The pathogenicity comparison of Lagovirus europaeus GI.1 and GI.2 strains in China by using relative quantitative assay.

Lagovirus europaeus GI.1 belongs to Lagovirus in the Caliciviridae family. GI.1 causes an acute, septic, and highly lethal disease in rabbits. Lagovirus europaeus GI.2, a new variant of GI.1, has caused explosive mortality in rabbits of all ages in Sichuan Province, China. To explore the differences in pathogenicity of rabbits infected with GI.1/GI.2, we investigated the virulence and disease progression of a naturally occurring GI.1/GI.2 in 4-week-old, 13-week-old, and 25-week-old New Zealand White laboratory rabbits after GI.1/GI.2 infection. Objective measures of disease progression were recorded using continuous body-temperature monitoring. We observed the kittens were infected with GI.2 during the most urgent course of the disease, and GI.1 was not lethal to kittens. We found that the target organ of both GI.1 and GI.2 was the liver, but the disease course of the two viruses was differed. Our study enriches the research on the pathogenicity of GI.1 and GI.2 under the same conditions.

Phylogenetic Analysis of European Brown Hare Syndrome Virus Strains from Poland (1992-2004).

European brown hare syndrome (EBHS) is lethal to several species of free-living hares worldwide. The genetic characterization of its virus (EBHSV) strains in European circulation and epidemiological knowledge of EBHSV infections is not yet complete. The study determined the nucleotide sequences of the genomes of EBHSV strains from Poland and analyzed their genetic and phylogenetic relationships to a group of hare lagoviruses. The genome of five virus strains detected in Poland between 1992 and 2004 was obtained by RT-PCR and sequencing of the obtained amplicons. The genetic relationships of the EBHSV strains were analyzed using the full genome and VP60 gene sequences. Additionally, the amino acid sequence of the VP60 gene was analyzed to identify mutations specific to recognized EBHSV subgroups. Partial amplification of the virus open reading frame (ORF)1 and ORF2 regions obtained nearly complete nucleotide genome sequences of the EBHSV strains. Phylogenetic analysis placed them in a GII.1 cluster with other European strains related to nonpathogenic hare caliciviruses. VP60 gene analysis allocated these EBHSV strains to the G1.2, G2.2-2.3 or G3 virus genetic groups. The amino acid sequence differences in the entire genome ranged from 1.1 to 2.6%. Compared to a reference French EBHSV-GD strain, 22 variable amino acid sites were identified in the VP60 region of the Polish strains, but only six were in VP10. Single amino acid changes appeared in different sequence positions among Polish and other European virus strains from different genetic groups, as well as in VP10 sequences of nonpathogenic hare caliciviruses. The results of the study showed a high genetic homogeneity of EBHSV strains from Poland despite their different location occurrence and initial detection times. These strains are also phylogenetically closely related to other EBHSV strains circulating in Europe, likely confirming the slow evolutionary dynamics of this lagovirus species.

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.

Epidemiology, control and management of an EBHS outbreak in captive hares.

Here we describe an outbreak of European brown hare syndrome (EBHS) in a captive hare population. The EBHS outbreak occurred in March 2009, at the beginning of the breeding season. Overall mortality was 53% out of an original population of 61 animals. Animals between five and eleven months showed a significantly higher mortality rate than other age classes. Pregnant females either aborted their foetuses and survived or died pregnant. All foetuses (n=10) of the pregnant hares were PCR positive for EBHSV. Only one offspring born during the outbreak survived. Shortly after the outbreak, the surviving hares developed a specific anti-EBHSV titre between 1:80 and 1:2560, which dropped to 1:10-1:160 nine months later. Hares between one and three years of age developed a significantly higher titre than hares younger than one year or older than four years. Offspring born after the outbreak showed a lower titre of 1:10, indicating passive antibody transfer via placenta and milk. After two months, the titre was not detectable any longer. In December 2009, the captive population was vaccinated against EBHS virus with inactivated virus prepared from the organs of infected hares. The titres after the first vaccination ranged from 1:10 to 1:640, and after the second vaccination from 1:10 to 1:320. To estimate the effect of EBHS on reproduction, we compared the breeding seasons 2008 and 2009. Several possible sources of infection of the colony are discussed, but the definite cause could not be determined.

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.