Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus)

Abstract

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.

Keywords: Adaptive evolution; Biological control; Calicivirus; Emerging disease; Genetic resistance; Rabbit; Transmission; Virulence.

Figure 1

Maximum likelihood (ML) tree showing the phylogenetic relationships among 40 complete genomes of RHDV. All branches are scaled according to the number of nucleotide substitutions per site, and relevant bootstrap values >90% are denoted by asterisks. The monophyletic grouping of RHDV from Australia and New Zealand, derived from the imported Czech CAPM V-351 strain, are shaded grey, while the three Turretfield viruses (TUR06, TUR07 and TUR09) are shown in bold italics. The tree is mid-point rooted for purposes of clarity only. The insert shows the mean d_N/d_S value and the amino acid (AA) changes on those branches associated with the evolution of TUR06, TUR07 and TUR09. The single site associated with positive selection across RHDV has a whole – amino acid residue 12 in the p16 protein – fixes a mutation on the branch leading to the three Turretfield viruses and is colored red.

Figure 2

A: Kaplan Meier Survival analysis of rabbits infected with different virus isolates. S: Surviving rabbits were euthanized 14 d.p.i.B: Increasingly shorter survival times and higher mortality rates for the more recent isolates are strongly associated.

Figure 3

Viral RNA copy numbers in liver tissue were quantified using qRT-PCR. Virus load is expressed as genome copies per mg tissue. B: Cumulative virus load of rabbits that died within a specified time. The cumulative virus load is the product of the number of rabbits that died and the viral genome copy number in their liver. Grey bars indicate the cumulative load of all dead rabbits, black bars the cumulative load of all rabbits that died within the first 60 hours, before the onset of adaptive immune responses.