Molecular epidemiology of European brown hare syndrome virus in France between 1989 and 2003.

Genetic diversity between French European brown hare syndrome (EBHS) viruses since the disease appeared has been evaluated. Nucleotide sequencing of the partial capsid protein genes of 169 EBHS viruses collected from various parts of France between 1989 and 2003, three reference strains, and a Greek EBHSV collected in 2002 revealed a maximum nucleotide divergence of 11.7%, indicating a high level of conservation between viruses. Two major groups were identified. The first group contained EBHS viruses collected since 1989 from different parts of France, the reference strains, and all of the viruses located in the far north of France. In this group, three genogroups were clearly identified as mainly related to their geographic origin. The distribution of the viruses suggests that the early viruses have not disappeared and have slowly evolved in their area of origin. The second group, supported by a significant bootstrap value, contained the Greek EBHSV with the French EBHS viruses collected between 1999 and 2003 from regions of southern France. It constitutes a newly identified genogroup. Our results demonstrate strong differences in genetic evolution between EBHSV and rabbit haemorrhagic disease virus, with persistence of the earlier EBHS viruses and interaction between the geographical and temporal distributions.

Phylogenetic analysis of rabbit haemorrhagic disease virus in France between 1993 and 2000, and the characterisation of RHDV antigenic variants.

The first molecular epidemiological study of Rabbit haemorrhagic disease virus undertaken in France between 1988 and 1995, identified three genogroups, two of which (G1, G2) disappeared quickly. We used immunocapture-RT-PCR and sequencing to analyse 104 new RHDV isolates collected between 1993 and 2000. One isolate was obtained in 2000 from a French overseas territory, the Reunion Island. The nucleotide sequences of these isolates were aligned with those of some French RHDV isolates representative of the three genogroups previously identified, of some reference strains and German and American RHDV antigenic variants. Despite the low degree of nucleotide sequence variation, three new genogroups (G4 to G6) were identified with significant bootstrap values. Two of these genogroups (G4 and G5) were related to the year in which the RHDV isolates were collected. Genogroup G4 emerged from genogroup G3, which has now disappeared. Genogroup G5 is a new independent group. The genogroup G6 contained an isolate collected in mainland France in 1999 and the isolate collected from the Reunion Island, as well as German and American RHDV variants. Multiple sequence alignments of the VP60 gene and antigenic analysis with monoclonal antibodies demonstrated that these French isolates are two new isolates of the RHDV variant.

Immunocapture-RT-PCR assay for detection and molecular epidemiology studies of Rabbit Haemorrhagic Disease and European Brown Hare Syndrome viruses.

Rabbit Haemorrhagic Disease Virus and European Brown Hare Syndrome Virus are two members of the genus Lagovirus in the family Caliciviridae. They are the causative agents of highly contagious and fatal diseases of rabbits and hares respectively. We adjusted one assay for the detection and the genomic characterisation of each virus, based on viral purification by immunocapture and genomic amplification by reverse transcription-polymerase chain reaction (IC-RT-PCR). It is carried out directly with the liver exudate obtained after thawing and suppresses the viral nucleic acid preparation step. This assay combines the advantages of an ELISA test (rapidity) because immunocapture and the RT reaction were carried out in the same microtitre plate, and the advantages of PCR (sensitivity). The procedure described allows the processing of large numbers of samples and is suitable for phylogenetic studies of lagomorphs caliciviruses. In addition, it was compared with sandwich-ELISA used for Rabbit Haemorrhagic Disease or European Brown Hare Syndrome diagnosis. A good correlation was found between ELISA and IC-RT-PCR results for Rabbit Haemorrhagic Disease diagnosis, whereas for European Brown Hare Syndrome diagnosis, the results confirmed the higher sensitivity of the molecular method.

Impact of viral hemorrhagic disease on a wild population of European rabbits in France.

An outbreak of rabbit viral hemorrhagic disease (RVHD) and of myxomatosis occurred in a free-living population of rabbits (Oryctolagus cuniculus) near Paris (France) in 1995. Annual mortality rates were 88% in adults and 99% in juveniles. There was no difference in mortality rates between males and females. Since most adults were protected with myxoma antibodies after May, they probably died of RVHD. Mortality lasted throughout the year despite high proportions of rabbits having developed myxomatosis and RVHD antibodies, which suggests that the combination of the two diseases and the immunosuppressive characteristics of myxoma virus could be responsible for the mortality caused by RVHD. The proportion of juveniles with RVHD antibodies increased with their weight. Seroconversion against RVHD occurred in spring and autumn.