Peter J. Kerr

Myxomatosis in Australia and Europe: a model for emerging infectious diseases.

Myxoma virus is a poxvirus naturally found in two American leporid (rabbit) species (Sylvilagus brasiliensis and Sylvilagus bachmani) in which it causes an innocuous localised cutaneous fibroma. However, in European rabbits (Oryctolagus cuniculus) the same virus causes the lethal disseminated disease myxomatosis. The introduction of myxoma virus into the European rabbit population in Australia in 1950 initiated the best known example of what happens when a novel pathogen jumps into a completely naïve new mammalian host species. The short generation time of the rabbit and their vast numbers in Australia meant evolution could be studied in real time. The carefully documented emergence of attenuated strains of virus that were more effectively transmitted by the mosquito vector and the subsequent selection of rabbits with genetic resistance to myxomatosis is the paradigm for pathogen virulence and host-pathogen coevolution. This natural experiment was repeated with the release of a separate strain of myxoma virus in France in 1952. The subsequent spread of the virus throughout Europe and its coevolution with the rabbit essentially paralleled what occurred in Australia. Detailed molecular studies on myxoma virus have dissected the role of virulence genes in the pathogenesis of myxomatosis and when combined with genomic data and reverse genetics should in future enable the understanding of the molecular evolution of the virus as it adapted to its new host. This review describes the natural history and evolution of myxoma virus together with the molecular biology and experimental pathogenesis studies that are informing our understanding of evolution of emerging diseases.

Immune Responses to Myxoma Virus

Myxoma virus causes the systemic disease myxomatosis in the European rabbit (Oryctolagus cuniculus). Originating in the South American rabbit Sylvilagus brasiliensis, where it causes a relatively localized fibroma, myxoma virus is a classic example of a virus that has jumped species to produce an exotic disease and then coevolved with its new host. Like other poxviruses, myxoma virus encodes multiple proteins capable of downregulating the host innate and acquired immune responses. Other virus-encoded proteins enable replication in host lymphocytes and monocytes, for example, by inhibiting apoptosis. Detailed studies on these proteins have demonstrated novel methods of interactions with the host immune system and added substantially to the understanding of the interaction of large DNA viruses with their hosts. Despite the increasingly detailed molecular knowledge of myxoma virus, relatively little is known about the dynamics of the interaction of the virus with the integrated host-immune system during infection and, in particular, about the evolution of resistance to the virus in wild rabbits or the species barrier. This review will focus on the detailed molecular studies that have been done with myxoma virus and discuss the more limited knowledge of the pathogenesis of myxoma virus in rabbits and the ways that the consolidated immune responses may determine genetic resistance to myxomatosis.

Origin and Phylodynamics of Rabbit Hemorrhagic Disease Virus

ABSTRACT To determine the origin, phylogenetic relationships, and evolutionary dynamics of rabbit hemorrhagic disease virus (RHDV), we examined 210 partial and complete capsid gene nucleotide sequences. Using a Bayesian Markov chain Monte Carlo approach, we estimated that these sequences evolved at a rate of 3.9 × 10−4 to 11.9 × 10−4 nucleotide substitutions per site per year. This rate was consistent across subsets of data, was robust in response to recombination, and casts doubt on the provenance of viral strains isolated from the 1950s to the 1970s, which share strong sequence similarity to modern isolates. Using the same analysis, we inferred that the time to the most recent common ancestor for a joint group of RHDV and rabbit calicivirus sequences was <550 years ago and was <150 years ago for the RHDV isolates that have spread around the world since 1984. Importantly, multiple lineages of RHDV were clearly circulating before the major Chinese outbreak of 1984, a finding indicative of an early evolution of RHDV virulence. Four phylogenetic groups within RHDV were defined and analyzed separately. Each group shared a common ancestor in the mid-1960s or earlier, and each showed an expansion of populations starting before 1984. Notably, the group characterized by the antigenic variant RHDVa harbors the greatest genetic diversity, compatible with an elevated fitness. Overall, we contend that the high virulence of RHDV likely evolved once in the early part of the 20th century, well before the documented emergence of rabbit hemorrhagic disease in 1984.

Evolutionary history and attenuation of myxoma virus on two continents.

The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.

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

ABSTRACT 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.

