Prior exposure to non-pathogenic calicivirus RCV-A1 reduces both infection rate and mortality from rabbit haemorrhagic disease in a population of wild rabbits in Australia.

Mortality caused by rabbit haemorrhagic disease virus (RHDV) in wild rabbits is reduced in parts of Australia where the related, non-pathogenic calicivirus RCV-A1 is endemic. Laboratory experiments previously showed that prior infection with RCV-A1 enabled rabbits to better withstand subsequent infection with highly virulent RHDV, and this was assumed to explain higher survival. Here, we analyse serological data from the field suggesting that reduced mortality rates among wild rabbits may also result from rabbits previously infected with RCV-A1 having a reduced likelihood of RHDV infection. We discuss the possible mechanisms underlying this finding and its implications. The methods we describe for analysing field data gave far greater insights into epidemiological processes and virus interactions than gained from reporting basic seroprevalence rates alone.

Arrival of rabbit haemorrhagic disease virus 2 to northern Europe: Emergence and outbreaks in wild and domestic rabbits (Oryctolagus cuniculus) in Sweden.

Incursion of rabbit haemorrhagic disease virus (RHDV) into Sweden was documented in 1990 and it is now considered endemic in wild rabbit (Oryctolagus cuniculus) populations. Rabbit haemorrhagic disease virus 2 (RHDV2), a new, related lagovirus was first detected in France in 2010, and has spread rapidly throughout Europe and beyond. However, knowledge of RHDV2 in northern Europe is sporadic and incomplete, and in Sweden, routinely available diagnostic methods to detect rabbit haemorrhagic disease (RHD) do not distinguish between types of virus causing disease. Using RHDV2-specific RT-qPCR, sequencing of the VP60 gene and immunological virus typing of archived and prospective case material from the National Veterinary Institute's (SVA) wildlife disease surveillance programme and diagnostic pathology service, we describe the emergence of RHDV2 in Sweden in both wild and domestic rabbits. The earliest documented outbreak occurred on 22 May 2013, and from May 2013 to May 2016, 10 separate incidents of RHDV2 were documented from six different municipalities in the southern half of Sweden. Phylogenetic analysis of the VP60 gene shows clear clustering of Swedish isolates into three separate clusters within two different clades according to geographic location and time, suggesting viral evolution, multiple introduction events or both. Almost all cases of RHD examined by SVA from May 2013 to May 2016 were caused by RHDV2, suggesting that RHDV2 may be replacing RHDV as the predominant cause of RHD in Sweden.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain.

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Detection of the new emerging rabbit haemorrhagic disease type 2 virus (RHDV2) in Sicily from rabbit (Oryctolagus cuniculus) and Italian hare (Lepus corsicanus).

Rabbit haemorrhagic disease virus (RHDV), a member of the genus Lagovirus, causes rabbit haemorrhagic disease (RHD), a fatal hepatitis of rabbits, not previously reported in hares. Recently, a new RHDV-related virus emerged, called RHDV2. This lagovirus can cause RHD in rabbits and disease and mortality in Lepus capensis (Cape hare). Here we describe a case of RHDV2 infection in another hare species, Lepus corsicanus, during a concurrent RHD outbreak in a group of wild rabbits. The same RHDV2 strain infected rabbits and a hare, also causing a RHD-like syndrome in the latter. Our findings confirmed the capability of RHDV2 to infect hosts other than rabbits and improve the knowledge about the epidemiology and the host range of this new lagovirus.

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.

A serological survey of antibodies to rabbit haemorrhagic disease virus (rabbit calicivirus disease) in two rural Central Otago communities.

AIMS: To determine whether individuals from two rural communities with heavy exposure to the Rabbit Haemorrhagic Disease Virus (RHDV) developed antibodies to this virus. METHODS: Sera were assayed using competition ELISA (cELISA) and solid phase ELISA (spELISA). Exposure estimates were based on answers to an interviewer administered questionnaire. RESULTS: Of the 104 participants, 79 were considered to have experienced high or medium exposure, many of whom described specific exposures. There were 58 people who reported contact with RHDV infected bait, organ homogenate mixtures or rabbit body fluids. A one-way analysis of variance (Kruskal Wallis) found that human cELISA results were differently distributed from both strongly RHDV positive rabbits (chi2(1) = 27.37, p < 0.001) and weakly RHDV positive rabbits (chi2(1) = 27.35, p < 0.001). The distribution of assay results in each exposure group did not differ in either cELISA (chi2(2) = 2.49, p = 0.29) or spELISA (chi2(2) = 1.70, p = 0.43). Relatively fewer results were categorised as reactive (two 'barely' positive and two doubtful) than in a previous survey of 493 unexposed people. None of the five positive results categorised by the less specific spELISA occurred in people described as 'barely' positive or doubtful by cELISA. CONCLUSIONS. No serological evidence of infection with RHDV was found in a cohort including many heavily exposed individuals.

