June Liu

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.

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.

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.

A sensitive and specific blocking ELISA for the detection of rabbit calicivirus RCV-A1 antibodies

BackgroundAntibodies to non-pathogenic rabbit caliciviruses (RCVs) cross-react in serological tests for rabbit hemorrhagic disease virus (RHDV) and vice versa, making epidemiological studies very difficult where both viruses occur. It is important to understand the distribution and interaction of the two viruses because the highly pathogenic RHDV has been used as a biocontrol agent for wild rabbits in Australia and New Zealand for the past 17 years. The presence of the benign RCV Australia 1 (RCV-A1) is considered a key factor for the failure of RHDV mediated rabbit control in cooler, wetter areas of Australia.ResultsA highly sensitive and specific blocking ELISA was developed for the detection of RCV-A1 antibodies. When sera from rabbits with a known infection history for either RCV-A1 or RHDV were tested, this assay showed 100% sensitivity and no cross-reactivity with RHDV sera (100% specificity).ConclusionsThis new ELISA not only allows the detection of RCV-A1 at a population level, but also permits the serological status of individual rabbits to be determined more reliably than previously described methods. This robust and simple to perform assay is therefore the tool of choice for studying RCV-A1 epidemiology in Australian wild rabbit populations.

Distribution and Prevalence of the Australian Non-Pathogenic Rabbit Calicivirus Is Correlated with Rainfall and Temperature

Background Australia relies heavily on rabbit haemorrhagic disease virus (RHDV) for the biological control of introduced European wild rabbits Oryctolagus cuniculus, which are significant economic and environmental pests. An endemic non-pathogenic rabbit calicivirus termed RCV–A1 also occurs in wild rabbits in Australian and provides partial protection against lethal RHDV infection, thus interfering with effective rabbit control. Despite its obvious importance for rabbit population management, little is known about the epidemiology of this benign rabbit calicivirus. Methods We determined the continent-wide distribution and prevalence of RCV-A1 by analysing 1,805 serum samples from wild rabbit populations at 78 sites across Australia for the presence of antibodies to RCV-A1 using a serological test that specifically detects RCV-A1 antibodies and does not cross-react with co-occurring RHDV antibodies. We also investigated possible correlation between climate variables and prevalence of RCV-A1 by using generalised linear mixed effect models. Results Antibodies to RCV-A1 were predominantly detected in rabbit populations in cool, high rainfall areas of the south-east and south-west of the continent. There was strong support for modelling RCV-A1 prevalence as a function of average annual rainfall and minimum temperature. The best ranked model explained 26% of the model structural deviance. According to this model, distribution and prevalence of RCV-A1 is positively correlated with periods of above average rainfall and negatively correlated with periods of drought. Implications Our statistical model of RCV-A1 prevalence will greatly increase our understanding of RCV-A1 epidemiology and its interaction with RHDV in Australia. By defining the environmental conditions associated with the prevalence of RCV-A1, it also contributes towards understanding the distribution of similar viruses in New Zealand and Europe.

Expression and partial characterisation of rabbit haemorrhagic disease virus non-structural proteins

The intracellular replication and molecular virulence mechanisms of Rabbit haemorrhagic disease virus (RHDV) are poorly understood, mainly due to the lack of an effective cell culture system for this virus. To increase our understanding of RHDV molecular biology, the subcellular localisation of recombinant non-structural RHDV proteins was investigated in transiently transfected rabbit kidney (RK-13) cells. We provide evidence for oligomerisation of p23, and an ability of the viral protease to cleave the p16:p23 junction in trans, outside the context of the nascent polyprotein chain. Notably, expression of the viral polymerase alone and in the context of the entire RHDV polyprotein resulted in a redistribution of the Golgi network. This suggests that, similar to other positive-strand RNA viruses, RHDV may recruit membranes of the secretory pathway during replication, and that the viral polymerase may play a critical role during this process.

Next step in the ongoing arms race between myxoma virus and wild rabbits in Australia is a novel disease phenotype

Significance When a pathogen emerges in a host population, will it evolve to do more or less harm to its host? A single strain of myxoma virus was released as a biocontrol agent against Australian rabbit populations in 1950. The subsequent coevolution has become a textbook classic, although there has been little experimental work on this topic since the early 1980s. Here, we show that the host–pathogen arms race continued with the evolution of highly lethal viruses that cause immune collapse. The possibility that pathogens can become highly immunosuppressive in response to increases in host resistance needs to be considered where genetic and immunologic manipulations are used to enhance host resistance, as, for instance, in agriculture. In host–pathogen arms races, increases in host resistance prompt counteradaptation by pathogens, but the nature of that counteradaptation is seldom directly observed outside of laboratory models. The best-documented field example is the coevolution of myxoma virus (MYXV) in European rabbits. To understand how MYXV in Australia has continued to evolve in wild rabbits under intense selection for genetic resistance to myxomatosis, we compared the phenotypes of the progenitor MYXV and viral isolates from the 1950s and the 1990s in laboratory rabbits with no resistance. Strikingly, and unlike their 1950s counterparts, most virus isolates from the 1990s induced a highly lethal immune collapse syndrome similar to septic shock. Thus, the next step in this canonical case of coevolution after a species jump has been further escalation by the virus in the face of widespread host resistance.

Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia

The co-evolution of myxoma virus (MYXV) and the European rabbit occurred independently in Australia and Europe from different progenitor viruses. Although this is the canonical study of the evolution of virulence, whether the genomic and phenotypic outcomes of MYXV evolution in Europe mirror those observed in Australia is unknown. We addressed this question using viruses isolated in the United Kingdom early in the MYXV epizootic (1954–1955) and between 2008–2013. The later UK viruses fell into three distinct lineages indicative of a long period of separation and independent evolution. Although rates of evolutionary change were almost identical to those previously described for MYXV in Australia and strongly clock-like, genome evolution in the UK and Australia showed little convergence. The phenotypes of eight UK viruses from three lineages were characterized in laboratory rabbits and compared to the progenitor (release) Lausanne strain. Inferred virulence ranged from highly virulent (grade 1) to highly attenuated (grade 5). Two broad disease types were seen: cutaneous nodular myxomatosis characterized by multiple raised secondary cutaneous lesions, or an amyxomatous phenotype with few or no secondary lesions. A novel clinical outcome was acute death with pulmonary oedema and haemorrhage, often associated with bacteria in many tissues but an absence of inflammatory cells. Notably, reading frame disruptions in genes defined as essential for virulence in the progenitor Lausanne strain were compatible with the acquisition of high virulence. Combined, these data support a model of ongoing host-pathogen co-evolution in which multiple genetic pathways can produce successful outcomes in the field that involve both different virulence grades and disease phenotypes, with alterations in tissue tropism and disease mechanisms.

DIFFERENT SEROLOGICAL PROFILES TO CO-OCCURRING PATHOGENIC AND NONPATHOGENIC CALICIVIRUSES IN WILD EUROPEAN RABBITS (ORYCTOLAGUS CUNICULUS) ACROSS AUSTRALIA

Abstract Rabbit hemorrhagic disease virus (RHDV) was released in Australia as a biocontrol agent for wild European rabbits (Oryctolagus cuniculus) in 1995–96; however, its effects were variable across Australia with the greatest population reductions seen in lower annual rainfall areas (<400 mm). There is speculation that the reduced effectiveness observed at higher annual rainfall sites is at least partially due to the presence of a nonpathogenic calicivirus (RCV-A1). The RCV-A1 is related to RHDV and confers partial and transient protection against lethal RHDV infection in laboratory tests. What is not well understood is where, how, and to what degree RCV-A1 impedes the effect of RHDV-mediated rabbit control under field conditions. We investigated seven wild rabbit populations across six states and territories representing different seasonal rainfall zones across Australia, four times during 2011–12, to investigate if the presence and prevalence of RCV-A1 coincided with a change in RHDV immunity status within these populations. Besides serology, tissue samples from both trapped and shot rabbits were collected for virus detection by reverse transcription PCR. Overall, 52% (n=258) of the total samples (n=496) tested positive for RHDV antibodies and 42% (n=208) positive for RCV-A1 antibodies; 30% (n=150) of the sera contained antibodies to both viruses. The proportion of rabbits with RHDV antibodies increased significantly at sites where RCV-A1 antibodies were present (χ21, α=0.1, P<0.001). Evidence that preinfection of RCV-A1 may lead to a higher proportion of sampled rabbits with antibodies to both viruses was found at only one site.

Reverse Engineering Field Isolates of Myxoma Virus Demonstrates that Some Gene Disruptions or Losses of Function Do Not Explain Virulence Changes Observed in the Field.

ABSTRACT The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics, we examined the roles of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole-gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here or that combinations of multiple mutations, possibly involving epistatic interactions or noncoding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies of a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined. IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti to the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for the attenuation of or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses isolated early and late in the Australian radiation of MYXV. Surprisingly, none of the candidate mutations that we identified as likely having roles in attenuation proved to be important for virulence. This indicates that considerable caution is warranted when interpreting the possible role of individual mutations during virulence evolution.