Myxoma Virus and the Leporipoxviruses: An Evolutionary Paradigm

Myxoma virus (MYXV) is the type species of the Leporipoxviruses, a genus of Chordopoxvirinae, double stranded DNA viruses, whose members infect leporids and squirrels, inducing cutaneous fibromas from which virus is mechanically transmitted by biting arthropods. However, in the European rabbit (Oryctolagus cuniculus), MYXV causes the lethal disease myxomatosis. The release of MYXV as a biological control for the wild European rabbit population in Australia, initiated one of the great experiments in evolution. The subsequent coevolution of MYXV and rabbits is a classic example of natural selection acting on virulence as a pathogen adapts to a novel host species. Slightly attenuated mutants of the progenitor virus were more readily transmitted by the mosquito vector because the infected rabbit survived longer, while highly attenuated viruses could be controlled by the rabbit immune response. As a consequence, moderately attenuated viruses came to dominate. This evolution of the virus was accompanied by selection for genetic resistance in the wild rabbit population, which may have created an ongoing co-evolutionary dynamic between resistance and virulence for efficient transmission. This natural experiment was repeated on a continental scale with the release of a separate strain of MYXV in France and its subsequent spread throughout Europe. The selection of attenuated strains of virus and resistant rabbits mirrored the experience in Australia in a very different environment, albeit with somewhat different rates. Genome sequencing of the progenitor virus and the early radiation, as well as those from the 1990s in Australia and Europe, has shown that although MYXV evolved at high rates there was no conserved route to attenuation or back to virulence. In contrast, it seems that these relatively large viral genomes have the flexibility for multiple pathways that converge on a similar phenotype.

Myxoma virus as a vaccine vector for rabbits: antibody levels to influenza virus haemagglutinin presented by a recombinant myxoma virus

To determine whether myxoma virus (MV) could be used as a vector for antigen presentation to European rabbits (Oryctolagus cuniculus), despite its immunosuppressive properties, a recombinant MV expressing the influenza virus haemagglutinin (HA) was constructed. Domestic rabbits, inoculated with the recombinant virus, developed high plasma antibody titres to the HA and IgG to HA was also detected in vaginal secretions. Antibody titres to HA obtained with recombinant MV were similar to titres obtained in rabbits inoculated with vaccinia virus expressing the HA. These results indicate that MV is an efficient vector for antigen presentation in rabbits.

Serological assays to discriminate rabbit haemorrhagic disease virus from Australian non-pathogenic rabbit calicivirus.

Serological cross reactivity between the virulent rabbit haemorrhagic disease virus (RHDV) and the closely related but non-pathogenic rabbit calicivirus (RCV) makes it difficult to study the epidemiology of each virus and the interaction between them when both viruses co-circulate in wild rabbit populations. ELISA methods for the diagnosis of RHDV infection are well characterized, but no specific serological tests for RCV have been developed. Following the characterization of Australian non-pathogenic RCV-A1 strains, we used virus-like-particles (VLPs) and anti-RCV-A1 specific antibodies to establish a set of isotype ELISAs for detection of IgG, IgA and IgM in rabbit sera and secretory mucosal IgA in rectal swabs, and two competition ELISAs. These assays were used to discriminate between anti-RCV-A1 and anti-RHDV antibodies in rabbits. The isotype ELISAs were highly sensitive for detection of anti-RCV-A1 antibodies, but varying levels of cross reactivity from anti-RHDV antibodies occurred in the isotype ELISAs and one competition ELISA. However, the second competition ELISA specifically detected antibodies to RCV-A1 and showed no cross reactivity to anti-RHDV sera. These ELISAs provide important tools to monitor RCV-A1 infection when it occurs alone, and to discriminate between RHDV and RCV-A1 infection when they occur in the same rabbit population. When used in parallel with RHDV serology, they could be used to monitor the dynamics of these two closely related but pathogenically distinct viruses in wild and domestic rabbit populations.

Genome Scale Evolution of Myxoma Virus Reveals Host-Pathogen Adaptation and Rapid Geographic Spread

ABSTRACT The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.

Comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens.

BackgroundOnly one strain (the Czech CAPM-v351) of rabbit haemorrhagic disease virus (RHDV) has been released in Australia and New Zealand to control pest populations of the European rabbit O. cuniculus. Antigenic variants of RHDV known as RHDVa strains are reportedly replacing RHDV strains in other parts of the world, and Australia is currently investigating the usefulness of RHDVa to complement rabbit biocontrol efforts in Australia and New Zealand. RHDV efficiently kills adult rabbits but not rabbit kittens, which are more resistant to RHD the younger they are and which may carry the virus without signs of disease for prolonged periods. These different infection patterns in young rabbits may significantly influence RHDV epidemiology in the field and hence attempts to control rabbit numbers.MethodsWe quantified RHDV replication and shedding in 4–5 week old rabbits using quantitative real time PCR to assess their potential to shape RHDV epidemiology by shedding and transmitting virus. We further compared RHDV-v351 with an antigenic variant strain of RHDVa in kittens that is currently being considered as a potential RHDV strain for future release to improve rabbit biocontrol in Australia.ResultsKittens were susceptible to infection with virus doses as low as 10 ID50. Virus growth, shedding and transmission after RHDVa infection was found to be comparable or non-significantly lower compared to RHDV. Virus replication and shedding was observed in all kittens infected, but was low in comparison to adult rabbits. Both viruses were shed and transmitted to bystander rabbits. While blood titres indicated that 4–5 week old kittens mostly clear the infection even in the absence of maternal antibodies, virus titres in liver, spleen and mesenteric lymph node were still high on day 5 post infection.ConclusionsRabbit kittens are susceptible to infection with very low doses of RHDV, and can transmit virus before they seroconvert. They may therefore play an important role in RHDV field epidemiology, in particular for virus transmission within social groups during virus outbreaks.