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Shah and colleagues [...].

In 2020/2021, several European brown hare syndrome virus (EBHSV) outbreaks were recorded in European hares (Lepus europaeus) from Catalonia, Spain. Recombination analysis combined with phylogenetic reconstruction and estimation of genetic distances of the complete coding sequences revealed that 5 strains were recombinants. The recombination breakpoint is located within the non-structural protein 2C-like RNA helicase (nucleotide position ~ 1889). For the genomic fragment upstream of the breakpoint, a non-pathogenic EBHSV-related strain (hare calicivirus, HaCV; GII.2) was the most closely related sequence; for the rest of the genome, the most similar strains were the European brown hare syndrome virus (EBHSV) strains recovered from the same 2020/2021 outbreaks, suggesting a recent origin. While the functional impact of the atypical recombination breakpoint remains undetermined, the novel recombinant strain was detected in different European brown hare populations from Catalonia, located 20-100 km apart, and seems to have caused a fatal disease both in juvenile and adult animals, confirming its viability and ability to spread and establish infection. This is the first report of a recombination event involving HaCV and EBHSV and, despite the recombination with a non-pathogenic strain, it appears to be associated with mortality in European brown hares, which warrants close monitoring., (© 2024. The Author(s).)

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.

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 is a viral disease that emerged in the 1980s and causes high mortality and morbidity in the European rabbit (Oryctolagus cuniculus). In 2010, a new genotype of the rabbit haemorrhagic disease virus emerged and replaced the former circulating Lagovirus europaeus/GI.1 strains. Several recombination events have been reported for the new genotype Lagovirus europaeus/GI.2, with pathogenic (variants GI.1a and GI.1b) and benign (genotype GI.4) strains that served as donors for the non-structural part while GI.2 composed the structural part; another recombination event has also been described at the p16/p23 junction involving GI.4 strains. In this study, we analysed new complete coding sequences of four benign GI.3 strains and four GI.2 strains. Phylogenetic and recombination detection analyses revealed that the first GI.2 strains, considered as non-recombinant, resulted from a recombination event between GI.3 and GI.2, with GI.3 as the major donor for the non-structural part and GI.2 for the structural part. Our results indicate that recombination contributed to the emergence, persistence and dissemination of GI.2 as a pathogenic form and that all described GI.2 strains so far are the product of recombination. This highlights the need to study full-genomic sequences of lagoviruses to understand their emergence and evolution.

Recombination is one of the major sources of genetic variation in viruses. RNA viruses, such as rabbit hemorrhagic disease virus (RHDV), are among the viruses with the highest recombination rates. Several recombination events have been described for RHDV, mostly as a consequence of their genomic architecture. Here, we undertook phylogenetic and recombination analyses of French and Swedish RHDV strains from 1994 to 2016 and uncovered a new intergenotypic recombination event. This event occurred in the late 1990s/early 2000s and involved nonpathogenic GI.3 strains as donors for the nonstructural part of the genome of these recombinants, while pathogenic GI.1d strains contributed to the structural part. These GI.3P-GI.1d recombinant strains did not entirely replace GI.1d (nonrecombinant) strains, but became the dominant strains in France and Sweden, likely due to a fitness advantage associated with this genomic architecture. GI.3P-GI.1d (P stands for polymerase) strains persisted until 2013 and 2016 in Sweden and France, respectively, and cocirculated with the new genotype GI.2 in France. Since strains from the first GI.2 outbreaks were GI.3P-GI.2, we hypothesize that GI.3P-GI.1d could be the parental strain. Our results confirm the outstanding recombination ability of RHDV and its importance in the evolution of lagoviruses, which was only revealed by studying complete genomic sequences.