Use of ELISAs in field studies of rabbit haemorrhagic disease (RHD) in Australia.

ELISA techniques developed for the veterinary diagnosis of Rabbit Haemorrhagic Disease (RHD) in domestic rabbits were used for studying the epidemiology of RHD in Australian wild rabbits. The combination of ELISA techniques that distinguished IgA, IgG and IgM antibody responses and a longitudinal data set, mainly based on capture-mark-recapture of rabbits, provided a reliable basis for interpreting serology and set the criteria used to classify rabbits' immunological status. Importantly, young with maternal antibodies, immune rabbits and rabbits apparently re-exposed to RHD were readily separated. Three outbreaks of RHD occurred in 1996-7. The timing of RHD outbreaks was mainly driven by recruitment of young rabbits that generally contracted RHD after they lost their maternally derived immunity. Young that lost maternal antibodies in summer were not immediately infected, apparently because transmission of RHDV slows at that time, but contracted RHD in the autumn when conditions were again suitable for disease spread.

Detection of rabbit haemorrhagic disease virus (RHDV) by in situ hybridisation with a digoxigenin labelled RNA probe.

An in-situ hybridisation (ISH) technique for the detection of rabbit haemorrhagic disease virus (RHDV) was developed. Thirteen seronegative adult rabbits were infected oro-nasally using the BS89 RHDV strain. Liver and spleen samples were collected from 4 h post infection (p.i.) and repeated every 4 h till 44 h p.i. Each sample was tested immunohistochemically, by sandwich ELISA and by ISH. A 2.482-kb RNA probe, matching the genomic fragment coding for the VP60 structural protein of RHDV, was arranged. Two RNA probes (sense and antisense) were transcribed in vitro and UTP-digoxigenin-labelled. The antisense probe clearly detected positivity in the cytoplasm of the hepatocytes at 8 h p.i. Labelled hepatocytes were scattered throughout the sections until 24 h p.i. followed by a more diffuse perilobular positive reaction. A much weaker signal of similar distribution was detected up to 24 h p.i. using the sense RNA probe. All spleen samples tested negative for both probes. Liver samples were positive at 32 h p.i. using both ELISA and the immunoperoxidase test. Spleen samples were positive using only the ELISA at 32 h p.i. This study showed that RHDV replication occurred almost immediately after inoculation and that the liver appears to be the main site of replication.

A further step in the evolution of rabbit hemorrhagic disease virus: the appearance of the first consistent antigenic variant.

Rabbit hemorrhagic disease virus (RHDV) is a noncultivable calicivirus that infects rabbits (Oryctolagus cuniculus) and causes epidemics of an acute fatal hepatitis. In 1997 we identified two RHDV isolates from geographically distant Italian regions, which differed antigenically from the reference strain RHDV.Bs89. In fact, they were not reactive with mAb 1H8, that is able to protect rabbits from RHD and showed a low reactivity with the rabbit convalescent serum raised against RHDV.Bs89. Experimental infection of rabbits with either RHDV isolates confirmed their high pathogenicity and their peculiar antigenic profile; nevertheless, rabbits vaccinated with the current vaccine were protected against challenge infection with these isolates. Sequence comparison definitely demonstrated that the two isolates originated from the same RHDV variant and that the similarity of their structural protein (VP60) sequences with the RHDV.Bs89 is equal to 93%. This variant was named RHDVa since shows consistent genetic and antigenic differences from the wild-type RHDV. In particular, 44% of amino acid substitutions in RHDVa VP60 were located between amino acids 344 and 370, where the similarity with RHDV.Bs89 drops to 70%, suggesting that this region probably contains the epitope recognized by mAb 1H8. In addition, this paper presents preliminary data concerning the amino acids of VP60 involved in the hemagglutination site of the virus.

Seroconversion in an industrial unit of rabbits infected with a non-pathogenic rabbit haemorrhagic disease-like virus.

A serological survey of 238 rabbits for antirabbit haemorrhagic disease virus (RHDV) antibodies was made in an industrial rabbitry where no signs of the disease had been reported for four years. Seroconversion was repeatedly detected and was due to a calicivirus antigenically related to RHDV but without its pathogenicity. There was a seroprevalence of 33.3 per cent among young animals at weaning at 31 days old, 27.6 per cent at five to seven days after weaning, 56.1 per cent at 13 to 14 days after weaning, 90.3 per cent at 19 to 20 days and 100 per cent at 32 to 33 days after weaning, and all the breeding rabbits were seropositive. In the last group and in the young at weaning, the anti-RHDV antibodies were mainly class IgG, but they were IgM and IgA at 13 to 14 days after weaning. In older fattening rabbits, there was a decrease of IgM and IgA and an increase of IgG confirmed seroconversion without any specific signs of rabbit haemorrhagic disease. On the basis of these results, the probable time of infection of the meat rabbits with this non-pathogenic virus was immediately after weaning.

Detection and preliminary characterization of a new rabbit calicivirus related to rabbit hemorrhagic disease virus but nonpathogenic.

