A new HaCV-EBHSV recombinant lagovirus circulating in European brown hares (Lepus europaeus) from Catalonia, Spain.

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.

Utilizing Molecular Epidemiology and Citizen Science for the Surveillance of Lagoviruses in Australia.

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses.

The genus Lagovirus of the family Caliciviridae contains some of the most virulent vertebrate viruses known. Lagoviruses infect leporids, such as rabbits, hares and cottontails. Highly pathogenic viruses such as Rabbit haemorrhagic disease virus 1 (RHDV1) cause a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 h of infection, killing over 95 % of susceptible animals. Research into the pathophysiological mechanisms that are responsible for this extreme phenotype has been hampered by the lack of a reliable culture system. Here, we report on a new ex vivo model for the cultivation of lagoviruses in cells derived from the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus). We show that three different lagoviruses, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids, but not in monolayer cultures derived from cat (Felis catus) or mouse (Mus musculus) organoids. Virus multiplication was demonstrated by (i) an increase in viral RNA levels, (ii) the accumulation of dsRNA viral replication intermediates and (iii) the expression of viral structural and non-structural proteins. The establishment of an organoid culture system for lagoviruses will facilitate studies with considerable implications for the conservation of endangered leporid species in Europe and North America, and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

Digital PCR (dPCR) Quantification of miR-155-5p as a Potential Candidate for a Tissue Biomarker of Inflammation in Rabbits Infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV).

MicroRNAs (miRNAs, miRs) are a group of small, 17-25 nucleotide, non-coding RNA sequences that, in their mature form, regulate gene expression at the post-transcriptional level. They participate in many physiological and pathological processes in both humans and animals. One such process is viral infection, in which miR-155 participates in innate and adaptive immune responses to a broad range of inflammatory mediators. Recently, the study of microRNA has become an interesting field of research as a potential candidate for biomarkers for various processes and disease. To use miRNAs as potential biomarkers of inflammation in viral diseases of animals and humans, it is necessary to improve their detection and quantification. In a previous study, using reverse transcription real-time quantitative PCR (RT-qPCR), we showed that the expression of ocu-miR-155-5p in liver tissue was significantly higher in rabbits infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV) compared to healthy rabbits. The results indicated a role for ocu-miR-155-5p in Lagovirus europaeus/RHDV infection and reflected hepatitis and the impairment/dysfunction of this organ during RHD. MiR-155-5p was, therefore, hypothesized as a potential candidate for a tissue biomarker of inflammation and examined in tissues in Lagovirus europaeus/RHDV infection by dPCR. The objective of the study is the absolute quantification of ocu-miR-155-5p in four tissues (liver, lung, kidney, and spleen) of rabbits infected with Lagovirus europaeus/RHDV by digital PCR, a robust technique for the precise and direct quantification of small amounts of nucleic acids, including miRNAs, without standard curves and external references. The average copy number/µL (copies/µL) of ocu-miRNA-155-5p in rabbits infected with Lagovirus europaeus GI.1a/Rossi in the liver tissue was 12.26 ± 0.14, that in the lung tissue was 48.90 ± 9.23, that in the kidney tissue was 16.92 ± 2.89, and that in the spleen was 25.10 ± 0.90. In contrast, in the tissues of healthy control rabbits, the average number of copies/µL of ocu-miRNA-155-5p was 5.07 ± 1.10 for the liver, 23.52 ± 2.77 for lungs, 8.10 ± 0.86 for kidneys, and 42.12 ± 3.68 for the spleen. The increased expression of ocu-miRNA-155-5p in infected rabbits was demonstrated in the liver (a fold-change of 2.4, p-value = 0.0003), lung (a fold-change of 2.1, p-value = 0.03), and kidneys (a fold-change of 2.1, p-value = 0.01), with a decrease in the spleen (a fold-change of 0.6, p-value = 0.002). In the study of Lagovirus europaeus/RHDV infection and in the context of viral infections, this is the first report that shows the potential use of dPCR for the sensitive and absolute quantification of microRNA-155-5p in tissues during viral infection. We think miR-155-5p may be a potential candidate for a tissue biomarker of inflammation with Lagovirus europaeus/RHDV infection. Our report presents a new path in discovering potential candidates for the tissue biomarkers of inflammation.

The potential role of scavenging flies as mechanical vectors of Lagovirus europaeus/GI.2.

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.

Immune response in the recombinant strain of Lagovirus europaeus GI.1a.

