Molecular and serological studies of Rabbit haemorrhagic disease virus in Otago

Abstract

Rabbit haemorrhagic disease virus (RHDV) is a member of the Caliciviridae with high morbidity and mortality rates in susceptible European rabbit (Oryctolagus cuniculus) populations. The pathogenicity and restricted host range of this virus has lead to its use as a biological control agent for wild rabbit populations in New Zealand and Australia. RHDV was discovered in New Zealand in late August 1997 on a property in Central Otago. Initial kill rates of this virus varied, but tended to be in the range of 70-90%, resulting in a substantial reduction in rabbit numbers.

The presence of a non-pathogenic rabbit calicivirus in New Zealand had been suggested by serological testing of rabbit serum samples collected prior to the known release of RHDV in this country. This possibility was investigated in the current study using molecular techniques. Tissue samples were collected from 75 shot rabbits and tested for the presence of the proposed non-pathogenic virus by reverse-transcriptase polymerase chain reaction. The nucleotide sequences of the resulting amplicons were determined. The derived nucleotide sequences showed high similarity (2:98.9%) to the V351 strain ofRHDV suggesting that the amplified fragments were likely to be from RHDV not the putative benign virus. Although no molecular evidence of a non-pathogenic rabbit calicivirus was obtained, the possibility that such a virus may exist cannot be excluded. It is possible that the agent responsible for causing the cross-reactive antibodies may not be a calicivirus, or may have a substantially different genome to the currently sequenced Lagovirus isolates.

Serological data were collected annually from 10 monitor sites in Otago in 1999 (390 samples), 2000 (396 samples) and 2001 (385 samples). These data were used to estimate the proportions of immune rabbits and to investigate factors that may influence the efficacy of RHDV in Otago. Additional serological data from a survey performed in late 1997 (507 samples) were included in comparisons where appropriate. The proportions of immune rabbits in each annual survey ranged from 44-56% overall. Between sites the proportions of immune rabbits ranged from 0-80%. In all surveys from 1999-2001 the proportion of immune individuals was greater among adult (27 weeks old or greater) rabbits than among young (26 weeks old or less) rabbits. No differences were identified between the proportions of immune male and female rabbits. Although the proportions of immune rabbits remained reasonably static in the four surveys considered in this study, the large variation in the proportions of immune rabbits between sites and also within individual sites from survey to survey indicates that there are site- or area-specific factors which may affect the exposure and kill-rate of RHDV in Otago. Effective use of secondary control was identified as a variable that appears to affect the efficacy ofRHDV. Sites with moderate to high secondary control (typically shooting) tended to have lower rabbit numbers and also fewer rabbits immune to RHDV than sites with low or no secondary control.

Three regions of the viral genome (helicase, polymerase and capsid) were amplified and sequenced to investigate the amount of genetic variation of RHDV in New Zealand. Analysis of the resulting nucleotide sequences revealed a maximum rate of change of 0.36%/year for the non-structural regions (helicase and polymerase) and 0.35%/year for the structural region (capsid). This suggests that the structural genes have not been subject to greater selection pressure from the host immune system than non-structural genes and therefore have a similar rate of change. The maximum rate of change over the 1719 nucleotides sequenced for any isolate was 0.33%/year. Phylogenetic analysis of the nucleotide sequences indicated a possible divergence of the North and South Island isolates. All regions of the genome examined produced similar phylogenetic trees.

The research presented in this thesis shows that RHDV in persisting in Otago and provides baseline data for future serological and molecular studies of RHDV in New Zealand. Topics that need to be addressed in future studies include determining whether the antibodies detected in serum collected prior to the discovery of RHDV in New Zealand can protect against challenge with RHDV, determining the role of insects in RHDV transmission in New Zealand and the development of a methodology for screening for the emergence of attenuated virus strains or genetically resistant rabbits.