Molecular epidemiology of Rabbit Haemorrhagic Disease Virus in Australia: when one became many

Abstract

Rabbit Haemorrhagic Disease Virus (RHDV) was introduced into Australia in 1995 as a biological control agent against the wild European rabbit (Oryctolagus cuniculus). We evaluated its evolution over a 16-year period (1995-2011) by examining 50 isolates collected throughout Australia, as well as the original inoculum strains. Phylogenetic analysis of capsid protein VP60 sequences of the Australian isolates, compared with those sampled globally, revealed that they form a monophyletic group with the inoculum strains (CAPM V-351 and RHDV351INOC). Strikingly, despite more than 3000 rereleases of RHDV351INOC since 1995, only a single viral lineage has sustained its transmission in the long-term, indicative of a major competitive advantage. In addition, we find evidence for widespread viral gene flow, in which multiple lineages entered individual geographic locations, resulting in a marked turnover of viral lineages with time, as well as a continual increase in viral genetic diversity. The rate of RHDV evolution recorded in Australia -4.0 (3.3-4.7) × 10(-3) nucleotide substitutions per site per year - was higher than previously observed in RHDV, and evidence for adaptive evolution was obtained at two VP60 residues. Finally, more intensive study of a single rabbit population (Turretfield) in South Australia provided no evidence for viral persistence between outbreaks, with genetic diversity instead generated by continual strain importation.

Keywords: European rabbit; Rabbit Haemorrhagic Disease Virus; biocontrol; epidemiology; evolution; phylogeny.

Fig. 1

Map showing the sampling location of field RHDV isolates in Australia. Numbers on the map correspond to the isolate reference numbers in Table 1 and colours to the state/territory of origin as documented in Fig. 3. No map reference points are given for the two inoculum strains; CAPM V-351/1988 (sequence 1) and RHDV351INOC/1995 (sequence 2).

Fig. 2

Phylogenetic relationships of RHDV sampled globally, including those from Australia. The major clusters of RHDV, as well as their geographic origins, are noted. Also shown are the phylogenetic positions of (i) the original Czech (CAPM V-351) strain, (ii) three viruses from New Zealand that fall within the phylogenetic diversity of the Australian viruses, and (iii) the background virus closest to the Australian cluster, Germany/M67473/1989. Bootstrap values (>70%) are shown for key nodes and all horizontal branches are drawn to a scale of nucleotide substitutions per site. Background RHDV sequences are listed in Table S1 (supporting information).

Fig. 3

Phylogenetic (Maximum Clade Credibility – MCC) tree depicting the evolutionary relationships among the Australian RHDV sequences. As the tree assumes a relaxed molecular clock, tip times reflect the date of sampling, and an estimated real-time (year) time-scale is given on the x-axis. Sequences are colour-coded according to their state/territory of sampling in Australia: Australian Capital Territory (ACT) = green, New South Wales (NSW) = blue, South Australia (SA) = red, Tasmania (TAS) = grey, Western Australia (WA) = pink, and those from outside Australia (i.e. inoculum strains) are unshaded. Numbers after the decimal place in the virus names denote the fractional year (e.g. 2008.75 = September 2008), and the date of the common ancestor of the Australian viruses (1991.4–1995.4) inferred from this analysis is shown next to the relevant node. Viruses sampled from Turretfield (SA) are numbered according to date of sampling. All branches supported by posterior probability values >0.95 are marked by a * symbol.

Fig. 4

Bayesian skyride analysis of RHDV in Australia depicting changing estimates of relative genetic diversity (y-axis) plotted against time from the youngest sampled sequence (x-axis). The solid black line represents the median value while the shaded region of the plot depicts the 95% HPD values.