The complete nucleotide sequence of rabbit haemorrhagic disease virus (Czech strain V351): use of the polymerase chain reaction to detect replication in Australian vertebrates and analysis of viral population sequence variation

Abstract

The complete nucleotide sequence of the Czech strain of rabbit haemorrhagic disease virus (RHDV) was determined to be 7437 nucleotides in length with a 5′-terminal non-coding region of 9 nucleotides and a 3′-terminal non-coding region of 59 nucleotides. Two open reading frames (ORFs) were found within this sequence coding for polypeptides of 2344 (nucleotides 10–7044) and 117 amino acids (nucleotides 7025–7378). The sequence of this isolate was approximately 1% different from that reported by Meyers et al., having 78 nucleotide changes which resulted in 30 amino acid differences, the majority of these clustering in the N-terminus of the large ORF and the middle of the viral coat protein. Only a single conservative amino acid change was seen in the smaller 3′-terminal ORF. Since the virus cannot at present be propagated in tissue culture, but isolated only after replication in rabbits, the reported sequence must be considered as a consensus sequence from the viral population. To gain some understanding of the possible sequence diversity within this virus population, 97 clones were sequenced from a polymerase chain reaction (PCR) fragment to determine the sequence diversity of the virus population. Four major classes of variant were described with mutations generally in the third base position of codons. A nested reverse transcriptase (RT)-PCR (using sequence derived for the coat protein of RHDV) was used to determine the presence or absence of RHDV inoculated into non-host animal species. No replication of the virus was detected in 28 different vertebrate species other than rabbits. PCR tests on both mosquitoes and fleas feeding on RHDV infected rabbits were positive. The RT-PCR test was more sensitive when compared with an antigen capture ELISA to detect the presence of genomic RNA/or virus in infected rabbits.