Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula.

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.

Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

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.

European and American subgroup C isolates of avian metapneumovirus belong to different genetic lineages.

The gene encoding the attachment glycoprotein (G) was sequenced in three French isolates of-subgroup C avian metapneumovirus (APV-C) from ducks. With 1771 nt, this gene proved as long as recently published for North-American APV-C isolates from turkeys. The nt sequences of the duck viruses shared 99% identity but proved only 75-83% identical with their North-American counterparts, viruses of both origins encoding 585 amino acid (aa)-long G proteins. Alignments revealed more homogeneity within the European and North-American groups (at least 98 and 79% aa identity, respectively) than between European and North-American viruses (at best 70% a identity), and confirmed the presence of an extracellular divergent domain (positions 302-484) in APV-C G. A phylogenetic analysis demonstrated that North-American and French isolates of APV-C belonged to significantly different genetic lineages, in agreement with the different geographical origin and host species of these viruses.

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.

Evidence of Muscovy duck parvovirus in Muscovy ducklings in California.

Muscovy duck parvovirus (MDPV) has been demonstrated in tissue samples from one- to four-week-old commercially reared Muscovy ducks that were weak, unable to walk and had a high mortality rate. On postmortem examination, the thigh and leg muscles, and the myocardium were found to be pale, and there was a fibrinous exudate on the capsule of the liver, and ascites. The parvovirus was isolated in embryonated Muscovy duck eggs and visualised by negative stain electron microscopy, detected by polymerase chain reaction (PCR) directly from the tissues, and antibodies to it were detected by immunoelectron microscopy, ELISA and immunofluorescence. In addition, the PCR products obtained that represented 1625 bp (74 per cent) of the capsid vP1 gene, including a hypervariable region between Derzsy's disease virus or goose parvovirus and MDPV, were sequenced and shown to be 100 per cent homologous with the MDPV 89384 reference strain, but only 82.3 per cent homologous with Derzsy's disease virus.