Rabbit haemorrhagic disease virus (RHDV) is a positive sense RNA virus in the family Caliciviridae. It causes a necrotizing hepatitis in European rabbits (Oryctolagus cuniculus), with a mortality rate of approximately 90% in susceptible adult rabbits. In Australia, the RHDV Czech strain V-351 has been widely used as a rabbit biocontrol agent since its release in 1995. However, the current effectiveness of this strain in the field is limited by widespread immunity, and also by emerging genetic resistance.

This study aims to develop an experimental platform for the isolation and characterisation of antigenically novel RHDV strains for ongoing sustainable rabbit biocontrol in Australia. As RHDV does not replicate in cell culture, antigenic variants were selected by serially passaging a highly virulent RHDV field isolate in laboratory rabbits that had been passively immunised with increasing monoclonal antibody concentrations. At each passage, viruses that replicated despite the presence of neutralising antibodies were recovered at necropsy. Virus load was quantified using qRT-PCR and whole genome sequencing was performed using the Illumina platform. Additionally, genetic variation within each viral isolate was investigated by whole genome SNP analysis.

There was clear phenotypic and genetic evidence of rapid viral evolution, at both the virus consensus and intra-host level, in response to selection with neutralising antibodies. Of particular note were two amino acid substitutions in the P2 domain of the capsid protein that rapidly became dominant within the virus population, likely because the changes conferred partial resistance to neutralising antibodies. Interestingly, there was also clear evidence for selection of synonymous substitutions, most notably in the viral polymerase and capsid genes.

These findings demonstrate proof of concept that RHDV evolution can be experimentally manipulated to select for variants with altered immunological characteristics. This may ultimately lead to the generation of novel RHDV serotypes that are able to overcome pre-existing immunity to currently used rabbit biocontrol strains.