Rabbit Hemorrhagic Disease Virus Isolate Research Articles

Molecular characterization of some isolates of rabbit viral hemorrhagic disease (VHD) in Egypt from 2014 to 2019.

Objective:Rabbit viral hemorrhagic disease (VHD) is a transmittable and lethal viral illness of rabbits. In this study, genetic identification and genetic analysis of the rabbit hemorrhagic disease virus (RHDV) was made in three governorates in Egypt from 2014 to 2019.Materials and Methods:Livers from 18 freshly dead rabbits, which was guessed to be VHD epidemics in Egypt (Giza, Menofia, and Fayoum governorates) from 2014 to 2019, were examined for RHDV. The examination was based on the hemagglutination assay (HA) test against different mammalian (human O-type and sheep) and avian (chicken and pigeon) erythrocytes, reverse transcriptase-polymerase chain reaction (RT-PCR), and sequencing of the segment of VP60.Results:33% of the examined samples’ virus titers were 5 log2 to 8 log2 hemagglutination of human O-type erythrocytes when compared to 28%, 11%, and 28% of sheep, chicken, and pigeon erythrocytes, respectively. Four RHDV isolates out of eight RT-PCR positives were sequenced and phylogenetically analyzed. Sequenced isolates were designed and submitted to GenBank with accession numbers MN904506, MN904507, MN904508, and MN904509. These four RHDV isolates were related to classic G3 (GI.1d/RHDV). Twelve amino acid differences were detected between the vaccine strain sequence (Giza-2006) and RHDV isolates. Amino acid differences at 416, 423, and 476 positions seem interesting as they changed polarity that could change the protein structure and affect host interaction.Conclusions:There is antigenic variation between circulating RHVD strains and the vaccinal strain. This may be the leading cause of vaccination failure and may increase the need to check out the vaccination program against RHVD.

Adaptive diversification between the classic rabbit hemorrhagic disease virus (RHDV) and the RHDVa isolates: A genome-wide perspective

Rabbit haemorrhagic disease virus (RHDV) is a highly infectious pathogen that causes high mortality in wild and domestic rabbits. RHDV could be divided into two subtypes, classic RHDV and RHDVa, which present clear genetic, antigenic, and epidemiological differences. To further understand the nature of the diversity, we performed a genome-wide evolutionary study on the classic RHDV and RHDVa isolates. The results show that RHDV had experienced adaptive diversification with the dividing process of these subtypes. Furthermore, amino acid changes relevant to the adaptive diversification mainly cluster in viral capsid protein VP60. These results might be beneficial for a further understanding the function of VP60 and provide helpful hints for the genetic basis of RHDV emergence and re-emergence.

Melatonin inhibits the sphingosine kinase 1/sphingosine-1-phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin.

The sphingosine kinase (SphK)1/sphingosine-1-phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2×10(4) hemagglutination units of a RHDV isolate and received 20mg/kg of melatonin at 0, 12, and 24hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV-infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll-like receptor (TLR)4, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, nuclear factor-kappa B (NF-κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.

Identification of novel rabbit hemorrhagic disease virus B-cell epitopes and their interaction with host histo-blood group antigens.

Rabbit haemorrhagic disease, caused by rabbit hemorrhagic disease virus (RHDV), results in the death of millions of adult rabbits worldwide, with a mortality rate that exceeds 90%. The sole capsid protein, VP60, is divided into shell (S) and protruding (P) domains, and the more exposed P domain likely contains determinants for cell attachment and antigenic diversity. Nine mAbs against VP60 were screened and identified. To map antigenic epitopes, a set of partially overlapping and consecutive truncated proteins spanning VP60 were expressed. The minimal determinants of the linear B-cell epitopes of VP60 in the P domain, N(326)PISQV(331), D(338)MSFV(342) and K(562)STLVFNL(569), were recognized by one (5H3), four (1B8, 3D11, 4C2 and 4G2) and four mAbs (1D4, 3F7, 5G2 and 6B2), respectively. Sequence alignment showed epitope D(338)MSFV(342) was conserved among all RHDV isolates. Epitopes N(326)PISQV(331) and K(562)STLVFNL(569) were highly conserved among RHDV G1-G6 and variable in RHDV2 strains. Previous studies demonstrated that native viral particles and virus-like particles (VLPs) of RHDV specifically bound to synthetic blood group H type 2 oligosaccharides. We established an oligosaccharide-based assay to analyse the binding of VP60 and epitopes to histo-blood group antigens (HBGAs). Results showed VP60 and its epitopes (aa 326-331 and 338-342) in the P2 subdomain could significantly bind to blood group H type 2. Furthermore, mAbs 1B8 and 5H3 could block RHDV VLP binding to synthetic H type 2. Collectively, these two epitopes might play a key role in the antigenic structure of VP60 and interaction of RHDV and HBGA.

