John Kovaliski

Emerging epidemiological patterns in rabbit haemorrhagic disease, its interaction with myxomatosis, and their effects on rabbit populations in South Australia

The impact of rabbit haemorrhagic disease (RHD) on wild rabbit populations was assessed by comparing population parameters measured before the introduction of RHD into Australia in 1995 with population parameters after RHD. We used data from an arid inland area and a moist coastal area in South Australia to examine the timing and extent of RHD outbreaks, their interaction with myxomatosis and their effect on breeding, recruitment and seasonal abundance of rabbits. From this we propose a generalised conceptual model of how RHD affects rabbit populations in southern Australia. RHD decreased long-term average numbers of rabbits by 85% in the arid area. In the coastal area, RHD decreased numbers of rabbits by 73% in the first year but numbers gradually recovered and were only 12% below pre-RHD numbers in the third year. Disease activity generally begins a month or two after the commencement of breeding in autumn or winter, peaks in early spring and ceases to be apparent in summer. Where the disease is most active, the pattern of population change is almost the inverse of the former pattern. During the breeding season, RHD severely suppresses rabbit numbers. Compensatory recruitment of late-born young, protected by maternal antibodies until the disease becomes inactive at the end of spring (also the end of breeding), allows the observed rabbit abundance to increase during summer, albeit to lower levels than before RHD. Maternal antibodies are lost during summer and the population becomes susceptible to RHD. The seasonal peak in myxomatosis activity is pushed back from late spring to early summer or autumn. Survivors of myxomatosis breed after opening rains in autumn but many succumb to RHD before raising their litters. The reduced abundance of rabbits and changed pattern of seasonal abundance have potential consequences for vegetation recovery.

The non-pathogenic Australian lagovirus RCV-A1 causes a prolonged infection and elicits partial cross-protection to rabbit haemorrhagic disease virus.

Two caliciviruses occur in Australian wild rabbits: rabbit calicivirus Australia 1 (RCV-A1) and rabbit haemorrhagic disease virus (RHDV), which is used in Australia as a biocontrol agent to reduce feral rabbit populations. There is concern that RCV-A1 acts as a natural vaccine and protects from lethal RHDV infection. To investigate this hypothesis, domestic rabbits were perorally infected with RCV-A1, monitored for 28 days and subsequently challenged with RHDV. We show that RCV-A1 causes a non-pathogenic infection and is shed in faeces for up to 7 days post-infection. RCV-A1 was detected in the bile 2 months post-inoculation, indicating a prolonged or possible persistent infection. All animals infected with RCV-A1 developed antibodies cross-reacting to RHDV. When challenged with RDHV, half of the rabbits (n=4) survived the infection. The results indicate that RCV-A1 is likely to persist in rabbit populations and can elicit partial cross-protection to lethal RHDV infection.

Molecular epidemiology of Rabbit Haemorrhagic Disease Virus in Australia: when one became many

Rabbit Haemorrhagic Disease Virus (RHDV) was introduced into Australia in 1995 as a biological control agent against the wild European rabbit (Oryctolagus cuniculus). We evaluated its evolution over a 16‐year period (1995–2011) by examining 50 isolates collected throughout Australia, as well as the original inoculum strains. Phylogenetic analysis of capsid protein VP60 sequences of the Australian isolates, compared with those sampled globally, revealed that they form a monophyletic group with the inoculum strains (CAPM V‐351 and RHDV351INOC). Strikingly, despite more than 3000 rereleases of RHDV351INOC since 1995, only a single viral lineage has sustained its transmission in the long‐term, indicative of a major competitive advantage. In addition, we find evidence for widespread viral gene flow, in which multiple lineages entered individual geographic locations, resulting in a marked turnover of viral lineages with time, as well as a continual increase in viral genetic diversity. The rate of RHDV evolution recorded in Australia −4.0 (3.3–4.7) × 10−3 nucleotide substitutions per site per year – was higher than previously observed in RHDV, and evidence for adaptive evolution was obtained at two VP60 residues. Finally, more intensive study of a single rabbit population (Turretfield) in South Australia provided no evidence for viral persistence between outbreaks, with genetic diversity instead generated by continual strain importation.

Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus)

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.

Antibody status and survival of Australian wild rabbits challenged with rabbit haemorrhagic disease virus

In Australia, the epidemiology of rabbit haemorrhagic disease virus (RHDV) is complicated by non-pathogenic forms of calicivirus (bCV) co-circulating with RHDV and providing variable protection from RHDV. Currently no bCV virus-specific antibody tests exist; however, a series of four ELISAs used to detect antibodies to RHDV provided an indirect means to detect antibodies to bCV, enabling antibody categories of seronegative, maternal RHDV, RHDV or bCV to be determined. Rabbits (188) from four locations were challenged with RHDV and logistic regression models determined that, for rabbits <15 months old, survival was dependent on antibody titres alone and the relationship did not vary with age, capture site, gender, liveweight or reproductive status. All rabbits survived challenge after reaching 15 months of age, irrespective of their antibody titres. Where bCV antibodies were prevalent in young rabbits, the bCV category did not adequately summarise all information about rabbit survival that can be obtained from antibody titres. Within antibody categories, 95% of rabbits with RHDV, 33% with bCV, 40% with maternal RHDV and 22% with seronegative antibodies survived. The high survival rate of adults implies that natural outbreaks or controlled releases of RHDV will have little impact on adult breeding rabbits. Therefore, where RHDV and bCV are endemic, conventional rabbit-control programs targeting the immune breeding populations should provide the most predictable outcome for long-term maintenance of low rabbit populations.

Comparative Phylodynamics of Rabbit Hemorrhagic Disease Virus in Australia and New Zealand

ABSTRACT The introduction of rabbit hemorrhagic disease virus (RHDV) into Australia and New Zealand during the 1990s as a means of controlling feral rabbits is an important case study in viral emergence. Both epidemics are exceptional in that the founder viruses share an origin and the timing of their release is known, providing a unique opportunity to compare the evolution of a single virus in distinct naive populations. We examined the evolution and spread of RHDV in Australia and New Zealand through a genome-wide evolutionary analysis, including data from 28 newly sequenced RHDV field isolates. Following the release of the Australian inoculum strain into New Zealand, no subsequent mixing of the populations occurred, with viruses from both countries forming distinct groups. Strikingly, the rate of evolution in the capsid gene was higher in the Australian viruses than in those from New Zealand, most likely due to the presence of transient deleterious mutations in the former. However, estimates of both substitution rates and population dynamics were strongly sample dependent, such that small changes in sample composition had an important impact on evolutionary parameters. Phylogeographic analysis revealed a clear spatial structure in the Australian RHDV strains, with a major division between those viruses from western and eastern states. Importantly, RHDV sequences from the state where the virus was first released, South Australia, had the greatest diversity and were diffuse throughout both geographic lineages, such that this region was likely a source population for the subsequent spread of the virus across the country. IMPORTANCE Most studies of viral emergence lack detailed knowledge about which strains were founders for the outbreak or when these events occurred. Hence, the human-mediated introduction of rabbit hemorrhagic disease virus (RHDV) into Australia and New Zealand from known starting stocks provides a unique opportunity to understand viral evolution and emergence. Within Australia, we revealed a major phylogenetic division between viruses sampled from the east and west of the country, with both regions likely seeded by viruses from South Australia. Despite their common origins, marked differences in evolutionary rates were observed between the Australian and New Zealand RHDV, which led to conflicting conclusions about population growth rates. An analysis of mutational patterns suggested that evolutionary rates have been elevated in the Australian viruses, at least in part due to the presence of low-fitness (deleterious) variants that have yet to be selectively purged.

