Phylogenetic analysis of rabbit haemorrhagic disease virus (RHDV) strains isolated between 1988 and 2003 in eastern Hungary

Summary. To define the genetic variability of RHDV strains collected in eastern Hungary, liver samples from rabbits that had died of RHD were collected between 1988 and 2003. The phylogenetic analysis of a 528-nucleotide-long portion of the gene encoding the VP60 capsid protein assigned the strains into three genogroups. The first group contained viruses from 1988–1993, and a second group comprised isolates from 1994–2002. A third group comprised all of the tested representatives of the RHDVa subtype and a Hungarian isolate from 2003. These findings were supported by the alignments of the deduced amino acid sequences of the VP60 gene and strongly suggest the presence of the RHDVa subtype in Hungary.     

Electron and immunoelectron microscopy of rabbit haemorrhagic disease virus (RHDV)

Summary Rabbit haemorrhagic disease virus (RHDV) had a calicivirus-like structure and a diameter of 31.5–33.0 nm. Antigenic relationship between the investigated RHDV strain and the causal agent of RHD in China was demonstrated by immunoelectron microscopy.     

Subacute liver necrosis after experimental infection with rabbit haemorrhagic disease virus (RHDV)

Rabbit haemorrhagic disease (RHD) is usually peracute to acute, while subacute to chronic disease is rare. This paper describes gross and histopathological findings in four out of 20 rabbits aged 14 weeks, experimentally infected with one of two German field isolates of RHD virus. Eight rabbits survived the infection for 10 days and were killed after four of them, infected with 100 to 10 000 haemagglutination units, had started to develop progressive jaundice. Histopathologically, icteric livers showed severe subacute centrilobular bridging necrosis with calcification, and proliferation of periportal hepatocytes and bile ducts. Positive-strand RHDV RNA was detected by in-situ hybridization, mainly in periportal macrophages. Loss of the normal hepatic architecture, reparation (fibrosis) and hepatocellular regeneration, together with moderate inflammatory reaction, are signs of liver cirrhosis. These signs, observed in young rabbits given small doses of RHD virus, are interpreted as an unusual outcome of experimental inoculation.     

Evidence for recombination in the major capsid gene VP60 of the rabbit haemorrhagic disease virus (RHDV)

Rabbit haemorrhagic disease (RHD) is a highly fatal disease caused by a virus of the family Caliciviridae. Whereas recombination is well documented in other members of this family, the extent of recombination has so far not been studied in RHDV. To reach a better evaluation of the possible role of recombination in the evolution of RHDV virulence, we have searched for recombination events in RHDV by analysing 43 complete sequences of the major capsid gene VP60. Phylogenetic analyses revealed two well separated groups. Clear evidence for recombination was found for the Hartmannsdorf strain which shows different phylogenetic profiles depending on the region of the capsid examined.     

Sequence analysis of rabbit haemorrhagic disease virus (RHDV) in Australia: alterations after its release

Summary.  Liver samples from rabbits killed by RHDV, collected from five States in Australia in 1996 and 1997 were analysed by RT-PCR. A 398 bp fragment of the capsid protein (VP60) gene was amplified by PCR and directly sequenced. The alignment of the nucleotide and amino acid sequences and their comparison with the original strain of the virus released in Australia indicated genetic changes after two years have been small with 98.2% to 100% identity. The constructed phylogenetic tree suggests slight differences in nucleotide substitutions in various States but there is no clear evidence of clustering of sequences according to their geographic origin. In practical terms, sequencing of viral RNA provides a means of testing the efficacy of further releases and subsequent spread of the virus if such a strategy is employed as a means of enhancing RHD as a biological control of the wild rabbit in Australia. Received March 5, 1998 Accepted June 26, 1998     

Detection of positive selection in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV)

Mutations were analysed in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV), a calicivirus responsible for high mortality rates in both wild and domestic European rabbits (Oryctolagus cuniculus). Likelihood of positive selection was estimated using the PAML software applied to 43 non-identical complete sequences of the major capsid protein. Three codons showed signs of positive selection (with posterior probabilities over 95%), one of them is located in the region containing the major antigenic determinants (region E). The presence of positively selected codons (PSCs) in other regions may suggest the existence of other antigenic regions on the major capsid protein that stimulate protective immune responses. At all the 3 PSCs, variation contributes to putative N-glycosylation sites of the protein. An N-glycosylation site is deleted in the non-pathogenic strain RCV. Some of the substitutions at PSCs may alter the polarity and the charge of the protein with possible implications in the protein structure and host interaction. The detection of PSCs should allow a better understanding of the interaction between RHDV and the rabbit immune system.     

Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains

This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.     

In vitro translation of a subgenomic mRNA from purified virions of the Spanish field isolate AST/89 of rabbit hemorrhagic disease virus (RHDV).

