Evolutionary history and molecular epidemiology of rabbit haemorrhagic disease virus in the Iberian Peninsula and Western Europe

Background  

Rabbit haemorrhagic disease virus (RHDV) is a highly virulent calicivirus, first described in domestic rabbits in China in 1984. RHDV appears to be a mutant form of a benign virus that existed in Europe long before the first outbreak. In the Iberian Peninsula, the first epidemic in 1988 severely reduced the populations of autochthonous European wild rabbit. To examine the evolutionary history of RHDV in the Iberian Peninsula, we collected virus samples from wild rabbits and sequenced a fragment of the capsid protein gene VP60. These data together with available sequences from other Western European countries, were analyzed following Bayesian Markov chain Monte Carlo methods to infer their phylogenetic relationships, evolutionary rates and demographic history.     

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.     

一株兔出血症病毒2型毒株的分离鉴定

【背景】我国于2020年4月突发兔出血症病毒2型(Rabbit Hemorrhagic Disease Virus 2,RHDV2)疫情,严重威胁兔养殖业和生态平衡,而且目前国内对RHDV2的病原学以及遗传特征等基础研究匮乏。【目的】分离鉴定RHDV2毒株,对分离株进行全基因测序与遗传进化分析。【方法】对成都某兔场疑似RHDV2感染致死的家兔进行病理剖检,通过RT-qPCR检测和动物回归试验,分离鉴定得到RHDV2毒株,进一步进行全基因测序及遗传进化分析。【结果】病死兔剖检表现为各实质器官出血肿大,以心、肺、肝脏尤为明显,经RT-qPCR确诊为RHDV2,而且不存在其他病原混合感染,试验感染家兔可致相似病变。将分离株命名为SCCN03,其基因序列全长为7464bp,与参考毒株(GenBank登录号为MN901451.1)一致性为99.21%。对比参考株氨基酸序列,分离株的非结构蛋白和结构蛋白氨基酸序列发生了多处错义突变,其中非结构蛋白p16和结构蛋白的几处错义突变可能与毒株变异有关。进化树显示毒株SCCN03属于GI.2基因型。【结论】分离鉴定出一株RHDV2毒株,获得其基因序列,丰富了RHDV2的全基因数据资料,为后续RHDV毒力相关研究和相关疫苗研发奠定了基础。     

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T = 3 capsid protein leads to larger T = 4 capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.     

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.     

Detection of Viral Proteins after Infection of Cultured Hepatocytes with Rabbit Hemorrhagic Disease Virus

The calicivirus rabbit hemorrhagic disease virus (RHDV), which replicates predominantly in the livers of infected rabbits, cannot be propagated in tissue culture. To enable the performance of in vitro studies, rabbit hepatocytes were isolated by liver perfusion and gradient centrifugation. After inoculation with purified RHDV, more than 50% of the cells proved to be infected. Protein analyses led to the detection of 13 RHDV-specific polypeptides within the infected cells. These proteins were assigned to defined regions of the viral genome, resulting in a refined model of RHDV genome organization.     

兔的一种新病毒：...

In the spring 1986, an acute infectious disease occurred in Wuhan Second Producing Medical Manufactory, and the rabbit almost died. We tested the mortal symptom and confirmed rabbit Hemorrhagic Disease (RHD) as same as Huang Yinyao report. Hubei Traditional Chinese Medicine Institute appear this RHD also. After we purified virus of above two source by low speed, high speed and sucrose density gradient centrifugation, they can react with antiserum of RHDV from Nanjing Agricultural University in agar gel immunodiffusion tests. These results proved that they belong to the same serotype. Data indicate RHDV have difference morphological superstructure, viral polypeptides and especially RHDV can't react with antiserum of standard Parvovirus of rabbit and so on, so we suggest RHDV is a new virus.     

表达兔出血症病毒VP60蛋白的重组兔粘液瘤病毒的构建与初步评价

兔出血症病毒 (Rabbit hemorrhagic disease virus,RHDV) 及兔粘液瘤病毒 (Myxoma virus,MYXV) 分别引起兔出血症 (兔瘟) 和兔粘液瘤病,是两种严重危害家兔养殖业以及导致原产地欧洲野兔-穴兔 (Oryctolagus cuniculus) 种群近濒危的重要病原。VP60为构成RHDV衣壳的主要抗原蛋白。为研制能同时免疫预防该两种疫病的重组二联疫苗,本研究分别以MYXV和其复制非必需基因——胸腺激酶 (Thymidine kinase,TK) 基因为重组载体和同源重组靶基因,构建穿梭载体p7.5-VP60-GFP。将p7.5-VP60-GFP载体转染被MYXV感染的兔肾细胞株RK13,经同源重组后,在荧光显微镜下筛选出表达GFP的重组病毒,并将其命名为rMV-VP60-GFP。通过PCR和Western blotting进行重组病毒vp60基因特异性插入和表达验证结果显示,vp60和gfp基因成功插入MYXV基因组中并且可成功表达,表明成功构建了表达RHDV衣壳蛋白基因vp60的重组MYXV。动物攻毒保护试验表明,制备的重组病毒能保护家兔抵抗MYXV的致死性攻击,这为后续疫苗的研发奠定了基础。     