Melatonin modulates the autophagic response in acute liver failure induced by the rabbit hemorrhagic disease virus

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 104 hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin‐1, ubiquitin‐like autophagy‐related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3‐I to autophagosome‐associated LC3‐II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome‐associated membrane protein (LAMP)‐1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase‐3 activity, and immunoglobulin‐heavy‐chain‐binding protein (BiP) and CCAAT/enhancer‐binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.

Autophagic response in the Rabbit Hemorrhagic Disease, an animal model of virally-induced fulminant hepatic failure

The Rabbit Hemorrhagic Disease Virus (RHDV) induces a severe disease that fulfils many requirements of an animal model of fulminant hepatic failure. However, a better knowledge of molecular mechanisms contributing to liver damage is required, and it is unknown whether the RHDV induces liver autophagy and how it relates to apoptosis. In this study, we attempted to explore which signalling pathways were involved in the autophagic response induced by the RHDV and to characterize their role in the context of RHDV pathogenesis. Rabbits were infected with 2 × 104 hemmaglutination units of a RHDV isolate. The autophagic response was measured as presence of autophagic vesicles, LC3 staining, conversion of LC3-I to autophagosome-associated LC3-II and changes in expression of beclin-1, UVRAG, Atg5, Atg12, Atg16L1 and p62/SQSTM1. RHDV-triggered autophagy reached a maximum at 24 hours post-infection (hpi) and declined at 30 and 36 hpi. Phosphorylation of mTOR also augmented in early periods of infection and there was an increase in the expression of the endoplasmic reticulum chaperones BiP/GRP78, CHOP and GRP94. Apoptosis, measured as caspase-3 activity and expression of PARP-1, increased significantly at 30 and 36 hpi in parallel to the maximal expression of the RHDV capsid protein VP60. These data indicate that RHDV infection initiates a rapid autophagic response, perhaps in an attempt to protect liver, which associates to ER stress development and is independent from downregulation of the major autophagy suppressor mTOR. As the infection continues and the autophagic response declines, cells begin to exhibit apoptosis.

Melatonin treatment reduces endoplasmic reticulum stress and modulates the unfolded protein response in rabbits with lethal fulminant hepatitis of viral origin

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress and unfolded protein response (UPR) inhibition is an underlying mechanism of melatonin anti-apoptotic effects in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2×10(4) hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10mg/kg and 20mg/kg at 0hr, 12hr and 24hr postinfection. RHDV infection induced increased expression of CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin heavy chain binding protein (BiP/GRP78), glucose-regulated protein 94 (GRP94), phospho-c-Jun N-terminal kinase (JNK) and caspase-12. These effects were attenuated by melatonin. Double immunofluorescence staining showed colocalization of CHOP and cleaved caspase-3 in liver sections of RHDV-infected rabbits, while immunostaining decreased markedly with melatonin treatment. RHDV infection resulted in significant increases in the mRNA levels of activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1 (IRE1), spliced X-box binding protein-1 (XBP1s) and tumor necrosis factor receptor-associated factor 2 (TRAF2). Melatonin attenuated the extent of the changes. Data obtained provide evidence that in rabbits with experimental infection by RHDV, reduction in apoptotic liver damage by melatonin is associated with attenuation of ER stress through a modulation of the three arms of UPR signaling and further support a potential hepatoprotective role of melatonin in FHF.

Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography

Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.

Protective immune responses in rabbits induced by a suicidal DNA vaccine of the VP60 gene of rabbit hemorrhagic disease virus

A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against rabbit hemorrhagic disease virus (RHDV). The VP60 gene of RHDV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/VP60, was transfected into BHK-21 cells, and the antigenicity of the expressed protein was confirmed using indirect immunofluorescence and a western blot assay. In addition, immunogenicity was studied in rabbits. Fifteen rabbits were injected intramuscularly twice with pSCA/VP60 at 2-week intervals. They were challenged with an RHDV isolate 2weeks after the second immunization. In all cases, anti-RHDV antibodies were detected by ELISA. Additionally, the lymphocyte proliferation response was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method, and neutralizing antibodies were measured by microneutralization tests. Our results showed that RHDV-specific antibodies and an RHDV-specific cell-mediated immune response were strongly induced in rabbits. Furthermore, all of the rabbits were protected against challenge with wild type RHDV. In conclusion, we demonstrated that the suicidal DNA vaccine is a promising vaccine candidate that facilitates the prevention of rabbit hemorrhagic disease caused by RHDV.