Lagovirus europaeus/GI.1 is the virus that causes severe and dangerous rabbit haemorrhagic disease (RHD) in rabbits. Recombination formation in RHD viruses is common. Recombination is thought to play a key role in the evolution of lagoviruses and therefore most likely influences the pathogenicity of L. europaeus/GI strains. Immunological events also play a key role in the control of RHD, and an in-depth knowledge of these phenomena provides insights into the characteristics of the infection, which can help implement appropriate infection control measures. To obtain a more complete picture of RHD caused by different GI.1 strains, it is necessary to correlate the genetic diversity within L. europaeus/GI.1 strains and the immune picture in response to infection. We performed a phylogenetic analysis of the L. europaeus/GI strains and compared the recombinant L. europaeus/GI.1 strain with the GI.1a strain on the basis of a thorough statistical analysis of immunological traits performed previously. Our phylogenetic analysis based on the sequence of the gene encoding the VP60 capsid protein of 34 strains of Lagovirus europaeus showed that the Hartmannsdorf strain forms a separate clade from the other GI.1a strains and is separate from the GI.1b-d strains. Next, we showed significant differences in the levels of individual parameters for non-specific cellular and humoral immunity in infection with the GI.1a strain and the Hartmannsdorf recombinant strain. Against the background of this study, our results indicate that the characteristics of each recombinant should be considered individually.

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.

European Brown Hare Syndrome in Poland: Current Epidemiological Situation.

European brown hare syndrome (EBHS) is one of the main causes of mortality in brown hares (Lepus europaeus) and mountain hares (Lepus timidus) in Europe. Since the mid-1990s, this highly lethal and contagious plague has been widespread in many European countries, contributing to a drastic decline in the number of free-living and farmed hares. A second lagovirus, able to infect some species of hares is rabbit haemorrhagic disease virus 2 (RHDV2; GI.2) recognised in 2010, a new viral emergence of RHDV (GI.1) which is known to be responsible for haemorrhagic disease in rabbits-RHD. The aim of this study was to evaluate the current EBHS epidemiological situation on the basis of the presence of antibodies to European brown hare syndrome virus (EBHSV) and anti-RHDV2 antibodies in sera collected from free-ranging hares in Central and Southeastern Poland in 2020-2021. Additionally, studies on the presence of EBHSV and RHDV2 antigens or their genetic material in the blood and internal organs taken from brown hares between 2014 - 2021 have been carried out. The results of the serological examination showed nearly 88% of tested blood samples were positive for EBHSV antibodies. No EBHSV was identified in the examined hares using virological and molecular tests. The positive results of EBHS serological studies confirmed the circulation and maintenance of EBHSV in free-living brown hares in Poland. However, no serological, virological or molecular evidence was obtained indicating that the brown hares tested had been in contact with RHDV2.

Occurrence of Lagovirus europaeus (Rabbit Hemorrhagic Disease Virus) in Domestic Rabbits in Southwestern Poland in 2019: Case Report.

Lagovirus europaeus (rabbit hemorrhagic disease virus [RHDV]) is a small, nonenveloped, single-stranded RNA virus that causes a severe, highly infectious, and fatal disease in rabbits (Oryctolagus cuniculus) called rabbit hemorrhagic disease (RHD). Since its discovery in the 1980s, it has posed a very serious threat to the global rabbit industry and the rabbit population in the wild. According to data from 2005 to 2018, the occurrence of RHD has been reported or suspected in 50 countries, with more than one-half of the reports being recorded in European countries. The main aim of the study was to detect Lagovirus europaeus (RHDV) strains found in domestic rabbits that died suddenly in the city of Wroclaw in southwest Poland. All animals (n = 14) tested in this study died naturally and showed macroscopic features at necropsy that indicated the possibility of death from RHD. As a result of the research, the presence of L. europaeus virus was confirmed in 8 samples of all 14 samples collected. All strains of Lagovirus europaeus isolated in the present study showed 100% nucleotide identity to L. europaeus GI.1 strain FRG and a strain isolated in New Zealand, as well as the L. europaeus GI.1a Erfurt strain. This suggests that it is likely that L. europaeus GI.2 strains have so far not displaced L. europaeus GI.1 strains from the environment in Poland. IMPORTANCE Lagovirus europaeus (RHDV) causes a severe, highly infectious, and fatal disease in rabbits called RHD. The disease is a very serious threat to the global rabbit industry and the rabbit population in the wild. The aim of the study was to detect Lagovirus europaeus (RHDV) strains in domestic rabbits that died suddenly in Poland. The presence of RHDV was confirmed in 8 samples of all 14 samples collected. This is one of the very few reports on the existence of this virus in pet rabbits in Poland.

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.

Genetic diversity and evolution of Hare Calicivirus (HaCV), a recently identified lagovirus from Lepus europaeus.

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.

Genetic variability and phylogenetic analysis of Lagovirus europaeus strains GI.1 (RHDV) and GI.2 (RHDV2) based on the RNA-dependent RNA polymerase (RdRp) coding gene.