Purified preparations of the Spanish field isolate of rabbit hemorrhagic disease virus AST/89 were found to contain the plus-stranded genomic RNA of more than 7.4 kilobases (kb) and large amounts of a subgenomic mRNA of 2.4 kb. The smaller RNA was translated in vitro and shown to code for a 60 kDa protein which was immunoprecipitated using anti-RHDV as well as anti-VP60 sera.     

Sequence and genomic organization of a rabbit hemorrhagic disease virus isolated from a wild rabbit

Rabbit hemorrhagic disease virus (RHDV) is a member of the caliciviridae family. The nucleotidic sequence of a full-length cDNA of one RHDV isolate (RHDV-SD) is reported. The genome is 7437 bases long and includes two ORFs, ORF1 (7034 b) and ORF2 (353 b), coding for the polyprotein and the Vp12, respectively. The coding sequence for the second structural protein (the capsid protein, Vp60) is located at the 3 end of ORF1. Comparison of RHDV-SD with the German RHDV isolate revealed 470 nucleotide substitutions (96% homology). The deduced amino acid sequences of the two isolates are closely related (98% identity), and no hypervariable region could be identified either in the structural or nonstructural proteins.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z29514.     

Phylogenetic analysis of rabbit haemorrhagic disease virus in France between 1993 and 2000, and the characterisation of RHDV antigenic variants

Summary.  The first molecular epidemiological study of Rabbit haemorrhagic disease virus undertaken in France between 1988 and 1995, identified three genogroups, two of which (G1, G2) disappeared quickly. We used immunocapture-RT-PCR and sequencing to analyse 104 new RHDV isolates collected between 1993 and 2000. One isolate was obtained in 2000 from a French overseas territory, the Reunion Island. The nucleotide sequences of these isolates were aligned with those of some French RHDV isolates representative of the three genogroups previously identified, of some reference strains and German and American RHDV antigenic variants. Despite the low degree of nucleotide sequence variation, three new genogroups (G4 to G6) were identified with significant bootstrap values. Two of these genogroups (G4 and G5) were related to the year in which the RHDV isolates were collected. Genogroup G4 emerged from genogroup G3, which has now disappeared. Genogroup G5 is a new independent group. The genogroup G6 contained an isolate collected in mainland France in 1999 and the isolate collected from the Reunion Island, as well as German and American RHDV variants. Multiple sequence alignments of the VP60 gene and antigenic analysis with monoclonal antibodies demonstrated that these French isolates are two new isolates of the RHDV variant. Received June 7, 2002; accepted August 12, 2002     

Isolation and identification of a non-haemagglutinating strain of rabbit hemorrhagic disease virus from China and sequence analysis for the VP60 Gene

A variant strain of rabbit hemorrhagic disease virus, designated “whn-1”, was isolated and identified in China. The virus lacked haemagglutinating activity at 25, 37 and 4°C, respectively, and gave negative results in the HAT after two passages in experimentally infected rabbits, but gave positive results in Agar Diffusion Reaction (ADR) and Counter Immunoelectrophoresis (CIE). Using electron microscopy, negatively stained particles of the RHDV isolate showed that the virions was approximately 35 nm in diameter. The capsid protein VP60 gene of whn-1 strain was cloned into pMD18-T vector by RT-PCR assays and sequenced. The obtained VP60 gene sequence has been submitted to GenBank with the accession number: DQ069280. The whole VP60 gene of whn-1 was 1740 nt in size and encodes 579 aa. Alignment with other 16 strains of RHDV in the world, including such “RHDVa” strains as France 99-05, France-Reu-00, Germany-Triptis and ChinaTP, in addition to RCV and EBHSV, showed that the homology of RHDV strains were 90.0–98.0% for nucleotide sequence, 94.3–99.0% for amino acid sequence, respectively. The results indicated that the sequences of VP60 gene of different RHDV isolates, including non-haemagglutinating whn-1 strain and low-haemagglutinating Rainham strain, were relatively highly homologous, and the major variant amino acid were located within region C (301–328 aa) and region E(344–434 aa), which were specific to “RHDVa” strains. Moreover, the molecular characterisation of VP60 protein of RHDV whn-1 strain, such as Hydrophilicity plot, Flexible regions, Antigenic index, etc., were compared with reference RHDV strains of Spanish-AST/89, France-99-5 and UK-Rainham in this article. From the experiment, it’s concluded that, the “whn-1” strain is probably an antigenic variant of “RHDVa”, and the 3 amino acids of Phe (304), Ala (305), Ser (309), and 5 amino acids of Gly (359), Asn (365), Ala (369), Ala (370), Asn (386), located in P2 region in the VP60 protein, probably played an important role in the haemagglutination activity.     

Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants

Summary.  Rabbit haemorrhagic disease virus (RHDV) isolates were obtained from several animals previously vaccinated with an inactivated vaccine. Seven isolates were analyzed by immunological and molecular biological methods and compared to reference strains. Antigenic characterization with monoclonal antibodies as well as haemagglutination assays demonstrated considerable differences between individual isolates. However, sequencing of the capsid protein genes revealed a high degree of homology between five of these isolates and the reference strain FRG. In contrast, two isolates specified remarkably different capsid proteins with a degree of variation not observed so far in RHDV. Amino acid alterations were found clustered between residues 301 and 328 (region C), 344 and 434 (region E) and also in the 3′ region of the capsid protein gene. Interestingly, experimental vaccination of rabbits followed by challenge with the heterologous variant strains showed restricted cross-protection against one of the strains. In summary, we found a level of antigenic variation not detected in RHDV so far, and describe two distinct new antigenic variants. Received November 3, 1997 Accepted October 19, 1998     

Recombination in rabbit haemorrhagic disease virus: possible impact on evolution and epidemiology

Rabbit haemorrhagic disease (RHD) was first recognised in 1984 following the introduction of apparently healthy rabbits into China from Germany. The aetiological agent Rabbit haemorrhagic disease virus (RHDV) has subsequently killed hundreds of millions of domestic and wild rabbits particularly in Europe, China and Australia. Previously, using phylogenetic analysis we have attempted to understand the underlying factors that determine why this virus emerged, and why it has such an unpredictable epidemiology. Here we report the use of tree congruency supported by bootscanning analysis to detect recombination amongst both closely related, and widely divergent strains of RHDV. We show that recombination occurs commonly and in several different regions of the RHDV genome. Moreover, the first identified strain of RHDV, i.e. from China in 1984, showed evidence of recombination in the capsid gene, with a virus or viruses containing lineages in German strains. These observations imply that recombination may play a significant role in the evolution, epidemiology and diversity of RHDV.     

Folding of the rabbit hemorrhagic disease virus capsid protein and delineation of N-terminal domains dispensable for assembly

Summary.  Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are caliciviruses that produce severe symptoms and are lethal to rabbits and hares. The folding of the capsid protein was studied by determination of the antigenic pattern of chimeric capsid proteins, composed of regions from RHDV and EBHSV capsid proteins. The anti-RHDV monoclonal antibody (MAb) E3, which is known to bind an external conformational epitope, recognized the RHDV C-terminal region. The anti-RHDV MAb A47, which binds a buried epitope, recognized the RHDV N-terminal region. Using a pGEX expression library, we more precisely mapped the MAb A47 epitope on a 31 residues length peptide, between residue 129 and 160 of the VP60, confirming its location in the N-terminal part of the protein. These results demonstrate that the C-terminal part of the protein is accessible to the exterior whereas the N-terminal domain of the protein constitutes the internal shell domain of the particle. With the aim of using virus-like particles (VLPs) of RHDV as epitope carriers or DNA transfer vectors, we produced in the baculovirus system three proteins, ΔN1, ΔN2 and ΔN3, truncated at the N terminus. The ΔN1 protein assembled into VLPs, demonstrating that the first 42 amino acid residues are not essential for capsid assembly. In contrast, ΔN2, from which the first 75 residues were missing, was unable to form VLPs. The small particles obtained with the ΔN3 protein lacking residues 31 to 93, located in the immunodominant region of the RHDV capsid protein, indicate that up to 62 amino acid residues can be eliminated without preventing assembly. Received September 6, 2001; accepted March 13, 2002 Published online June 21, 2002     

Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus.

A rapid and sensitive one-tube RT-PCR assay using a fluorogenic (TaqMan) probe was developed to improve the diagnosis of Hendra virus (HeV) infection. The TaqMan assay was developed to rapidly and specifically identify Hendra virus. The sensitivity of the new TaqMan-based PCR assay compared favourably with conventional RT-PCR. The major advantage of the TaqMan-based assay was the speed of diagnosis with results available within minutes of completing the PCR, and within 4 h of receiving the specimen. This test greatly reduces the chance of false positives through the elimination of second-round PCR and the requirement for agarose gel. Recombinant primer controls consisting of the Hendra virus primer sequence flanking a rodent GADPH probe sequence and recombinant probe controls consisting of the rodent GADPH primer sequence flanking the Hendra virus probe sequence were designed, cloned and transcribed in vitro to generate RNA. This has alleviated the requirement for viral RNA to be used as positive controls, thus reducing the chance of producing a false positive, at the same time eliminating the biosafety risk associated with handling live virus. This assay will provide a rapid diagnosis of future outbreaks of Hendra virus.     

Development of a real-time RT-PCR assay for improved detection of Borna disease virus

Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan probes and primers specific for the BDV P and BDV L genes were designed by aligning the sequences of known BDV strains. After optimisation, the sensitivity and specificity of the rRT-PCR were established. The detection limit was set to 10-100 viral genomic copies per reaction and the assay detects the BDV strains V and He/80, as well as the most divergent BDV strain known so far, No/98. Furthermore, the system detected feline BDV variants in five naturally infected cats and a feline isolate used in experimental infection of cats. This rRT-PCR assay will be a powerful tool in further studies of BDV, including epidemiological screening and diagnosis.