Adenovirus-based oral vaccine for rabbit hemorrhagic disease

Vaccine antigens for rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from the livers of experimentally infected rabbits or from several recombinant immunogens. However, the application of these vaccine antigens has been restricted because of biosecurity and immunity characteristics. In the current study, a recombinant adenovirus expressing the RHDV capsid protein (VP60) was constructed and the expression of the recombinant protein was identified through western blot analysis using RHDV-positive rabbit sera. Eighteen rabbits were immunized by injection, direct oral instillation, or using bait. They were challenged with RHDV isolate three weeks after boost immunization. In all cases, the rabbits immunized with the recombinant adenovirus developed RHDV-specific antibodies and cell immune response. The rabbits injected with the recombinant adenovirus were completely protected against RHDV challenge. The adenovirus expression system may provide a strategy for the immunization of rabbits, particularly for the control of RHDV in wild rabbits.

Histo-Blood Group Antigens Act as Attachment Factors of Rabbit Hemorrhagic Disease Virus Infection in a Virus Strain-Dependent Manner

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50–90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level.

Use of VP60 RT-PCR to overcome the limitation of haemagglutination inhibition diagnosis of rabbit viral haemorrhagic disease

Rabbit viral haemorrhagic disease (RVHD) is a highly contagious, highly fatal, peracute and acute viral disease of both wild and domestic rabbits caused by rabbit haemorrhagic disease virus (RHDV). Testing for haemagglutination (HA) activity in processed liver samples is one of the cornerstones for rapid diagnosis of RHDV outbreaks in national rabbitries. However, RHDV isolates exhibiting no HA activity are increasingly being reported. The extent of deviation from classical HA activity patterns for RHDV strains in Egypt has not been investigated. This study compared the HA activity patterns of samples collected from 61 RHDV outbreaks that occurred between 1999 and 2005 to determine whether dependence on HA test (HAT) for diagnosis of RHDV outbreaks needs to be reviewed. All samples were confirmed RHDV positive using SDS-PAGE and western blotting. Using slide HAT, only 36.1% of samples were positive (22 samples). Plate HAT conducted at 4 0 C detected an additional 16 positive samples bringing the total HA-positive samples to 38 (62.3%). Plate HAT conducted at 22 0 C failed to detect additional positive samples. The majority of samples detected after plate HA testing (62.5%) had HA titres comparable to those obtained from slide-HA-positive samples, indicating that the difference in HA activity is dependent on the nature of the HA antigen rather than its presence. Direct detection of HA activity failed in 37.7% of samples despite the presence of classical signs, pathology, and being RT-PCR positive for three different VP60 regions. Experimental infection of seronegative rabbits with 9 HA negative RHDV samples showed that 5 isolates were in-fact HA positive, while only 4 isolates remained HA negative. The increased detection of viruses lacking HA activity and the low HAT sensitivity mandates the use of molecular techniques for rapid confirmation of RHDV diagnosis in the Egyptian environment.

Melatonin attenuates apoptotic liver damage in fulminant hepatic failure induced by the rabbit hemorrhagic disease virus

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate the antiapoptotic effect of melatonin in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 and 20 mg/kg at 0, 12, and 24 hr postinfection. RHDV infection induced liver apoptosis, with increased caspase-3 immunoexpression and activity and poly(ADP-ribose)polymerase-1 (PARP-1) proteolysis. These effects were attenuated by melatonin in a concentration-dependent manner. Antiapoptotic effects of melatonin were related to a reduced expression of Bax and cytosolic cytochrome c release, increased expression of Bcl-2 and Bcl-xL, and inhibition of caspase-9 activity. Increased thiobarbituric reactive acid substances concentration and oxidized-to-reduced glutathione ratio were significantly prevented by melatonin administration. Melatonin treatment also resulted in a reduction in caspase-8 activity, tumor necrosis factor receptor-1 (TNF-R1) expression, and phosphorylated Janus kinase (JNK) expression, and increased expression of cellular FLICE-inhibitory protein (c-FLIP). Our findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by RHDV and supports a potential hepatoprotective role of melatonin in FHF.

