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An in vivo system for directed experimental evolution of rabbit haemorrhagic disease virus.

ABSTRACT: The calicivirus Rabbit haemorrhagic disease virus (RHDV) is widely used in Australia as a biocontrol agent to manage wild European rabbit (Oryctolagus cuniculus) populations. However, widespread herd immunity limits the effectiveness of the currently used strain, CAPM V-351. To overcome this, we developed an experimental platform for the selection and characterisation of novel RHDV strains. As RHDV does not replicate in cell culture, variant viruses were selected by serially passaging a highly virulent RHDV field isolate in immunologically naïve laboratory rabbits that were passively immunised 18-24 hours post-challenge with a neutralising monoclonal antibody. After seven passages, two amino acid substitutions in the P2 domain of the capsid protein became fixed within the virus population. Furthermore, a synonymous substitution within the coding sequence of the viral polymerase appeared and was also maintained in all subsequent passages. These findings demonstrate proof-of-concept that RHDV evolution can be experimentally manipulated to select for virus variants with altered phenotypes, in this case partial immune escape.

SUBMITTER: Hall RN

PROVIDER: S-EPMC5348035 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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An in vivo system for directed experimental evolution of rabbit haemorrhagic disease virus.

Hall Robyn N RN Capucci Lorenzo L Matthaei Markus M Esposito Simona S Kerr Peter J PJ Frese Michael M Strive Tanja T

PloS one 20170313 3

The calicivirus Rabbit haemorrhagic disease virus (RHDV) is widely used in Australia as a biocontrol agent to manage wild European rabbit (Oryctolagus cuniculus) populations. However, widespread herd immunity limits the effectiveness of the currently used strain, CAPM V-351. To overcome this, we developed an experimental platform for the selection and characterisation of novel RHDV strains. As RHDV does not replicate in cell culture, variant viruses were selected by serially passaging a highly v ...[more]

PMID: 28288206

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Project description:BackgroundRabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00-21).ResultsWe performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10- 1 viral genome copies), low (104), high (107) and very high (1011) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00-21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5' untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00-21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 104 gRNA copies, whereas between 104 and 107 gRNA copies were required to also induce mortality.ConclusionsThese results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5' untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00-21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies.

Project description:The aim of this study was to characterise the nucleotide and amino acid sequence of complete genomes (7.5 kb) from RHDV strains isolated in Poland and estimate the genetic variability in different elements of the viral RNA. In addition, the sequence of Polish RHDV isolates isolated from 1988-2015 was compared with the sequences of other European RHDV, including the RHDVa and RHDV2/RHDVb subtypes. The complete sequence was developed by the compilation of partial nucleotide sequences. This sequence consisted of approximately 7428 nucleotides. For comparison of nucleotide sequences and the development of phylogenetic trees of Polish RHDV isolates and reference RHDV strains representing the main phylogenetic groups of classical RHDV, RHDVa and RHDV2 as well as the non-pathogenic rabbit lagovirus RCV, the BLAST software with blastn and MEGA6 with neighbour-joining method was applied. The complete nucleotide sequence of Polish isolates of RHDV has also been entered into GenBank. For comparative analysis, nineteen complete sequences representing the main RHDV genetic types available in GenBank were used. The results of phylogenetic analysis of Polish RHDV strains reveals the presence of three classical RHDV genogroups (G2, G4 and G5) and an RHDVa variant (G6). The oldest RHDV isolates (KGM 1988, PD 1989 and MAL 1994) belong to genogroup G2. It can be assumed that the elimination of these strains from the environment probably occurred at the turn of 1994 and 1995. Genogroup G2 was replaced by the phylogenetically younger BLA 1994 and OPO 2004 strains from genogroup G4, which probably originated from the G3 lineage, represented by the Italian strains BS89. The last representatives of classical RHDV in Poland are isolates GSK 1988 and ZD0 2000 from genogroup G5. A single clade contains the Polish RHDV strains from 2004-2015 (GRZ 2004, KRY 2004, L145 2004, W147 2005, SKO 2013, GLE 2013, RED1 2013, STR 2012, STR2 2013, STR 2014, BIE 2015) identified as RHDVa, which clustered into genogroup G6, as represented by the RHDV strain Triptis 1996. All recent isolated RHDV isolates belong exclusively to RHDVa and no RHDV2 was diagnosed in Poland.

Project description:Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD.

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An in vivo system for directed experimental evolution of rabbit haemorrhagic disease virus.

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An in vivo system for directed experimental evolution of rabbit haemorrhagic disease virus.

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