Stéphane Bertagnoli

Detection of a new variant of rabbit haemorrhagic disease virus in France

WE wish to report the detection of a new variant of rabbit haemorrhagic disease virus (RHDV) (Lagovirus, Caliciviridae) which is circulating in France and has been causing high mortality in domestic and wild rabbit populations since the end of the summer of 2010.nnRabbit haemorrhagic disease (RHD)

Emergence of a new lagovirus related to Rabbit Haemorrhagic Disease Virus

Since summer 2010, numerous cases of Rabbit Haemorrhagic Disease (RHD) have been reported in north-western France both in rabbitries, affecting RHD-vaccinated rabbits, and in wild populations. We demonstrate that the aetiological agent was a lagovirus phylogenetically distinct from other lagoviruses and which presents a unique antigenic profile. Experimental results show that the disease differs from RHD in terms of disease duration, mortality rates, higher occurrence of subacute/chronic forms and that partial cross-protection occurs between RHDV and the new RHDV variant, designated RHDV2. These data support the hypothesis that RHDV2 is a new member of the Lagovirus genus. A molecular epidemiology study detected RHDV2 in France a few months before the first recorded cases and revealed that one year after its discovery it had spread throughout the country and had almost replaced RHDV strains. RHDV2 was detected in continental Italy in June 2011, then four months later in Sardinia.

Characterisation of a non-pathogenic and non-protective infectious rabbit lagovirus related to RHDV

The existence of non-pathogenic RHDV strains was established when a non-lethal virus named rabbit calicivirus (RCV) was characterised in 1996 in Italy. Since then, different RNA sequences related to RHDV have been detected in apparently healthy domestic and wild rabbits, and recently a new lagovirus was identified in Australia. We have characterised from seropositive healthy domestic rabbits a non-lethal lagovirus that differs from RHDV in terms of pathogenicity, tissue tropism and capsid protein sequence. Phylogenetic analyses have revealed that it is close to the Ashington strain and to the RCV, but distinct. We proved experimentally that it is infectious but non-pathogenic and demonstrated that, contrary to the other described non-pathogenic lagoviruses, it induces antibodies that do not protect against RHDV. Our results indicate the existence of a gradient of cross-protection between circulating strains, from non-protective, partially protective to protective strains, and highlight the extent of diversity within the genus Lagovirus.

Risk of zoonotic transmission of HEV from rabbits

n Abstractn n Hepatitis E virus strains from rabbits indicate that these mammals may be a reservoir for HEVs that cause infection in humans. Further issues remain to be clarified, including whether the genotype of rabbit HEV differs from human and swine HEV genotype 3 and whether rabbit HEV can infect human and other animals.n HEV was found in farmed rabbits in several geographic areas of China, in USA and more recently in France. The prevalence of antibodies against HEV was 36%, 57% and 55% in rabbits from Virginia (USA), Gansu Province and Beijing (China), respectively. HEV RNA was detected in 16.5% of serum samples from farmed rabbits in Virginia, 7.5% in Gansu Province and 7.0% in Beijing. HEV RNA was detected in 7% of bile samples from farmed rabbits and in 23% of liver samples from wild rabbits in France. The full-length genomic sequences analysis indicates that all the rabbit strains belong to the same clade. Nucleotide sequences were 72.2–78.2% identical to HEV genotypes 1–4. Comparison with HEV sequences of human strains circulating in France and reference sequences identified a human strain closely related to rabbit HEV. A 93-nucleotide insertion in the X domain of the ORF1 of the human strain and in all the rabbit HEV strains was found. Moreover, the ability of rabbit HEV to cause cross-species infection in a pig model has recently been demonstrated. Rabbit HEV can replicate efficiently in human cell lines. Collectively, these data support the possibility of zoonotic transmission of HEV from rabbits.n n

Proposal for a unified classification system and nomenclature of lagoviruses

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970-1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision.

Emergence of Pathogenicity in Lagoviruses: Evolution from Pre-existing Nonpathogenic Strains or through a Species Jump?

1 InBIO—Research Network in Biodiversity and Evolutionary Biology, CIBIO, Campus de Vairão, Universidade do Porto, Vairão, Portugal, 2 Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal, 3 CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Gandra, Portugal, 4 UMR 1225, INRA, Toulouse, France, 5 INP-ENVT, University of Toulouse, Toulouse, France, 6 Proteomic Unit, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini”, Brescia, Italy, 7 Department of Pathology andWildlife Diseases, National Veterinary Institute, Uppsala, Sweden, 8 Department of Studies and Research, National Hunting andWildlife Agency (ONCFS), Nantes, France, 9 Virology Unit, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini”, Brescia, Italy, 10 Avian and Rabbit Virology Immunology Parasitology Unit, Ploufragan-Plouzané Laboratory, French Agency for Food, Environmental and Occupational Health & Safety (Anses), Ploufragan, France, 11 European University of Brittany, Rennes, France, 12 Inserm U892; CNRS, UMR 6299University of Nantes, Nantes France

Myxomavirus as a vector for the immunisation of sheep: protection study against challenge with bluetongue virus.

Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.

Possible interaction between myxomatosis and calicivirosis related to rabbit haemorrhagic disease affecting the European rabbit

Serological data on myxoma virus, rabbit haemorrhagic disease (RHD) virus and RHD-like viruses in juvenile rabbits (Oryctolagus cuniculus) trapped in 1995, 1996 and 1997 in two areas of France were analysed. For each disease, the effects of bodyweight, year, month and seropositivity for the other disease were modelled by using logistic regressions. In one area, a model including RHD seropositivity was selected to explain the myxoma virus seropositivity. Models including myxoma virus seropositivity were selected to explain the RHD seropositivity in both areas, and the odds of a rabbit being seropositive to both viruses were 5˙1 and 8˙4 times higher than the odds of a rabbit being seronegative to myxoma virus and seropositive to RHD. The year and bodyweight had significant effects for myxomatosis in one area and for RHD in both areas.

Diversity of avipoxviruses in captive-bred Houbara bustard

Implementation of conservation breeding programs is a key step to ensuring the sustainability of many endangered species. Infectious diseases can be serious threats for the success of such initiatives especially since knowledge on pathogens affecting those species is usually scarce. Houbara bustard species (Chlamydotis undulata and Chlamydotis macqueenii), whose populations have declined over the last decades, have been captive-bred for conservation purposes for more than 15 years. Avipoxviruses are of the highest concern for these species in captivity. Pox lesions were collected from breeding projects in North Africa, the Middle East and Central Asia for 6 years in order to study the diversity of avipoxviruses responsible for clinical infections in Houbara bustard. Molecular and phylogenetic analyses of 113 and 75 DNA sequences for P4b and fpv140 loci respectively, revealed an unexpected wide diversity of viruses affecting Houbara bustard even at a project scale: 17 genotypes equally distributed between fowlpox virus-like and canarypox virus-like have been identified in the present study. This suggests multiple and repeated introductions of virus and questions host specificity and control strategy of avipoxviruses. We also show that the observed high virus burden and co-evolution of diverse avipoxvirus strains at endemic levels may be responsible for the emergence of novel recombinant strains.

Safety and immunogenicity of myxoma virus as a new viral vector for small ruminants

Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.