Evolution and Phylogeography of the Nonpathogenic Calicivirus RCV-A1 in Wild Rabbits in Australia

Despite its potential importance for the biological control of European rabbits, relatively little is known about the evolution and molecular epidemiology of rabbit calicivirus Australia 1 (RCV-A1). To address this issue we undertook an extensive evolutionary analysis of 36 RCV-A1 samples collected from wild rabbit populations in southeast Australia between 2007 and 2009. Based on phylogenetic analysis of the entire capsid sequence, six clades of RCV-A1 were defined, each exhibiting strong population subdivision. Strikingly, our estimates of the time to the most recent common ancestor of RCV-A1 coincide with the introduction of rabbits to Australia in the mid-19th century. Subsequent divergence events visible in the RCV-A1 phylogenies likely reflect key moments in the history of the European rabbit in Australia, most notably the bottlenecks in rabbit populations induced by the two viral biocontrol agents used on the Australian continent, myxoma virus and rabbit hemorrhagic disease virus (RHDV). RCV-A1 strains therefore exhibit strong phylogeographic separation and may constitute a useful tool to study recent host population dynamics and migration patterns, which in turn could be used to monitor rabbit control in Australia.Lagoviruses form a genus within the Caliciviridae family of RNA viruses (16). All representatives of this genus are highly species specific and infect only their respective hosts, i.e., rabbits and hares. The prototype species of the genus Lagovirus is rabbit hemorrhagic disease virus (RHDV), which was first described in China in 1984 when a severe infectious necrotizing hepatitis with mortality rates up to 90% was observed in angora rabbits (22). However, phylogenetic analysis indicates that the pathogenic strains of RHDV likely evolved from nonpathogenic lagoviruses several decades before they were initially described (20). Today, RHDV is found on most continents, causing ongoing damage to the rabbit meat industry (24) and threatening wild native rabbit populations in Europe. On the Iberian peninsula the rabbit is considered an endangered species and is itself a staple food of endangered predators, such as the imperial eagle and the Iberian lynx (10).In marked contrast, since 1995 Australia has been using RHDV as a successful viral biocontrol agent for rabbits (7), which cause severe environmental and economic damage in this country. The use of a viral biocontrol agent for a vertebrate species has been and remains controversial, but there is little doubt that since its release RHDV has generated close to $6 billion of savings to the Australian agricultural industry (41), as well as some much needed relief for the regeneration of many native plant species (35).Notably, RHDV-induced mortality is lower in certain areas of Australia, namely, the cooler and more humid southeast region of the continent, which is believed to be in part due to the presence of related but nonpathogenic lagoviruses circulating in the population (8). Recently, such a virus was identified in Australian wild rabbits (38). This new member of the genus Lagovirus, termed rabbit calicivirus Australia 1 (RCV-A1), is a nonpathogenic virus causing a predominantly enteric infection in rabbits. Other benign or moderately pathogenic RCV strains have also been described in the United States and Europe (1, 5, 14, 15). While the Italian nonpathogenic RCV provides complete cross-immunity to RHDV, only partial protection is conveyed by the Australian virus RCV-A1 (37) although this may be sufficient to reduce overall RHDV-induced mortality.Like all lagoviruses, RCV-A1 has a single-stranded positive-sense RNA genome of approximately 7.5 kb that is polyadenylated and has a viral protein (VpG) covalently bound to its 5′ end (25). The genome is organized into two open reading frames (ORFs) (6, 39). ORF1, which represents the majority (7 kb) of the genome, encodes a polyprotein that is auto-proteolytically cleaved during posttranslational processing into several smaller proteins, including the helicase, protease, and polymerase (26). In contrast, ORF2 is only 351 nucleotides (nt) long, and the function of its VP10 gene product is unknown although it is present in small amounts in the virion (39).Given the potential interactions between RCVs and pathogenic RHDV, which could impact rabbit survival in both Australia and Europe, it is clearly important to understand the evolutionary history, genetic diversity, and geographic distribution of RCV-A1 and other benign lagoviruses. In Australia, growing reports of rising rabbit numbers mean that there is a clear need for improved strategies to monitor and control rabbit populations. In addition, because RCV-A1 does not cause any fitness decrease in its rabbit host (37), it is possible that this virus could be used as a population genetic marker to track movements and changes in the distribution of the European rabbit across the Australian continent. Indeed, other microorganisms have proven to be useful indicators of the population dynamics of their hosts (3, 42). Microbial markers are particularly informative for the study of population processes in the very recent past since genetic changes will not have had sufficient time to be recorded in the more slowly evolving host genome (2).The host-pathogen interaction of Australian rabbits and their viruses is unique in that both were introduced only once (or possibly a few times in a limited period of time). In addition, 150 years of historical records are available documenting the introduction, spread, and control efforts of rabbits in Australia. Herein, we explore the evolutionary history and dynamics of RCV-A1 in Australia, with a particular focus on revealing the phylogeographic distribution of the virus across this continent, the evolutionary processes that have shaped its diversity, and its suitability as a marker to study host distribution and migration patterns.