Polymerase chain reaction amplification and gene sequence analysis of a calicivirus from a feral rabbit

A reverse transcription-polymerase chain reaction (RT-PCR) amplification assay was used to detect calicivirus gene sequences in a liver tissue derived from a feral rabbit which died of a recent outbreak of rabbit haemorrhagic disease (RHD) in New Zealand. Five pairs of primers were designed to amplify five complementary DNA genomic sequence stretching from nucleotide positions 1594 to 7071, yielding amplified fragments of 361, 340, 805,670 and 386 bp for the primer pairs RC-1/RC-2, RC-3/RC-4, RC-5/RC-6, RC-7/RC-8 and RC-9/RC-10 respectively. The identity of the amplified fragments was confirmed by chemiluminescence Southern blot hybridization and direct cycle sequencing. The nucleotide sequences of the five amplified fragments were determined and comparisons of the nucleotide and deduced amino acid sequences revealed a close genetic relationship of the New Zealand isolate 97-10372 with overseas strains of RHD virus.