Lagovirus europaeus GI.1 (RHDV-rabbit haemorrhagic disease virus) and GI.2 (RHDV2-rabbit haemorrhagic disease virus 2), family Caliciviridae, genus Lagovirus, are etiological factors of the rabbit haemorrhagic disease (RHD). This small RNA virus is a great model for tracking the variability and evolution of RNA viruses, because it uses an RNA-dependent RNA polymerase (RdRp) to replicate its own genetic material. This polymerase determines the fidelity and the rates of replication and mutation of the virus, conditioning its adaptation to the environment and even to a new host, and thus influencing evolution of the virus. The aim of this study was to determine the genetic variability and phylogenetic relationships of 105 Lagovirus europaeus strains with different genotypes based on the RdRp gene. The strains came from around the world in the years of 1987-2017. The aforementioned group of 105 strains included 14 strains whose RdRp sequences were obtained and analysed in this study, and the rest were retrieved from GenBank: 74 strains classified as genotype GI.1 (RHDV), 14 as GI.2 (RHDV2), 2 strains of Lagovirus europaeus not assigned to any genotype, and a MRCV strain, the sequences of which were collected from GenBank. Among the 14 strains whose RdRp sequences were obtained in this study, the highest variability was presented in the Austrian 237 strain from 2004. The genetic distance between the Austrian 237 strain and the remaining thirteen analysed strains ranged from 0.117 to 0.123 (from 11.7% to 12.3% nucleotide substitutions). The lowest variability, however, was recorded for Hungarian, Czech and Austrian strains. On the phylogenetic tree, the 14 analysed strains were allocated into GI.1c (G2), GI.1d (G3-G5) and GI.1a (RHDVa). Analysis of the genetic variability of the 105 strains of Lagovirus europaeus indicated a growing genetic distance between the strains, both in time and location. Phylogenetic analysis showed a division of the strains into seven groups, dictated by the chronology, geographical location and evolutionary events in the history of the virus, such as mutations and recombinations.

GI.1b/GI.1b/GI.2 recombinant rabbit hemorrhagic disease virus 2 (Lagovirus europaeus/GI.2) in Morocco, Africa.

Rabbit hemorrhagic disease virus (RHDV) is highly lethal to the European rabbit (Oryctolagus cuniculus). It was first reported in 1984 in China, but in 2010, a new variant of the virus was detected (GI.2) in France. Several recombination events with pathogenic and non-pathogenic strains have been described. Here, we report the first sequences of RHDV in Africa, isolated from Moroccan rabbits, and these resemble GI.1b/GI.1b/GI.2 recombinants found in the Iberian Peninsula. Monitoring and characterization of strains from future outbreaks are advised to guarantee the success of current programs on small-rabbit production for poverty alleviation in African countries.

Large-scale lagovirus disease outbreaks in European brown hares (Lepus europaeus) in France caused by RHDV2 strains spatially shared with rabbits (Oryctolagus cuniculus).

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.

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.

Comparative analysis of rabbit hemorrhagic disease virus (RHDV) and new RHDV2 virus antigenicity, using specific virus-like particles.

In 2010 a new Lagovirus related to rabbit haemorrhagic disease virus (RHDV) emerged in France and has since rapidly spread throughout domestic and wild rabbit populations of several European countries. The new virus, termed RHDV2, exhibits distinctive genetic, antigenic and pathogenic features. Notably, RHDV2 kills rabbits previously vaccinated with RHDV vaccines. Here we report for the first time the generation and characterization of RHDV2-specific virus-like particles (VLPs). Our results further confirmed the differential antigenic properties exhibited by RHDV and RHDV2, highlighting the need of using RHDV2-specific diagnostic assays to monitor the spread of this new virus.

Molecular evolution and antigenic variation of European brown hare syndrome virus (EBHSV).

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.

The genetic analysis of new Polish strains of European brown hare syndrome virus.

In this paper we describe recently occurring outbreaks of European brown hare syndrome (EBHS) in a captive hare population. The aim of our study was to evaluate the phylogenetic position of detected Polish strains compared to other European strains of EBHSV. Investigations were undertaken in hares from different provinces of Poland. Liver or spleen samples were tested for viral RNA using the RT-nested PCR method and the products were subsequently sequenced. The genetic analysis was based on the fragment of gene encoding viral capsid protein; it revealed a high homology and close relationship between Polish and European EBHSV strains isolated between 2001 and 2011.

Polarisation of major histocompatibility complex II host genotype with pathogenesis of European Brown Hare syndrome virus.

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead), collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1%) hares; 35 (20.6%) had liver lesions not typical of the syndrome, 50 (29.4%) had lesions in other tissues and 61 (35.9%) had no lesions. Sixty five (38.2%) of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively). Within the peptide binding region codons the number of nonsynonymous substitutions (dN) was much higher than synonymous substitutions (dS), which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006) frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027). These data reveal a polarisation between EBHSV pathogenesis and MHC class II genotype within the European brown hare in Denmark.

Complete coding sequences of European brown hare syndrome virus (EBHSV) strains isolated in 1982 in Sweden.

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.