Cryo-electron microscopy reconstructions of two types of wild rabbit hemorrhagic disease viruses characterized the structural features of Lagovirus

Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection. The etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the Caliciviridae family. Compared to other calicivirus, such as rNV and SMSV, the structure of Lagovirus members is not well characterized. In this report, structures of two types of wild RHDV particles, the intact virion and the core-like particle (CLP), were reconstructed by cryo-electron microscopy at 11Å and 17Å, respectively. This is the first time the 3D structure of wild caliciviruses CLP has been provided, and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus. Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated. In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus, the capsomers of RHDV virion exhibited unique structural features and assembly modes. Both P1 and P2 subdomains have interactions inside the AB capsomer, while only P2 subdomains have interaction inside CC capsomer. The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting, and the rotation of RHDV VP60 P domain with respect to its S domain, compared with SMSV, was observed. Collectively, our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus, which is important for functional analysis and better vaccine development in the future.     

Rabbit haemorrhagic disease: applying Occam's razor to competing hypotheses

Rabbit haemorrhagic disease virus (RHDV) is a highly virulent lagovirus endemic in Europe and Australasian populations of the European rabbit, Oryctolagus cuniculus. It has also caused several unexplained disease outbreaks in domestic European rabbits in North America. Non-pathogenic spread of RHDV leading to persistent infection which later reactivated has recently been proposed as the cause of overt disease and death of a pet rabbit in Canada, the first confirmed case of Rabbit haemorrhagic disease in that country. We suggest that there is little evidence to support non-pathogenic spread of virulent RHDV, some evidence that is contradictory, and evidence to support a simpler alternative hypothesis. RHDV can be spread over long distances between sparse rabbit populations by fomites or flying insects. Although highly pathogenic, RHDV can be limited in its spread within rabbit populations, or its presence masked by closely related but non-pathogenic lagoviruses which can provide protection against acute disease. In the absence of any evidence from clinical studies to support reactivation of persistent RHDV infection, the simpler explanation seems more likely to be correct.     

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.     

Oxidative stress in wild European rabbits naturally infected with myxoma virus and rabbit haemorrhagic disease virus

The European rabbit (Oryctolagus cuniculus) is one of the most important vertebrate species in the Mediterranean Basin ecosystem. Over the last 60 years, the arrival of two viral diseases, myxomatosis and rabbit haemorrhagic disease, have led to dramatic declines in wild rabbit populations across the Iberian Peninsula. These diseases are currently endemic. Periodic outbreaks occur and have significant impacts on wild populations. Both infection types have diverse physiological effects on their hosts that are rooted in aerobic metabolic processes. To fight off these viruses, rabbits activate their immune systems. However, the production of immune defences generates reactive oxygen species that may consequently damage host tissues. Hypothesising that immune responses increase oxidative stress, we examined whether wild rabbits naturally infected with myxoma virus (MV) and rabbit haemorrhagic disease virus (RHDV) had high oxidative stress. Using blood samples, we measured anti-MV and anti-RHDV antibody concentrations and different oxidative stress markers (i.e., glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, and malondialdehyde). Our results show that rabbits that were seropositive for both MV and RHDV had high concentrations of malondialdehyde. Age and body condition were also positively related to dual seropositivity. No significant relationships were observed between serostatus and the concentrations of the other oxidative stress markers. Although we expected infection with MV and RHDV to be correlated with oxidative stress, the influence of external sources of oxidative stress (e.g., climatic conditions) likely made it more difficult to detect such relationships in wild rabbits.     

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus

We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.     

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.     

Recombinant VP60 in the form of virion-like particles as a potential vaccine against rabbit hemorrhagic disease virus

Rabbit hemorrhagic disease virus (RHDV) which causes a highly contagious disease of wild and domestic rabbits belongs to the family Caliciviridae. It is a small, positive single-stranded RNA virus with a genome of 7.5 kb and has a diameter of approximately 40 nm. In negatively stained electron micrographs the virus shows typical calicivirus morphology with regularly arranged cup-shaped structures on the surface. It is a major pathogen of rabbits in many countries. Vp60 - a coat protein of molecular mass around 60 kDa is the major antigen of RHDV. It is present as 90 dimeric units per virion particle. We have expressed VP60 gene in the baculovirus system with the aim to use it as a potential vaccine against RHDV and a diagnostic reagent in immunological tests. cDNA of the vp60 gene of strain SGM, was cloned into a baculovirus transfer vector as full-length gene, as well as truncated gene lacking 600 5'-terminal nucleotides. The sequence of SGM VP60 differed markedly from that of the reference strain. Full-length recombinant VP60 protein from the SGM strain self-assembled to form virus-like particles (VLPs). These particles observed by electron microscopy were morphologically similar to native virions and were able to agglutinate human group 0 erythrocytes. After immunization the recombinant particles induced RHDV-specific antibodies in rabbits and guinea pigs. Rabbits immunized with the VLPs were fully protected against challenge with a virulent RHDV.     

Processing of rabbit hemorrhagic disease virus polyprotein.

Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.     

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.     

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

ABSTRACT: BACKGROUND: Antibodies 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. RESULTS: A 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). CONCLUSIONS: This 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.