First recognized as highly pathogenic viruses, hare lagoviruses belonging to genotype GII.1 (EBHSV) infect various Lepus species. Genetically distinct benign lagoviruses (Hare Calicivirus, HaCV) have recently been identified but few data have been available so far on these strains. The analysis of 199 samples from hunted hares collected throughout France allowed the detection of 20 HaCV and showed that they were widely distributed in this country. Ten HaCV capsid protein gene sequences were characterized. A first HaCV capsid protein structural model was proposed, revealing a global structure similar to that of a pathogenic GII.1 strain. The HaCV sequences showed an even higher genetic diversity than previously appreciated, with the characterization of two genotypes (GII.2, GII.3) and several additional putative genotypes. The most recent common ancestor for HaCV VP60 gene was estimated to be much older than that for GII.1 pathogenic strains. These results give new insights into the phylogenetic relationships of HaCV within the Lagovirus genus., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The first full-genome sequence of a hare calicivirus (HaCV), recently characterized as a novel member of the Caliciviridae , is described. This presumed nonpathogenic lagovirus is 7,433 nucleotides long, shows the same genomic organization as that of other lagoviruses, and has the highest nucleotide identity (79%) with pathogenic European brown hare syndrome viruses.

We report the full genome sequence of the non-pathogenic rabbit lagovirus Lagovirus europaeus/GI.3/O cun/FR/2006/06-11 (GI.3/06-11), collected from a healthy French domestic rabbit in 2006, and initially described as 06-11 strain. The sequence reveals a genomic organization similar to lagoviruses. It was 7,436 bases long and contained two open reading frames (ORF). A dipeptide variation at the potential p23/2C-like helicase cleavage site (EE instead of ED) was observed, a feature only shared with non-recombinant pathogenic lagoviruses in GI.2 and with two European brown hare syndrome viruses (EBHSV) collected in 1982 in Sweden. GI.3/06-11 has only one initiation codon at the beginning of the ORF2 like the avirulent Italian rabbit calicivirus (RCV) and EBHSV. Previous genetic analyses based on the capsid gene sequences showed that GI.3/06-11 was closer to the RCV and pathogenic lagoviruses GI.1 strains than other lagoviruses. This study, by revealing that GI.3/06-11 genome sequence significantly clustered with pathogenic GI.2 strains, gives prominence of new genetic relationship among lagoviruses and should contribute to understand the emergence of pathogenic strains.

Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus , respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged, and many nonpathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, nonpathogenic lagoviruses, and EBHSV potentially play a role in determining the host range and virulence of lagoviruses. We observed binding to A, B, or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits ( Oryctolagus cuniculus ), which have recently been classified as GI strains. However, we could not explain the emergence of virulence, since similar glycan specificities were found in several pathogenic and nonpathogenic strains. In contrast, EBHSV, recently classified as GII.1, bound to terminal β-linked N -acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species ( Oryctolagus cuniculus , Lepus europaeus , and Sylvilagus floridanus ) showed species-specific patterns regarding susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagovirus host specificity and range. IMPORTANCE Lagoviruses constitute a genus of the family Caliciviridae comprising highly pathogenic viruses, RHDV and EBHSV, that infect rabbits and hares, respectively. Recently, nonpathogenic strains were discovered and new pathogenic strains have emerged. In addition, host jumps between lagomorphs have been observed. The mechanisms responsible for the emergence of pathogenicity and host species range are unknown. Previous studies showed that RHDV strains attach to glycans expressed in the upper respiratory and digestive tracts of rabbits, the likely portals of virus entry. Here, we studied the glycan-binding properties of novel pathogenic and nonpathogenic strains looking for a link between glycan binding and virulence or between glycan specificity and host range. We found that glycan binding did not correlate with virulence. However, expression of glycan motifs in the upper respiratory and digestive tracts of lagomorphs revealed species-specific patterns associated with the host ranges of the virus strains, suggesting that glycan diversity contributes to lagovirus host ranges., (Copyright © 2018 American Society for Microbiology.)

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.

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.

