Restricted Host Specificity of Rabbit Hemorrhagic Disease Virus Is Supported by Challenge Experiments in Immune-compromised Mice (Mus musculus) Open Access

Rabbit hemorrhagic disease virus (RHDV; Caliciviridae) is a highly pathogenic virus of domestic and wild European rabbits (Oryctolagus cuniculus; Abrantes et al. 2012). In Australia and New Zealand, RHDV has been successfully employed since the mid-1990s to control wild rabbit populations, resulting in substantial benefits to agricultural industries and the environment (Cooke 2002). Although there is no evidence that RHDV replicates in species other than lagomorphs, the repeated detection of RHDV-specific antibodies and viral RNA in animals sympatric with European rabbits (Frölich et al. 1998; Parkes et al. 2004; Merchán et al. 2011) warrants further investigation.

Genetically targeted interferon (IFN) −α/β receptor knockout (IFNAR^−/−) mice (Mus musculus) were originally generated to investigate the physiologic role of type I IFNs (Müller et al. 1994). These mice lack the IFNAR β subunit and thus do not possess a functional receptor for type I IFNs, which largely prevents upregulation of IFN-stimulated genes during virus infection, despite higher than normal IFN protein levels. Because they lack an effective innate immune response, IFNAR^−/− mice can be a useful model for caliciviruses (Thackray et al. 2012) and other viruses that struggle to replicate in immune competent mice (Müller et al. 1994; Hefti et al. 1999; Orozco et al. 2012). We inoculated IFNAR^−/− mice with RHDV to test whether animals with a severely compromised innate immune system can support virus replication and develop disease. The laboratory mouse is a close relative of Mus spretus, a species sympatric with rabbits in Europe, that reportedly supports RHDV replication and dissemination (Merchán et al. 2011).

Seventeen IFNAR^−/− mice and seven IFNAR^+/+ control mice were challenged with 0.1 mL of a commercial virus preparation containing 9×10⁷ RHDV capsid gene copies, equating to >300 rabbit lethal dose 50% (as titrated in susceptible adult rabbits; stock virus was obtained from the Elizabeth Macarthur Agricultural Institute, Menangle, New South Wales, and was guaranteed to contain >3,000 infectious dose 50% per milliliter suspension). Concurrently, five IFNAR^−/− mice and three IFNAR^+/+ control mice were mock infected. Animals were monitored daily for weight loss and signs of disease until they were killed by cervical dislocation on d 1, 2, 3, 4, 7, or 21 postinoculation (three mice at each time point, except for two mice at the last time point). Animal experiments were carried out at the University of Canberra in accordance with the Australian Animal Research Act and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes; researchers involved in the work were authorized, and procedures were approved by the University of Canberra's animal ethics committee (authorization reference no. CEAE 13-02; date of approval, 8 March 2013). Liver and spleen samples were collected, and the virus load was measured by quantitative real-time (RT)-PCR. Tissue samples (50 mg) were homogenized with 1-mm glass beads in a Precellys Dual 24 tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France), RNA was extracted by using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and quantitative RT-PCR was performed by using the One-Step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, California, USA). Reactions were set up manually in 96-well plates with duplicate 10-μL reactions that included 1 μL of the extracted RNA or previously described standards (Matthaei et al. 2014) and forward (5′-ACCCAGTACGGCACRGGCTCCCAACCAC-3′) and reverse (5′-CTATCTCCATGAAACCAGATGCAAAGGT-3′) primers. Conditions for each reaction step were 50 C for 10 min for reverse transcription, 95 C for 5 min for initial denaturation, 41 cycles at 95 C for 10 s, 63 C for 40 s, and 78 C for 10 s for DNA amplification and data acquisition, and 65–95 C (5 s increments with 5 s per increment) for the melt curve analysis.

None of the inoculated animals developed any signs of disease or lost more than 5% of body weight. However, trace amounts of viral RNA were found in some spleen and liver samples from RHDV-inoculated mice up to 3 d (spleen) and 7 d (liver) postinoculation (Fig. 1a, b, respectively). The detected viral load was very low, close to the detection limit of the assay (4×10⁵ capsid gene copies per g tissue; Matthaei et al. 2014), and did not significantly increase over time. Furthermore, there was no significant difference in viral load between the IFNAR^−/− mice and IFNAR^+/+ control mice. For example, viral loads in samples taken 2 d postinoculation were not significantly different between IFNAR^−/− mice and IFNAR^+/+ mice, in either liver (P=0.197) or spleen samples (P=0.562). Unpaired t-tests were performed by using GraphPad Prism 7.00 (GraphPad Software, La Jolla, California, USA). At the conclusion of the experiment (21 d postinoculation), even trace amounts of viral RNA were no longer detectable.

