate of the third codon position. The overall and synonymous evolution rates of the CP gene of RYMV were compared to the evolution rates of 50 RNA animal viruses (24, 26). Experimentally, the number of changes was calculated by comparing the full sequence of each isolate at inoculation with that 1 to 6 months later. Then the number of changes was compared to that estimated from the evolution rate. Altogether, we found that the overall and synonymous evolution rates of RYMV were within the ranges of those of RNA animal viruses. This shows that an RNA plant virus such as RYMV evolves as rapidly as most RNA animal viruses.

D Fargette et al., 'Rice Yellow Mottle Virus, an RNA Plant Virus, Evolves as Rapidly as Most RNA Animal Viruses', Journal of Virology, vol. 82(7), pp.3584-3589, 2008