European brown hare syndrome virus (EBHSV) is the aetiological agent of European brown hare syndrome (EBHS), a disease affecting Lepus europaeus and Lepus timidus first diagnosed in Sweden in 1980. To characterize EBHSV evolution we studied hare samples collected in Sweden between 1982 and 2008. Our molecular clock dating is compatible with EBHSV emergence in the 1970s. Phylogenetic analysis revealed two lineages: Group A persisted until 1989 when it apparently suffered extinction; Group B emerged in the mid-1980s and contains the most recent strains. Antigenic differences exist between groups, with loss of reactivity of some MAbs over time, which are associated with amino acid substitutions in recognized epitopes. A role for immune selection is also supported by the presence of positively selected codons in exposed regions of the capsid. Hence, EBHSV evolution is characterized by replacement of Group A by Group B viruses, suggesting that the latter possess a selective advantage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Lagovirus is an emerging genus of Caliciviridae, which includes the Rabbit Hemorrhagic Disease Virus (RHDV) of rabbits and the European brown hare syndrome virus (EBHSV) of hares that cause lethal hepatitis. In 2010, a new RHDV related virus (RHDV2) with a unique genetic and antigenic profile and lower virulence was identified in France in rabbits. Here we report the identification of RHDV2 as the cause in Sardinia of several outbreaks of acute hepatitis in rabbits and Cape hare (Lepus capensis mediterraneus). This is the first account of a lagovirus that causes fatal hepatitis in both rabbits and hares.

European brown hare syndrome (EBHS) is characterised by high mortality of European brown hares (Lepus europaeus) and mountain hares (Lepus timidus). European brown hare syndrome virus (EBHSV) and the closely related rabbit haemorrhagic disease virus (RHDV) comprise the genus Lagovirus, family Caliciviridae. In contrast to RHDV, which is well studied, with more than 30 complete genome sequences available, the only complete genome sequence available for EBHSV was obtained from a strain isolated in 1989 in France. EBHS was originally diagnosed in Sweden in 1980. Here, we report the complete coding sequences of two EBHSV strains isolated from European brown hares that died with liver lesions characteristic of EBHS in Sweden in 1982. These sequences represent the oldest complete coding sequences of EBHSV isolated from the original area of virus diagnosis. The genomic organisation is similar to that of the published French sequence. Comparison with this sequence revealed several nucleotide substitutions, corresponding to 6 % divergence. At the amino acid level, the Swedish strains are 2 % different from the French strain. Most amino acid substitutions were located within the major capsid protein VP60, but when considering the amino acid sequence length of each protein, VP10 is the protein with the highest percentage of amino acid differences. The same result was obtained when Swedish strains were compared. This evolutionary pattern has not been described previously for members of the genus Lagovirus.

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.

Since 2006, the members of the molecular epidemiological working group of the European "EPIZONE" network of excellence have been generating sequence data on avian influenza and avian paramyxoviruses from both European and African sources in an attempt to more fully understand the circulation and impact of these viruses. This review presents a timely update on the epidemiological situation of these viruses based on sequence data generated during the lifetime of this project in addition to data produced by other groups during the same period. Based on this information and putting it all into a European context, recommendations for continued surveillance of these important viruses within Europe are presented., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50-90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level.

Developing vaccines for livestock provides researchers with the opportunity to perform efficacy testing in the natural hosts. This enables the evaluation of different strategies, including definition of effective antigens or antigen combinations, and improvement in delivery systems for target antigens so that protective immune responses can be modulated or potentiated. An impressive amount of knowledge has been generated in recent years on vaccine strategies and consequently a wide variety of antigen delivery systems is now available for vaccine research. This paper reviews several antigen production and delivery strategies other than those based on the use of live viral vectors. Genetic and protein subunit vaccines as well as alternative production systems are considered in this review., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Infections with H5/H7 low-pathogenic avian influenza (LPAI) viruses are now notifiable because such viruses can mutate into highly pathogenic avian influenza viruses, leading to serious problems for both animal and public health. Domestic ducks can play a crucial role in the transmission of H5 LPAI viruses to other poultry. Although prime boost vaccination using, respectively, a recombinant vaccine and an inactivated vaccine was shown to be protective in ducks against H5N1 highly pathogenic avian influenza, vaccination of domestic ducks against H5 LPAIV is poorly documented. However, substituting inactivated vaccines with subunit vaccines might be more advantageous. In this context, we generated a triple recombinant baculovirus composed of HA and NA proteins derived from a French H5N3 LPAI virus strain and the M protein derived from an Italian H7N1 LPAI virus strain. We describe a molecular construction strategy that enabled the development of virus-like particles (VLPs). Western blot analyses and neuraminidase inhibition assay of cell supernatants purified by sucrose density gradient ultracentrifugation showed that HA, NA and M1 proteins were expressed and co-released. Electron microscopy examination revealed VLPs that were morphologically identical to wild-type virus. Immunogold electron microscopy demonstrated that H5 and N3 proteins were present on the VLP surface, and haemagglutination and neuraminidase assays showed that the H and N proteins, respectively, were biologically active. In addition, VLP immunogenicity (induction of haemagglutination-inhibiting antibodies) was demonstrated in specific pathogen free Muscovy ducks. According to our successful previous experimental results of protection in ducks following vaccination with the three baculovirus-expressed proteins, the present results make feasible the reliable use of H5N3 VLPs as a subunit vaccine in this species.