Antibodies against lagoviruses or viral RNA or both have repeatedly been detected in animals, such as foxes (Vulpes vulpes) and mice (M. spretus, Apodemus sylvaticus) that live sympatrically with rabbit populations (Frölich et al. 1998; Merchán et al. 2011; Rocha et al. 2017). The RHDV replicates to high titers in rabbits, and virions are environmentally stable; the virus can survive for at least 3 mo in animal tissues and for at least 10 d in the environment (Henning et al. 2005). Environments are heavily contaminated during outbreaks, and it is likely that nonhost species ingest virions through predation, scavenging, grazing, or grooming activities. In addition, scratching and biting may introduce viral antigens and trigger adaptive immune responses in the absence of virus replication, which could explain seroconversion in foxes and other sympatric predatory species. Alternatively, yet undetected caliciviruses may exist that cross-react antigenically with RHDV (Frölich et al. 1998).

During lagovirus outbreaks, infected rabbits and hares (Lepus europaeus and related species) are likely to be consumed by predators, such as foxes (Frölich et al. 1998; Chiari et al. 2016). Because lagovirus particles are extremely robust (Lieberman et al. 1992), they may resist degradation in the gastrointestinal tract of predators and scavengers. Simón et al. (1994) fed dogs (Canis lupus familiaris) a liver homogenate prepared from RHDV-infected rabbits and showed that feces collected from these dogs contained enough infectious virus particles to infect rabbits and cause disease. The dogs remained healthy throughout the duration of the study, suggesting that predators and scavengers can disseminate infectious lagoviruses in their territories without becoming infected themselves.

The detection of trace amounts of RHDV RNA using highly sensitive laboratory PCR techniques cannot be considered sufficient evidence for a productive infection and virus replication. While characterizing a new DNA vaccine against RHDV, Yuan et al. (2013) found that at 10 d postinoculation, four out of six control rabbits that had received the inactivated virus control contained residual amounts of viral RNA in the liver. Their finding suggested that it takes considerable time to clear RHDV RNA if it is delivered in genuine virus particles. Claims of RHDV replication in animals other than lagomorphs—let alone claims of RHDV-induced morbidity—should be substantiated by additional data, for example, an increase in viral RNA over time, the presence of replicative intermediates, and the detection of viral antigens in tissues suspected to support replication of the virus. To date, no such evidence has been reported. Therefore, all claims of an extended host range for RHDV, especially if they include species outside the order Lagomorpha, should be treated with caution. Before the release of RHDV as a biocontrol agent in Australia, a wide range of species, including one reptile, six birds, eight marsupials, and 14 placental mammal species were experimentally inoculated with RHDV but in no case was evidence for virus replication found (Buddle et al. 1997; Gould et al. 1997).

The most likely explanation for the data we present and similar findings (Zheng et al. 2003) is that sensitive PCR methods detect traces of the virus inoculum that persist for some time before being cleared. Because we found no evidence for virus replication or disease in either normal or severely immune-compromised laboratory mice, we conclude that RHDV cannot replicate in this species or, alternatively, that replication is abortive and does not yield detectable RNA levels. This confirms the widespread but not universally accepted notion that RHDV does not replicate or cause disease in animals outside the order Lagomorpha. The reason for the high degree of host specificity of RHDV is presently unknown. The existence of lagomorph-specific attachment and/or entry receptors that are expressed in a tissue- and age-dependent manner may explain the ability of RHDV to infect only certain species and tissues and the inability of some strains to cause disease in young rabbits. Histo-blood group antigens (HBGA) have been described as attachment receptors for RHDV (Ruvoën-Clouet et al. 2000), and certain rabbit HBGA can confer increased susceptibility to RHDV in a strain-specific manner (Nyström et al. 2011). However, HBGA with the capacity to bind RHDV are expressed in a variety of tissues and mammalian species, and their expression pattern does not always correlate with susceptibility to RHDV infection. A true RHDV entry receptor has not yet been identified; therefore, one can only speculate about the role of entry receptors in the species specificity of RHDV and other lagoviruses. Innate immune responses have been reported to represent a species barrier for some viruses (Wang et al. 2004). However, our results suggest that the host specificity of lagoviruses is not a consequence of restrictive innate immune responses present in mice but absent in lagomorphs.

Our results confirm that lagoviruses possess a very narrow host range. During RHDV outbreaks, predators and scavengers (and small rodents) are frequently exposed to large amounts of infectious virus, which may lead to seroconversion and has enabled researchers to use PCR-based methods to detect RHDV RNA in a variety of species. Animal species other than rabbits, in particular, animals with large territories, such as foxes or wild dogs, may serve as passive carriers and distribute RHDV over large distances. However definitive evidence for virus replication in nonlagomorph species remains elusive.

Funding for this work was obtained from the Invasive Animals Cooperative Research Centre (IACRC), grant IACRC 3.L.4. The IACRC and a University of Canberra International Postgraduate Research Scholarship supported N.U. We thank Stéphanie Haboury and Jacqui Richardson for technical assistance, and Jackie Mahar and Matthew Neave for critical reading of the manuscript.