Capsid gene divergence in rabbit hemorrhagic disease virus

The emergence and rapid global spread of rabbit hemorrhagic disease virus (RHDV) remains enigmatic despite two decades of study, largely due to the difficulties associated with modelling substitution processes of the RNA genome for phylogenetic inference. We used maximum-likelihood and Bayesian methods to investigate rates of molecular evolution in the capsid gene, finding evidence of positive selection and of variable substitution rates between nucleotide sites and between lineages. The maximum-likelihood and Bayesian analyses produced fully congruent topologies; however, strong support for older nodes of the phylogeny was only obtained from the Bayesian analyses that utilized the additional information of collection dates for RHDV isolates spanning 22 years. These dates also allowed calibration of the RHDV phylogenetic tree in a calendar year timescale and estimation of dates for the most recent common ancestors of virulent and benign strains. These dates suggested the divergence of RHDV approximately 20 years prior to the first report of haemorrhagic disease in rabbits.

Origin and Phylodynamics of Rabbit Hemorrhagic Disease Virus

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 x 10(-4) to 11.9 x 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.

N-acetyl-cysteine protects liver from apoptotic death in an animal model of fulminant hepatic failure

This work was undertaken to investigate whether treatment with N-acetyl-cysteine (NAC) prevents oxidative stress and inhibits the apoptotic pathways in an animal model of fulminant hepatic failure. Rabbits were experimentally infected with 2x10(4) hemagglutination units of a rabbit hemorrhagic disease virus isolate. The spontaneous mortality rate of infected animals was 67% at 36 h post infection (pi) and 90% at 48 h pi. This percentage decreased significantly in animals receiving an i.p. injection of NAC (150 mg/kg body way/daily), for 7 days prior to infection. From 36 h pi marked increases were detected in blood levels of transaminases, lactate dehydrogenase, bilirubin and the oxidised/reduced glutathione ratio. All these effects were significantly prevented by NAC treatment. The Bax to Bcl-2 relative expression, the expression of FasL, cytochrome c and PARP-1, and the activity of caspase 3 were significantly increased at 36 and 48 h pi in infected animals. These changes were markedly reduced in animals treated with NAC, with the exception of FasL. Our results suggest a potential hepatoprotective role of NAC in fulminant hepatic failure, mediated partially through the modulation of the intrinsic pathway of apoptosis.

Rabbit hemorrhagic viral disease: Characterization of a new animal model of fulminant liver failure

In this study we sought to characterize a novel model of fulminant liver failure (FLF) by means of experimental infection of rabbits with the rabbit hemorrhagic disease virus (RHDV). Thirty-seven 9-week-old rabbits were injected intramuscularly with 2 × 104 hemagglutination units of an RHDV isolate. Eighty-five percent of rabbits died 36 to 54 hours after infection. From 36 hours after infection we noted marked increases in transaminases, lactate dehydrogenase, and total bilirubin. The rabbits exhibited hypoglycemia and coagulation abnormalities, with a significant decrease in factor V, factor VII, and prothrombin. Plasma aromatic amino acids and taurine showed progressive increases, and the Fischer index was significantly reduced. Expression of hepatocyte growth factor messenger RNA was inhibited from 36 hours after infection. Prostration and side recumbency were present at later stages, and neurologic symptoms rapidly progressed to coma. Onset of brain death was associated with a significant increase in intracranial pressure and blood ammonia. RHDV infection reproduces clinical, biochemical, and histologic features of the FLF syndrome and satisfies criteria for a suitable animal model. Rabbit hemorrhagic viral disease could provide a useful tool for the study of FLF and the evaluation of new liver-support technologies in human subjects. (J Lab Clin Med 2003;141:272-8)

Molecular epidemiology of Rabbit haemorrhagic disease virus.

Millions of domestic and wild European rabbits (Oryctolagus cuniculus) have died in Europe, Asia, Australia and New Zealand during the past 17 years following infection by Rabbit haemorrhagic disease virus (RHDV). This highly contagious and deadly disease was first identified in China in 1984. Epidemics of RHDV then radiated across Europe until the virus apparently appeared in Britain in 1992. However, this concept of radiation of a new and virulent virus from China is not entirely consistent with serological and molecular evidence. This study shows, using RT-PCR and nucleotide sequencing of RNA obtained from the serum of healthy rabbits stored at 4 degrees C for nearly 50 years, that, contrary to previous opinions, RHDV circulated as an apparently avirulent virus throughout Britain more than 50 years ago and more than 30 years before the disease itself was identified. Based on molecular phylogenetic analysis of British and European RHDV sequences, it is concluded that RHDV has almost certainly circulated harmlessly in Britain and Europe for centuries rather than decades. Moreover, analysis of partial capsid sequences did not reveal significant differences between RHDV isolates that came from either healthy rabbits or animals that had died with typical haemorrhagic disease. The high stability of RHDV RNA is also demonstrated by showing that it can be amplified and sequenced from rabbit bone marrow samples collected at least 7 weeks after the animal has died.

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.

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.