First detected case of rabbit Haemorrhagic disease virus 2 (RHDV2) in the Irish hare (Lepus timidus hibernicus)

Background Rabbit haemorrhagic disease virus (RHDV) is a Lagovirus, a subgroup of the family Caliciviridae. RHDV2 is a variant first described in France in 2010, and has since spread globally. It has been reported in several Lagomorph species (rabbits, hares, and their relatives) as well as other mammals including voles and shrews. The disease has raised international concerns for its potential impact on population abundance trajectories, particularly as 25% of Lagomorphs are currently Red-Listed by the International Union for the Conservation of Nature (IUCN). The Irish hare (Lepus timidus hibernicus) is a subspecies of the mountain hare, L. timidus, and is endemic to Ireland, making it an Evolutionarily Significant Unit of intrinsic value. Case presentation The first case of RHDV2 was detected in a wild Irish hare in July 2019. The individual exhibited atypical neurological behaviour (running in circles) prior to death. On necropsy, pink tinged foam was seen in the trachea and congestion was noted in the lungs, but there was no evidence of haemorrhages in any other organ. Both the liver and spleen were tested by reverse transcription real time qPCR confirming high levels of RHDV2 RNA. Histopathology confirmed multifocal necrotising hepatitis. Conclusion The Irish hare is susceptible to RHDV2 infection. Further investigation is warranted to explore the clinical, epidemiological, and population biology implications.

Frequent Intergenotypic Recombination Between the Non-Structural and Structural Genes is a Major Driver of Epidemiological Fitness in Caliciviruses

The diversity of lagoviruses (Caliciviridae) in Australia has increased considerably in recent years. By the end of 2017, five variants from three viral genotypes were present in populations of Australian rabbits, while prior to 2014 only two variants were known. To understand the evolutionary interactions among these lagovirus variants we monitored their geographical distribution and relative incidence over time in a continental-scale competition study. Within three years of the incursion of rabbit haemorrhagic disease virus 2 (RHDV2, denoted genotype GI.1bP-GI.2 [polymerase genotype]P-[capsid genotype]) into Australia, two novel recombinant lagovirus variants emerged: RHDV2-4e (genotype GI.4eP-GI.2) in New South Wales and RHDV2-4c (genotype GI.4cP-GI.2) in Victoria. Although both novel recombinants contain non-structural genes related to those from benign, rabbit-specific, enterotropic viruses, these variants were recovered from the livers of both rabbits and hares that had died acutely. This suggests that the determinants of host and tissue tropism for lagoviruses are associated with the structural genes, and that tropism is intricately connected with pathogenicity. Phylogenetic analyses demonstrated that the RHDV2-4c recombinant emerged independently on multiple occasions, with five distinct lineages observed. Both the new RHDV2-4e and 4c recombinant variants replaced the previous dominant parental RHDV2 (genotype GI.1bP-GI.2) in their respective geographical areas, despite sharing an identical or near-identical (i.e. single amino acid change) VP60 major capsid protein with the parental virus. This suggests that the observed replacement by these recombinants was not driven by antigenic variation in VP60, implicating the non-structural genes as key drivers of epidemiological fitness. Molecular clock estimates place the RHDV2-4e recombination event in early to mid-2015, while the five RHDV2-4c recombination events occurred from late 2015 through to early 2017. The emergence of at least six viable recombinant variants within a two-year period highlights the high frequency of these events, detectable only through intensive surveillance, and demonstrates the importance of recombination in lagovirus evolution.

Chimeric VLPs Bearing VP60 from Two Serotypes of Rabbit Haemorrhagic Disease Virus Are Protective against Both Viruses

The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.

Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d strain: genome sequencing, in vivo virus replication kinetics, and viral dose effect

Background Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00–21). Results We performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10− 1 viral genome copies), low (104), high (107) and very high (1011) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00–21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5′ untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00–21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 104 gRNA copies, whereas between 104 and 107 gRNA copies were required to also induce mortality. Conclusions These results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5′ untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00–21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies.

Microbial metagenomic approach uncovers the first rabbit haemorrhagic disease virus genome in Sub-Saharan Africa

Rabbit Haemorrhagic Disease (RHD) causes high morbidity and mortality in rabbits and hares. Here, we report the first genomic characterization of lagovirus GI.2 virus in domestic rabbits from sub-Saharan Africa. We used an unbiased microbial metagenomic Next Generation Sequencing (mNGS) approach to diagnose the pathogen causing the suspected outbreak of RHD in Ibadan, Nigeria. The liver, spleen, and lung samples of five rabbits from an outbreak in 2 farms were analyzed. The mNGS revealed one full and two partial RHDV2 genomes on both farms. Phylogenetic analysis showed close clustering with RHDV2 lineages from Europe (98.6% similarity with RHDV2 in the Netherlands, and 99.1 to 100% identity with RHDV2 in Germany), suggesting potential importation. Subsequently, all the samples were confirmed by RHDV virus-specific RT-PCR targeting the VP60 gene with the expected band size of 398 bp for the five rabbits sampled. Our findings highlight the need for increased genomic surveillance of RHDV2 to track its origin, understand its diversity and to inform public health policy in Nigeria, and Sub-Saharan Africa.

Retrospective studies on rabbit haemorrhagic disease outbreaks caused by RHDV GI.2 virus on farms in France from 2013 to 2018

Rabbit haemorrhagic disease (RHD) is a critical health threat to the rabbit industry in Europe. In 2018, the French rabbit industry adopted a voluntary control plan against this disease. In this context, two epidemiological studies were conducted on RHD outbreaks that occurred between 2013 and 2018 in France. The objectives were to describe the spread of RHD due to the new genotype RHDV GI.2 (rabbit haemorrhagic disease virus GI.2) and to identify rearing factors influencing the occurrence of the disease in order to guide the prevention measures recommended in the control plan. An analysis of cases on 295 farms between 2013 and 2017 showed that 32% of farms were affected at least once; the incidence of the disease increased in 2016-2017 compared to 2013-2015. Farms already affected in 2013-2015 had a higher risk of being infected in 2016-2017 than those that remained unaffected until 2015 (Relative Risk and 95% Confident Interval 1.7 [1.1-2.7]). A case-control study carried out between 2016 and 2018 on 37 outbreaks and 32 control farms revealed variability in biosecurity and decontamination practices between farms. The risk of being infected tends to be linked to these practices, but certain structural factors (e.g. the manure disposal system, transfer of rabbits at weaning) could also influence the risk of virus introduction into farms. In the context of a limited vaccination coverage of the farms (only females are vaccinated), these hypotheses will be studied further, using information from the RHD outbreak monitoring system implemented at the same time as the control plan in 2018.