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.

Importance: The 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.

FIG 1

Schematic representation of the RHDV and RCV-A1 genomes. (Top) Both genomes are organized into two ORFs (open boxes), with short 5′ and 3′ untranslated regions (UTRs) (lines), and are VpG linked and polyadenylated. (Bottom) ORF 1 encodes a polyprotein that is proteolytically cleaved into multiple proteins (open boxes), including the 2C-like helicase, the protease, RdRp, and VP60 (capsid protein). As indicated and separated by dotted lines, VP60 has three structural domains, the N-terminal arm (N), the shell domain (S), and the protruding (P) domain, which is split into P1 and P2 subdomains. ORF 2 encodes a minor structural protein, VP10.

FIG 2

Distribution of RHDV and RCV-A1 in Australia and sampling locations. Areas where RHDV occurs in Australia are shaded in pink (based on rabbit distribution) (22, 60, 61). Antibodies to RHDV have been identified in almost every Australian rabbit population tested, such that the distribution of RHDV is likely to be very similar to the distribution of rabbits (22, 60). The distribution of RCV-A1 is limited to the more temperate climate zones in the southeast of the continent and always overlaps with RHDV, as shaded in purple (17). Collection sites for Australian samples sequenced in this study are marked with blue dots and red dots for RCV-A1 and RHDV, respectively. Australian state and territory abbreviations are as follows: WA, Western Australia; NT, Northern Territory; SA, South Australia; QLD, Queensland; NSW, New South Wales; ACT, Australian Capital Territory; VIC, Victoria; and TAS, Tasmania.

FIG 3

Phylogenetic trees of RHDV and RCV nonstructural genes (n = 94) and the capsid gene, VP60 (n = 112). Maximum-likelihood trees of the nonstructural genes (A) and the VP60 (capsid) gene (B) are shown. Samples newly sequenced in this study are shown in bold, and Australian RCV-A1 taxa are colored according to the state in which they were isolated. The accession numbers for previously published sequences are indicated in the taxon name. The European (RCV-E) sequences and additional previously published RCV-A1 sequences were included in the VP60 tree only, as the full genomes were not available. The putative recombinants detected in this study, Gudg-26, Gore-425A (from New Zealand) (RCV-A1), and MRCV (semipathogenic) are highlighted yellow and boxed in blue, red, and green boxes, respectively (although the Gore-425A breakpoints are not between the RdRp and capsid, these trees are still clearly incongruent). The phylogenies were rooted using an early European EBHSV strain (not shown), and the scale bar is proportional to the number of nucleotide substitutions per site. Bootstrap support values are indicated at the major nodes.

FIG 4

RCV-A1 temporal structure, rates of evolution, and time scale. (A) Temporal structure of the RCV-A1 full genome data set assessed by root-to-tip regression. The root-to-tip genetic distances (y axis) from the RCV-A1 full genome ML phylogeny were plotted against time (x axis), and a linear regression was conducted using Path-O-Gen v1.4. (B and C) Nucleotide substitution rate (B) and TMRCA (C) estimates for RCV-A1 sequences as measured using the Bayesian MCMC method for the complete genome data set (7,342 nt, n = 43), the nonstructural gene data set (5,274 nt, n = 44), and the VP60 gene data set (1,740 nt, n = 60). The y axis indicates the binned kernel density estimates of the posterior distribution.