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.

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 &0A and 17 &0A, 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.

Recovery of infectious rabbit hemorrhagic disease virus from rabbits after direct inoculation with in vitro-transcribed RNA.

We report the first full-length infectious clone of strain JX/CHA/97 of rabbit hemorrhagic disease virus (RHDV). The transcripts from the full-length cDNA clones were infectious when they were directly injected into rabbits. The sequence of the virus recovered from the rabbits was identical to that of the injected RNA transcripts. The cDNA clone was engineered to contain one silent nucleotide change to create an EcoRV site (A to T at nucleotide 2908). The genetic marker was retained in the recovered progeny virus. The transfection of RNA transcripts into RK-13 cells resulted in the synthesis of viral antigens, indicating that the cDNA clones were replication competent. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of RHDV.

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.

Liver enzymes and ultrastructure in rabbit haemorrhagic disease (RHD).

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24-72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150-200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.

When two into one won't go: fitting in the presence of steric hindrance and partial occupancy.

Combining structural data from cryo-electron microscopy (cryo-EM) and X-ray crystallography to give pseudo-atomic models of large molecular complexes has proved particularly suitable for studying viruses and viral complexes. Several groups are developing programs to fit X-ray data to EM data. These programs are in general tailored to particular problems with regard to size, symmetry, number of rigid bodies, resolution etc. Here, two approaches are described to fitting X-ray data to EM data in the presence of steric interference and their relative merits and limitations are indicated. These fitting techniques are applied to the case of the rotavirus double-layered particle (DLP) in complex with antibodies which inhibit the transcription of mRNA by the DLP. This is a particularly good test case, as the cryo-electron microscopy map of the DLP-Fab complex, the X-ray structure of the viral protein (VP6) and also that of the VP6-Fab complex are available. The estimation of partial occupancy is also considered.

Characterization of rabbit hemorrhagic disease virus field isolates in Taiwan.

Liver tissues from animals that were suspected to have died of rabbit hemorrhagic disease (RHD) were used for isolation and characterization of the causative agent. Three strains of RHD virus were isolated as the supernatants of liver homogenates reacted positively by hemagglutination (HA) assays and were infective for rabbits after second passage in animals. Following extraction of liver homogenates from animals infected with each of three isolates, each virus strain was purified by CsCl density gradient ultracentrifugation for further characterization. In negative-stained preparations, the purified virions were icosahedral, measured approximately 40 nm in diameter, and were without an envelope. Morphologically, the three isolates were identical. By immunoblotting, a protein with a molecular weight of 60,000 was identified as the major structural protein in each isolate. Furthermore, two sets of primer framed two different regions within RHD virus genome and could amplify two fragments of the expected size, respectively, from each isolate, whereas, none were obtained from uninfected control samples. The identity of the amplified products was confirmed further using different restriction endonucleases. Among three isolates of RHD virus, neither protein migration patterns of the virions nor cleavage patterns of the amplified product by restriction enzymes were found to differ.

Bivalent binding of a neutralising antibody to a calicivirus involves the torsional flexibility of the antibody hinge.

The structure of a complex between rabbit haemorrhagic disease virus (RHDV) virus-like particles (VLPs) and a neutralising monoclonal antibody mAb-E3 has been determined at low resolution by cryo-electron microscopy and three-dimensional (3-D) reconstruction techniques. The atomic co-ordinates of an Fab were fitted to the cryo-electron microscope density map to produce a binding model. The VLP has a T = 3 icosahedral lattice consisting of a hollow spherical shell with 90 protruding arches. Each dimeric arch presents two mAb binding sites; however, steric hindrance between the variable domains of the Fabs prevents the occupation of both sites simultaneously. Thus the maximum mAb occupation is 50%. Once a mAb is bound to one site it may bind to either of two neighbouring sites related by a local 3-fold axis. The mAbs are bound bivalently on epitopes not related by a 2-fold symmetry axis. This binding geometry implies a torsional flexibility of the mAb hinge region, involving a 60 degrees rotation of one Fab arm with respect to the other. Owing to extreme flexibility of the hinge region, the Fc domains occupy random orientations and are not visible in the reconstruction. The bivalent attachment of mAb-E3 to RHDV suggests that the neutralisation mechanism(s) involves inhibition of viral decapsidation and/or the inhibition of binding to the receptor.

Rabbit hemorrhagic disease virus (RHDV): ultrastructure and biochemical studies of typical and core-like particles present in liver homogenates.

Calicivirus particles isolated from rabbits suffering from acute RHD were compared with virions found in rabbits with chronic disease. Liver homogenates of rabbits with the protracted disease display no hemagglutinating activity and contain viral particles with diameters of 25-27 nm. These virions contain only one structural protein of 30 kDa and are distinctly smaller than intact rabbit hemorrhagic disease virus (RHDV) (32-40 nm). To prove the RHDV identity of the smaller virions, their reactivity with RHDV specific antibodies was investigated by immunoblots of the virion protein and by immunoelectron microscopy. Proteolytic digestion of RHDV particles with alpha-chymotrypsin did not transform RHDV into the smaller form. We assume that these core-like particles (CLPs) are not a result of proteolytic digestion but arise from a truncated RHDV genome or defective expression.

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.

Development of a diagnostic approach to the identification of rabbit haemorrhagic disease.

Haemagglutination and ELISA tests, and negative contrast electron microscopy, have been used to identify rabbit haemorrhagic disease virus in naturally occurring cases of the disease and in experimentally infected rabbits in the United Kingdom. Haemagglutination tests alone are not satisfactory for the diagnosis because non-haemagglutinating isolates of the virus, otherwise indistinguishable from others, have been found in some outbreaks. Haemagglutination inhibition tests have shown that a proportion of both commercial laboratory and wild rabbits in the UK are seropositive to the virus although they have not been associated with clinical disease. This observation, made previously in other parts of Europe, may indicate the longstanding circulation of a related but non-pathogenic strain of virus. Naturally occurring antibody appears to afford a high degree of protection against experimental challenge with virulent virus.

Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus.

Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.

Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection.

VP60, the unique component of rabbit hemorrhagic disease virus capsid, was expressed in the baculovirus system. The recombinant VP60, released in the supernatant of infected insect cells, assembled without the need of any other viral component to form viruslike particles (VLPs), structurally and immunologically indistinguishable from the rabbit hemorrhagic disease virion. Intramuscular vaccination of rabbits with the VLPs conferred complete protection in 15 days; this protection was found to be effective from the fifth day after VLP injection and was accompanied by a strong humoral response.

Clinical evolution and diagnosis of an outbreak of European brown hare syndrome in hares reared in captivity.

The authors studied an outbreak of an acute form of European brown hare syndrome (EBHS) in captive hares. The farm involved had shown negative results in a previous serological test for EBHS conducted on approximately 8% of the animals. Hares which succumbed during the outbreak were submitted to an anatomo-pathological examination and the livers of these animals were collected for laboratory analysis. Examination by immunoelectron microscopy and enzyme-linked immunosorbent assay confirmed the diagnosis of EBHS virus (EBHSV). An initial serological survey conducted on the survivors twenty-two days after the outbreak demonstrated an immunological response against EBHSV. During the outbreak, data were collected on morbidity, mortality, incidence of the disease in various age groups, and also on the antigenic characteristics of the virus responsible for the outbreak.