In Asia, domestic ducks have been shown to play a pivotal role in H5 high-pathogenicity avian influenza virus transmission. We have also observed that the same situation may exist for H5 low-pathogenicity avian influenza (LPAI) virus. No data are available regarding the protection afforded by commercial inactivated vaccines against H5 LPAI virus infection in ducks, and two preliminary experiments using commercial inactivated vaccines gave poor results. Virus-like particles (VLPs) have been shown to be immunogenic in different species. With regard to the influenza model, the matrix (M) protein has been shown to be necessary for the formation of VLPs. In order to attempt to develop a VLP influenza vaccine expressing hemagglutinin and neuraminidase (NA) of interest, we generated a triple recombinant baculovirus (rB) expressing three structural proteins: H5, N3, and M, derived from a recent French LPAI virus strain. Although the three proteins were successfully expressed in rB-infected cells and displayed the expected biological activity, no VLPs were observed. Despite this result, the protection afforded to ducks by rB-infected cell lysates was assessed and was compared with the protection afforded by an inactivated commercial H5N9 vaccine. For this purpose, specific-pathogen-free Muscovy ducks (15 per group) received rB-infected cell lysates (3 wk apart), while a second group received the H5N9 vaccine. Ten days after the boost, a homologous virus challenge was implemented. Both vaccines induced positive hemagglutination inhibition titers and M immune response, whereas lysates of rB-infected cells elicited NA immune response. Tracheal and cloacal sheddings were measured using M-based real-time-reverse transcription-polymerase chain reaction and were compared with the sheddings of vaccinated and unvaccinated infected controls. Lysates of rB-infected cells afforded a significant decrease of cloacal shedding and a delayed peak of tracheal shedding, whereas the inactivated commercial vaccine afforded a significant decrease of tracheal shedding only.

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.

Although muscovy duck reovirus (DRV) shares properties with the reovirus isolated from chicken, commonly named avian reovirus (ARV), the two virus species are antigenically different. Similar to the DRV sigmaB-encoded gene (1201 bp long) previously identified, the three other double-stranded RNA small genome segments of DRV have been cloned and sequenced. They were 1325, 1191 and 1124 bp long, respectively, and contained conserved terminal sequences common to ARVs. They coded for single expression products, except the smallest (S4), which contained two overlapping open reading frames (ORF1 and ORF2). BLAST analyses revealed that the proteins encoded by the 1325 and 1191 bp genes shared high identity levels with ARV sigmaA and sigmaNS, respectively, and to a lesser extent with other orthoreovirus counterparts. No homology was found for the S4 ORF1-encoded p10 protein. The 29.4 kDa product encoded by S4 ORF2 appeared to be 25% identical to ARV S1 ORF3-encoded sigmaC, a cell-attachment oligomer inducing type-specific neutralizing antibodies. Introduction of large gaps in the N-terminal part of the DRV protein was necessary to improve DRV and ARV sigmaC amino acid sequence alignments. However, a leucine zipper motif was conserved and secondary structure analyses predicted a three-stranded alpha-helical coiled-coil feature at this amino portion. Thus, despite extensive sequence divergence, DRV sigmaC was suggested to be structurally and probably functionally related to ARV sigmaC. This work provides evidence for the diversity of the polycistronic S class genes of reoviruses isolated from birds and raises the question of the relative classification of DRV in the Orthoreovirus genus.