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Background: Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00-21)., Results: We performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10 - 1 viral genome copies), low (10 4 ), high (10 7 ) and very high (10 11 ) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00-21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5' untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00-21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 10 4 gRNA copies, whereas between 10 4 and 10 7 gRNA copies were required to also induce mortality., Conclusions: These results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5' untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00-21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies., (© 2021. The Author(s).)

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that causes rabbit haemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus). In 2010, a new genotype called RHDV2 emerged in France. It exhibits a larger host range than classical RHDV strains by sporadically infecting different hare species, including the European hare (Lepus europaeus). Phylogenetic analyses revealed that closely related RHDV2 strains circulate locally in both hares and rabbits, and therefore that RHDV2 strains infecting hares do not belong to a lineage that has evolved only in this species. We showed that RHDV2 is widely distributed in France and that it was responsible for more than a third of cases of lagovirus disease in European hare populations in 2015. The oldest RHDV2 positive hare was sampled in November 2013 and we reported two hares co-infected by EBHSV and RHDV2. All together, our results raise important epidemiological and evolutionary issues. In particular, along with the potential emergence of recombinant EBHSV/RHDV2 strains in hares, the enlargement of the host range changes the host population structure of RHDV2 and may alter the impact of the virus on rabbit and hare populations.

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.

The data were recorded during a Rabbit haemorrhagic disease outbreak that occurred in France in 2001 in a wild population of rabbits that we have been monitoring since 2000. These data suggested the existence of non-protective antibodies due to a putative RHDV-like virus. Twenty-one blood and 22 liver samples were taken from the 26 corpses of recently dead rabbits that were found. RHDV was found in all liver samples. A first screening for RHD antibodies, carried out using an ELISA based on the detection of VP60-RHDV antigen, showed that 20 of the rabbits were seropositive. Moreover, we determined antibody titres for 13 of these 20 seropositive samples. All were > or = 1/400. Such titres normally indicate antibody levels sufficient to confer protection to all known RHDV or RHDV-like strains. For 16 samples, we determined whether these rabbits had died of a chronic or an acute form of the disease, by employing monoclonal antibody (Mabs)--based differential ELISA. All had died of an acute form of RHD. Because the antibodies detected by this VP60-ELISA test are known to appear 5-6 days after infection and since acute RHD generally kills the rabbits 2-3 days after infection, we assumed that the detected antibodies must have been present before the exposure to the virus that killed these rabbits. A second detection of antibodies was made with Mabs that are specific for RHDV. The results were negative, showing that the antibodies detected with the VP60 ELISA test were not specific for RHDV. We sequenced a portion of the VP60 gene of viruses isolated in 17 rabbits. All RHDV isolates were very similar to the RHDV strains commonly isolated in France during this period, suggesting that this viral strain was not a putative variant that is not neutralised by antibodies. Therefore we conclude that the detected antibodies were probably due to a RHDV-like virus that induces the production of detectable but non-protective antibodies.

Simple Summary: Rabbit Haemorrhagic Disease is caused by a virus that affects the liver, the spleen and the lungs of rabbits, causing hepatitis, splenomegaly and haemorrhages. A new genotype of the virus was first reported in France in 2010 and has spread globally since then, replacing most of the circulating former viruses in many countries. The detection of the virus and the differentiation of both genotypes is of crucial importance for disease surveillance. In this article, a rapid test for antigen detection is described and evaluated, providing the first description of a quick and easy-to-use test that allows for the simultaneous detection and differentiation of the genotypes. A total of 136 samples, rabbit liver samples and liver exudates (liquid collected after freeze–thawing) classified as infected and non-infected, were analysed, with good results. These data confirm that the developed rapid test can be used as a reliable diagnostic test for disease surveillance, especially in farms and the field. Rabbit Haemorrhagic Disease Virus 2 (RHDV2, recently named Lagovirus europaeus/GI.2) was first reported in France in 2010 and has spread globally since then, replacing most of the circulating former RHDV (genotype GI.1) in many countries. The detection and differentiation of both genotypes is of crucial importance for the surveillance of the disease. In this article, a duplex lateral flow assay (LFA) for antigen detection is described and evaluated, providing the first description of a quick and easy-to-use test that allows for the simultaneous detection and differentiation of RHDV genotypes GI.1 and GI.2. A panel of GI.1- or GI.2-infected and non-infected rabbit liver samples and liver exudates (136 samples) was analysed, obtaining a total sensitivity of 94.4% and specificity of 100%. These data confirm that the developed duplex LFA can be used as a reliable diagnostic test for RHD surveillance, especially in farms and the field. [ABSTRACT FROM AUTHOR]