Does a benign calicivirus reduce the effectiveness of rabbit haemorrhagic disease virus (RHDV) in Australia? Experimental evidence from field releases of RHDV on bait

Context. European rabbits are serious environmental and agricultural pests throughout their range in Australia. Rabbit haemorrhagic disease virus (RHDV) greatly reduced rabbit numbers in arid central Australia but had less impact in cooler, higher-rainfall areas. RHDV-like benign caliciviruses (bCVs) have been implicated in limiting the impact of RHDV in the higher-rainfall regions of Australia and also in Europe. Aims. Experimental releases of RHDV on bait were tested as a means of initiating disease outbreaks. Serological evidence of antibodies to bCVs was examined to determine whether they reduce mortality rates and/or spread of the released RHDV, and how that might influence the effectiveness of future RHDV releases for rabbit management. Methods. Four experimental releases were conducted in high-rainfall and coastal regions of southern Australia. Virus activity was implied from recapture rates and serological changes in marked rabbits, and genetic sequencing of virus recovered from dead rabbits. Changes in rabbit abundance were estimated from spotlight transect counts. Key results. Release of RHDV on bait produced disease outbreaks that challenged almost all animals within the general release area and spread up to 4 km beyond the release sites. Recapture rates were high in marked rabbits that possessed antibodies from previous exposure to RHDV and extremely low amongst rabbits that lacked any detectable antibodies. Rabbits carrying antibodies classified as being due to previous infection with bCVs had recapture rates that were dependent on circulating antibody titre and were ~55% of recapture rates in rabbits with clear antibodies to RHDV. Conclusions. This is the first quantified evidence that antibodies produced against bCVs provide significant protection against RHD outbreaks in field populations of rabbits. Implications. bCVs can greatly reduce the impact of RHDV on wild-rabbit populations in Australia and presumably elsewhere. RHDV can be effectively released on bait although further releases are likely to be of minor or inconsistent benefit for controlling rabbit numbers where bCVs are common.

Rabbit haemorrhagic disease: virus persistence and adaptation in Australia.

In Australia, the rabbit haemorrhagic disease virus (RHDV) has been used since 1996 to reduce numbers of introduced European rabbits (Oryctolagus cuniculus) which have a devastating impact on the native Australian environment. RHDV causes regular, short disease outbreaks, but little is known about how the virus persists and survives between epidemics. We examined the initial spread of RHDV to show that even upon its initial spread, the virus circulated continuously on a regional scale rather than persisting at a local population level and that Australian rabbit populations are highly interconnected by virus‐carrying flying vectors. Sequencing data obtained from a single rabbit population showed that the viruses that caused an epidemic each year seldom bore close genetic resemblance to those present in previous years. Together, these data suggest that RHDV survives in the Australian environment through its ability to spread amongst rabbit subpopulations. This is consistent with modelling results that indicated that in a large interconnected rabbit meta‐population, RHDV should maintain high virulence, cause short, strong disease outbreaks but show low persistence in any given subpopulation. This new epidemiological framework is important for understanding virus–host co‐evolution and future disease management options of pest species to secure Australias remaining natural biodiversity.

Proposal for a unified classification system and nomenclature of lagoviruses

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970-1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision.

Detection of RHDV2 in European brown hares (Lepus europaeus) in Australia

RABBIT haemorrhagic disease virus 2 (RHDV2) belongs to the family Caliciviridae, genus Lagovirus , along with RHDV , European brown hare syndrome virus (EBHSV) and other unassigned rabbit caliciviruses (RCVs). RHDV2 was first detected in European rabbits ( Oryctolagus cuniculus ) in France in 2010 (Le Gall-Recule and others 2011). It spread rapidly throughout Europe (Dalton and others 2012, Abrantes and others 2013, Le Gall-Recule and others 2013, Baily and others 2014, Westcott and others 2014) and was detected in Australia in May 2015 (Hall and others 2015). In contrast to RHDV and EBHSV, which are strictly species-specific and restricted to Oryctolagus (rabbit) and Lepus (hare) genera, respectively (Lavazza and others 1996), RHDV2 causes a fatal hepatitis in European rabbits (Le Gall-Recule and others 2011), Sardinian Cape hares ( Lepus capensis mediterraneus ; Puggioni and others 2013), and in Italian hares ( Lepus corsicanus ) (Camarda and others 2014). Recently, RHDV2 has also been detected in European brown hares ( Lepus europaeus ) in Italy and Spain (Velarde and others, 2016). Australia has only two species of lagomorphs, O cuniculus and L europaeus , both introduced as game species in the mid-nineteenth century. The distribution of hares is limited to the south-east of the continent, mostly sympatric with rabbits, while rabbits inhabit an area covering 70 per cent of …