A new rabbit calicivirus related to the rabbit hemorrhagic disease virus (RHDV) was identified. The new virus contains significant differences from the previously characterized RHDV isolates in terms of pathogenicity, viral titer, tropism, and primary sequence of the structural protein. Cross-protection experiments, antigenic data, and sequence comparisons demonstrate that the new virus is more closely related to RHDV than to the European brown hare syndrome virus, another member of the caliciviruses of the lagomorph group. The existence of a nonpathogenic calicivirus, which we propose to name rabbit calicivirus (RCV), provides an explanation for the early discrepancies found in the course of serological surveys of the rabbit population in European countries.

Susceptibility of hares and rabbits to the European brown hare syndrome virus (EBHSV) and rabbit haemorrhagic disease virus (RHDV) under experimental conditions.

The European brown hare syndrome virus (EBHSV) and the rabbit haemorrhagic disease (RHDV) virus were inoculated in hares and rabbits to discover whether the homologous and heterologous host could be infected. The aims were to confirm the results of previous studies that showed the existence of antigenic differences between these two viruses, and also to define the role attributed to the hare in transmission to rabbits of a disease, EBHS, initially mistaken for RHD. During the trials, clinical symptoms and pathological lesions were noted, and virological and serological analysis were conducted, using specific tests set up for both diseases. The hares infected with EBHSV died of an acute form of EBHS, whereas the rabbits remained healthy. The low serological response in these rabbits towards the EBHSV did not protect them against RHDV. Similarly, hares inoculated with RHDV remained healthy and showed a low anti-RHDV antibody titre but died when challenged with EBHSV.

Preliminary characterization of a non-haemagglutinating strain of rabbit haemorrhagic disease virus from the United Kingdom.

A variant strain of rabbit haemorrhagic disease virus, designated "Rainham', originally isolated from a small localized outbreak of the disease in southern England, has been further examined and compared with conventional reference strains. The virus originally failed to haemagglutinate in standard conditions at normal temperature and consistently lacked HA activity after two passages in experimental rabbits. It did haemagglutinate at 4 degrees C. It reacted with hyperimmune sera to normal isolates of RHDV from the UK and Italy, and showed no differential binding activity with a panel of monoclonal antibodies prepared from an Italian reference strain. The Rainham strain appears to be antigenically indistinguishable from other known isolates and this suggests that it may differ by only a few amino acid changes in its capsid protein sequence.

Antibody response to rabbit viral hemorrhagic disease virus in red foxes (Vulpes vulpes) consuming livers of infected rabbits (Oryctolagus cuniculus).

Six red foxes (Vulpes vulpes) were given oral doses of homogenized liver from rabbits (Oryctolagus cuniculus) that died from rabbit viral hemorrhagic disease (RVHD) and four control foxes were given liver from uninfected rabbits. Antibodies to RVHD virus were monitored over 6 months. There was a pronounced antibody response 7 days after exposure which persisted to 14 days and then diminished. Low titers still were evident in three foxes at the end of the experiment. Based on these results, fox serum may be useful as an index of the prevalence of RVHD in sympatric rabbit populations.

Two independent pathways of expression lead to self-assembly of the rabbit hemorrhagic disease virus capsid protein.

The rabbit hemorrhagic disease virus capsid protein was expressed in insect cells either as an individual protein species, from a mRNA analogous to the viral subgenomic RNA, or as part of a polyprotein that included the viral 3C-like protease and the RNA polymerase. Both pathways of expression led to the assembly of viruslike particles morphologically and antigenically similar to purified virus.

Antigenicity of the rabbit hemorrhagic disease virus studied by its reactivity with monoclonal antibodies.

A panel of anti-rabbit hemorrhagic disease virus (anti-RHDV) monoclonal antibodies was produced and characterized. The ability of the MAbs to recognize epitopes present on RHDV capsids, European brown hare syndrome virus capsids and RHDV subunits was determined. Preliminary results on the neutralizing capacity of the MAbs were obtained by in vivo protection experiments. The antigenic map of RHDV obtained by this study is consistent with the current models of the calicivirus structure.

European brown hare syndrome in northern Italy: results of a virological and serological survey.

Between August 1988 and August 1991, 456 carcasses of captive or sylvatic hares from several areas of northern Italy, and 931 sera taken from adult hares in farms, in hunting and natural reserves and on importation were examined using virological (sandwich enzyme-linked immunosorbent assay [ELISA] and immuno-electron microscopy) and serological (competition ELISA) tests. The epidemiological data presented relate to the incidence of European brown hare syndrome (EBHS) in various provinces of northern Italy, the mortality caused by EBHS and the seasonal frequency of this disease. The endemic character of EBHS in Italy is proved by the large number of samples testing positive for EBHS virus (EBHSV) (47.6%) and by the results of the seroepidemiological survey, in which approximately 95% of samples tested positive for specific anti-EBHSV antibodies, showing varying titres according to the different environmental conditions.