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

Abstract

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.

Evaluation of the usefulness of laboratory diagnostic methods in RHD outbreak

“…The study showed the lack of amplification of STR 2012 genetic material with P1 primer (5182-5201) from the C-terminus of the RHDV RdRp polymerase gene. The primer set P1/P2, originally proposed by Guittré et al (21), has been used for many years in many laboratories for routine amplification of classic RHDV strains, including HA-negative phenotypic variants (23,26). Similar difficulties were encountered in the amplification of cDNAs of the samples L1-L6 with the pair of primers N3-N4 encoding the N-terminal part of the VP60 gene.…”

“…Previous studies have shown the presence of the classic form of RHDV strains with an altered haemagglutination (HA) profile (phenotypic HAnegative variant), RHDV strains known as antigenic and genetic variants (RHDVa), a moderately pathogenic calicivirus (MRCV), a recently new French RHDV variant, and non-pathogenic caliciviruses (RCV, RCV-A1, Lambay, 06-11) (1,4,5,6,24,26,27,34,36). Pathogenic RHDV and RHDVa isolates belong to one serotype (2,3,10,36).…”

Diversity of RHD virus: epidemiological, diagnostic and immunological importance

“…Stopniowy zanik występowania poszczególnych grup genetycznych na świecie obserwuje się od końca lat 90. (44,54). Enklawą klasycznego RHDV w Europie był do niedawna Półwysep Pirenejski, gdzie diagnozowano go jeszcze 2008 r. (3,4,22).…”

“…Na zakażenie podatne są jedynie króliki Oryctolagus cuniculus w takim samym przedziale wiekowym, jak w przypadku RHDV, głównie hodowlane, ale także dzikie (14, 62). Wszystkie izolaty podtypu RHDVa wyosobnione dotąd na świecie należą do genogrupy G6 (11,36,44,48,54). W porównaniu do klasycznego wirusa RHD, podtyp RHDVa wykazuje znaczne zmiany genetyczne, sprzyjające selekcji i zwiększające moż-liwości przetrwania.…”

Serological evidence for a non-protective RHDV-like virus

“…For this purpose, RNA was extracted and purified from 100 µL of liver exudate obtained after thawing by an immunocapture assay [17] or with the RNeasy Kit (Qiagen, Venlo, The Netherlands). Then, a 559 bp fragment from the 3' end of the gene was amplified by RT-PCR with RHDVAU and RHDVAL degenerated primers and for two isolates, the complete VP60 encoding sequence was amplified [18]. The amplified products were analysed by conventional electrophoresis on agarose gel and purified (Geneclean II kit, Bio 101, Qbiogene, Montreal, Canada).…”