Benign Rabbit Caliciviruses Exhibit Evolutionary Dynamics Similar to Those of Their Virulent Relatives

Abstract

Two closely related caliciviruses cocirculate in Australia: rabbit hemorrhagic disease virus (RHDV) and rabbit calicivirus Australia 1 (RCV-A1). RCV-A1 causes benign enteric infections in the European rabbit (Oryctolagus cuniculus) in Australia and New Zealand, while its close relative RHDV causes a highly pathogenic infection of the liver in the same host. The comparison of these viruses provides important information on the nature and trajectory of virulence evolution, particularly as highly virulent strains of RHDV may have evolved from nonpathogenic ancestors such as RCV-A1. To determine the evolution of RCV-A1 we sequenced the full-length genomes of 44 RCV-A1 samples isolated from healthy rabbits and compared key evolutionary parameters to those of its virulent relative, RHDV. Despite their marked differences in pathogenicity and tissue tropism, RCV-A1 and RHDV have evolved in a very similar manner. Both viruses have evolved at broadly similar rates, suggesting that their dynamics are largely shaped by high background mutation rates, and both exhibit occasional recombination and an evolutionary environment dominated by purifying selection. In addition, our comparative analysis revealed that there have been multiple changes in both virulence and tissue tropism in the evolutionary history of these and related viruses. Finally, these new genomic data suggest that either RCV-A1 was introduced into Australia after the introduction of myxoma virus as a biocontrol agent in 1950 or there was drastic reduction of the rabbit population, and hence of RCV-A1 genetic diversity, perhaps coincident with the emergence of myxoma virus. IMPORTANCEThe comparison of closely related viruses that differ profoundly in propensity to cause disease in their hosts offers a powerful opportunity to reveal the causes of changes in virulence and to study how such changes alter the evolutionary dynamics of these pathogens. Here we describe such a novel comparison involving two closely related RNA viruses that cocirculate in Australia, the highly virulent rabbit hemorrhagic disease virus (RHDV) and the nonpathogenic rabbit calicivirus Australia 1 (RCV-A1). Both viruses infect the European rabbit, but they differ in virulence, tissue tropism, and mechanisms of transmission. Surprisingly, and despite these fundamental differences, RCV-A1 and RHDV have evolved at very similar (high) rates and with strong purifying selection. Furthermore, candidate key mutations were identified that may play a role in virulence and/or tissue tropism and therefore warrant further investigation. R evealing the factors that determine the evolution of virulence in viruses and identifying how changes in virulence might impact viral evolutionary dynamics are key queries in the study of infectious disease evolution. Although there is a large body of work on revealing the relationship between virulence and transmissibility, most studies of virulence evolution involve the retrospective analysis of single viruses (1-4). However, the comparison of closely related...

Benign Rabbit Calicivirus in New Zealand

“…In the current study, evolutionary analysis of the VP60 gene for all published RCV-A1 isolates and the New Zealand RCV variants produced a mean evolutionary rate of 5.57 ϫ 10 Ϫ3 subs/ site/year (95% HPD interval, 4.61 ϫ 10 Ϫ3 to 6.85 ϫ 10 Ϫ3 subs/site/year) and hence equivalent to those rates previously estimated in RCV-A1 (19). Interestingly, these rates are higher than those observed for the pathogenic RHDV (mean, 2.77 ϫ 10 Ϫ3 subs/ site/year) (29) and hence may be inflated because of the presence of transient deleterious mutations that are yet to be removed by purifying selection (19). The VP60 data set from this study placed the common ancestor of all RCV-A1s in the late 1980s, whereas dating based on the nonstructural gene data set (19) gave a TMRCA closer to the early 1980s.…”

“…3), confirming that these viruses sit within the level of genetic diversity of RCV-A1 that was isolated from rabbits in Australia (19).…”

“…The capsid region of Gore-425 was amplified using two primer sets (Table 2), RCV-4/ALR-6 and capsRCV-A1[HindIII]/capsRCV-A1rev[NotI], using Platinum Taq DNA polymerase high-fidelity kit (Invitrogen), as per the manufacturer's instructions. Cycling conditions for RCV-4/ALR-6 were performed as described previously (19). Cycling conditions for capsRCV-A1[HindIII]/capsRCV-A1rev[NotI] were 94°C for 2 min, 16 cycles of 15 s at 94°C, 68°C (Ϫ0.5°C per cycle) for 20 s, and 68°C for 3 min, 24 cycles of 15 s at 94°C, 60°C for 20 s, and 68°C for 3 min, followed by a final extension step of 68°C for 5 min.…”

“…Low-quality reads were removed, and reads were trimmed and merged as described previously (19). The final cleaned reads were mapped to MIC-07 RCV-A1 (accession no.…”

“…Since the discovery of this first nonpathogenic RCV, other nonpathogenic and moderately pathogenic strains have been isolated. A rabbit calicivirus (RCV-A1) was discovered in Australia in 2009 (11), and further viruses have since been isolated from rabbitries and wild populations across Australia (18,19). The distribution and prevalence of Australian RCV-A1 are associated with cool high-rainfall areas found in the south of the continent (20), similar to conditions that prevail in New Zealand.…”