Evaluation of the virulence of RHDV and mechanisms of host resistance – CALILAGO

Rabbit haemorrhagic Disease Virus (RHDV) is a highly lethal pathogen of the calicivirus family that emerged in the 1980s causing dramatic mortalities in rabbits (Oryctolagus cuniculus) and locally threatening other species that depend on rabbits for survival. The origin and evolution of RHDV are not well understood. Phylogenetic analyses showed that RHDV strains cluster chronologically, a pattern common in RNA viruses that likely results from the strong selective pressure exerted by the host adaptive immunity. Detection of positive selection in the RHDV capsid within the major antigenic determinants supports this hypothesis. Interestingly, the selected codons are associated with putative N-glycosylation sites, suggesting that glycosylation might also have a role in RHDV infection and virulence. Recombination within the RHDV genome appears common. However, most phylogenetic studies have focused on partial sequences of the capsid that may not allow a full assessment of the virus evolution. Host ligands of RHDV have been characterized. Thus, pathogenic strains bind to histo-blood group antigen (HBGAs) H/A/B type 2/Ley present on the surface of epithelial cells of the respiratory and digestive tracts. Synthesis of these antigens requires several glycosyltransferases; an a1,2-fucosyltransferase which in rabbits is encoded by three genes, Fut1, Fut2 and Sec1 that underwent multiple events of gene conversion, an a1,3-fucosyltransferase and either A or B transferases encoded by Abo genes. Preliminary data suggest that there are at least 6 Abo genes arranged in tandem in the rabbit genome. Previously, a link between a Sec1 allele and resistance to RHDV was observed. Furthermore, the expression of H/A/B type 2/Ley is highly variable in wild rabbits and an evolution of RHDV strains in their ability to recognize these host glycans leading to recognition of distinct individuals was documented. This suggested a progressive adaptation to the host HBGA polymorphism. Caliciviruses exhibit high levels of host switching, but cross-species has not been observed in RHDV. Nevertheless, RHDV RNA was isolated from micromammals living in sympatry with RHDV-infected rabbits opening the possibility of a wider host range. RHDV has also the ability to recognize HBGAs in likely points of entry into human cells.
In this project, we propose to sequence the complete genomes of strains isolated in the Iberic Peninsula and France since the first reported RHDV outbreaks. With this, we intend to study if different host genetic backgrounds have contributed to different viral evolutionary rates and if these are constant along the genome. These sequences will also allow the full assessment of the role of recombination in shaping viral evolution. In light of previous results, the role of glycosylation in RHDV virulence will be also be evaluated.
The genetics underlying rabbit HBGAs diversity will be evaluated by studying polymorphisms in the glycosyltransferases genes. Functional studies will determine if these polymorphisms affect enzyme expression and/or activity and their potential role in RHDV resistance. Availability of historical rabbit samples will allow study the evolution of their frequency in parallel with the evolution of the virus HBGAs recognition ability in order to document a co-evolution of the host-pathogen pair.
Cross-species transmission of RHDV to other species will be evaluated by screening species that might be in contact with RHDV-infected rabbits such as rodents, carnivores, artiodactyla, humans and hares. With this, we hope to assess the role of other species in RHDV epidemiology and the importance of RHDV as a zoonotic disease.