Poster Presentation 8th Australasian Virology Society Meeting and 11th Annual Meeting of the Australian Centre for Hepatitis & HIV Virology Meeting 2015

RHD-Accelerator: a pipeline for directed evolution of Rabbit haemorrhagic disease virus to generate antigenic variants. (#142)

Robyn N Hall 1 2 , Nadya Urakova 1 2 3 , Markus Matthaei 2 , Stephanie Haboury 2 , Michael Frese 1 2 3 4 , Lorenzo Capucci 5 , Peter J Kerr 6 , Tanja Strive 1 2 4
  1. Invasive Animals Cooperative Research Centre, University of Canberra, Canberra, ACT, Australia
  2. CSIRO Health and Biosecurity, CSIRO, Canberra, ACT, Australia
  3. Health Research Institute, University of Canberra, Canberra, ACT, Australia
  4. Institute for Applied Ecology, University of Canberra, Canberra, ACT, Australia
  5. IZSLER, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini”, Brescia, Italy
  6. School of Biological Sciences, The University of Sydney, Sydney, NSW, Australia

Rabbit haemorrhagic disease virus (RHDV) is a positive sense RNA virus in the family Caliciviridae. It causes a necrotising 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 biocontrol agent since its release in 1995. However, the current effectiveness of this strain in the field is limited by widespread immunity and emerging genetic resistance.

This study aims to develop an experimental platform for the selection and characterisation of antigenically novel RHDV strains. 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 a neutralising monoclonal antibody. Virus load was quantified 3-4 days after infection using qRT-PCR. Whole genome sequencing of each virus was performed using the Illumina platform and the genetic variation within each viral isolate was investigated by whole genome SNP analysis.

There was genetic evidence of rapid viral evolution, at both the virus consensus and intra-host level, over serial passage. Of particular interest are 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. Furthermore, several synonymous substitutions were detected that were maintained in subsequent passages, most notably in the viral polymerase and capsid genes.

Later virus passages were increasingly able to overcome shorter incubation times prior to the application of the selection pressure. This suggests that the virus may be adapting by increasing replication speed, in addition to antigenic variation. These findings demonstrate proof of concept that RHDV evolution can be experimentally manipulated to select for variants with altered characteristics.