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This focus article was prepared by Paul Duff of the APHA Wildlife Expert Group, with support from David Everest (APHA Pathology Department), Lucy Martindale (APHA Surveillance Intelligence Unit), Alex Barlow (Wildlife Network for Disease Surveillance), Mara Rocchi (Moredun Research Institute) and Antonio Lavazza (Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Italy)., (© 2024 British Veterinary Association.)

A new form of the rabbit haemorrhagic disease virus, RHDV2, first observed in European rabbits, has spread widely among different species of hares in Europe, jackrabbits and cottontails in North America, and hares in southern Africa. However, only limited surveillance studies have been undertaken so far. It is suggested that methods developed for controlling the disease in farmed rabbits in Europe and studying the efficacy of RHDV as a biological control agent in Australia could facilitate epidemiological research on those recently affected lagomorph species. This would enable the assessment of the risk of RHDV2 to native lagomorphs, including endangered species, and the determination of the main host species of RHDV2. Because RHDV2 has not spread equally through all lagomorph species, epidemiological studies could give insights into factors important for determining host susceptibility.

The myxoma virus species jump from European rabbits (Oryctolagus cuniculus) to Iberian hares (Lepus granatensis) has raised concerns. We assess the decline suffered by Iberian hare populations on the Iberian Peninsula and discuss the association between the effect of myxomatosis and the average abundance index, which we estimated by using hunting bags.

Rabbit haemorrhage disease virus 2 (RHDV2) is a highly pathogenic lagovirus that causes lethal disease in rabbits and hares (lagomorphs). Since its first detection in Europe in 2010, RHDV2 has spread worldwide and has been detected in over 35 countries so far. Here, we provide the first detailed report of the detection and subsequent circulation of RHDV2 in New Zealand. RHDV2 was first detected in New Zealand in 2018, with positive samples retrospectively identified in December 2017. Subsequent time-resolved phylogenetic analysis suggested a single introduction into the North Island between March and November 2016. Genetic analysis identified a GI.3P-GI.2 variant supporting a non-Australian origin for the incursion; however, more accurate identification of the source of the incursion remains challenging due to the wide global distribution of the GI.3P-GI.2 variant. Furthermore, our analysis suggests the spread of the virus between the North and South Islands of New Zealand at least twice, dated to mid-2017 and around 2018. Further phylogenetic analysis also revealed a strong phylogeographic pattern. So far, no recombination events with endemic benign New Zealand rabbit caliciviruses have been identified. This study highlights the need for further research and surveillance to monitor the distribution and diversity of lagoviruses in New Zealand and to detect incursions of novel variants.

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.

An outbreak of rabbit hemorrhagic disease virus 2 (RHDV2)-associated disease occurred in the southwestern United States following its first detection in New Mexico in March 2020. The disease spread throughout several states and was diagnosed for the first time in California on May 11, 2020, in a black-tailed jackrabbit ( Lepus californicus ). The following day, the California Department of Food and Agriculture (CDFA) issued an order banning the entrance into California of several lagomorph species and their products from any state in which the disease had been detected in the last 12 mo. RHDV2 is a threat to wild lagomorph species in California, including the endangered riparian brush rabbit ( Sylvilagus bachmani riparius ). Therefore, the California Department of Fish and Wildlife (CDFW) started tracking any mortality event in wild lagomorph populations. As of August 9, 2020, RHDV2 had been detected in wild and domestic lagomorphs of several counties in southern California that were submitted to the California Animal Health and Food Safety laboratory system by the CDFA or the CDFW. These positive cases included 2 additional black-tailed jackrabbits and 3 desert cottontail rabbits ( Sylvilagus audubonii ). In addition, the infection spilled over to domestic populations, whereby it was confirmed on July 10, 2020, in a domestic rabbit ( Oryctolagus cuniculus ).

Hepatitis E is an emerging viral disease that is the leading cause of viral hepatitis in the world. The vast majority of hepatitis E cases in developed countries are caused by zoonotic genotypes 3 and 4 of hepatitis E virus (HEV) for which pig and wild boar and to lesser extent rabbits are the main reservoir. According to recent reports rabbits are a source of human HEV infection and highlight the risk of zoonotic foodborne transmission. Here we report the molecular analysis of a novel HEV strain identified in a rabbit during a countrywide surveillance of rabbits and hares in Germany, 2016. The analysis of the complete genome reveals characteristics of a putative novel recombinant subtype of the species Orthohepevirus A within the clade of genotype 3 but not closely related to any known subtypes. Importantly, the genome of this strain possesses a nucleotide exchange in the overlapping region of open reading frames ORF2/ORF3 interfering with a broadly applied diagnostic real-time RT-PCR. In conclusion, a new type of HEV strain was identified in a German rabbit with atypical and novel sequence characteristics.

In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.

Myxoma virus (MYXV) causes morbidity and mortality in European wild rabbits (Oryctolagus cuniculus) worldwide, and recently in Iberian hares (Lepus granatensis) in Spain. We aimed to assess the presence of MYXV-specific DNA in ixodid ticks collected from both hosts. A total of 417 ticks harvested from 30 wild lagomorphs, including wild rabbits and Iberian hares were collected from southern Spain. Enzyme-linked immunosorbent assay and PCR-sequencing were used to detect virus exposure and presence, respectively. Antibodies to MYXV were detected in 68% (17/25) of wild rabbits and in 67% (2/3) of Iberian hares. We detected MYXV DNA in 50.7% of pools of two different tick species (nymphs and adults of Rhipicephalus pusillus, and nymphs of Hyalomma lusitanicum) parasitizing rabbits and hares. The obtained partial sequence of the viral major envelope protein gene showed a mutation (G383A) within the MYXV_gp026 locus between the rabbit strain and Iberian hare strain (recently isolated in tissues of infected hares from Spain). However, in our study, the viral DNA presence was detected for the first time using tick DNA as the PCR-template, but the possible role of ticks as vectors of MYXV still needs to be elucidated., (© Wildlife Disease Association 2021.)

The Lagovirus genus comprises both pathogenic viruses as European brown hare syndrome virus (EBHSV- GII.1) and rabbit hemorrhagic disease viruses (RHDV-GI.1 and RHDV2-GI.2), that principally infect European brown hares (Lepus europeaus) and European rabbits (Oryctolagus cuniculus), respectively, causing severe necrotic hepatitis, spleen enlargement and disseminated haemorrhage. This genus includes also non-pathogenic agents, such as rabbit calicivirus (RCV-E1 - GI.3) and the non-pathogenic hare Lagovirus, provisionally named hare calicivirus (HaCV - GII.2). The latter had been identified for the first time in 2012 in the gut contents and faeces of healthy young hares raised in a breeding farm. In this study, we further investigated the presence of HaCV by testing the intestinal tract of 621 wild hares collected between 2010 and 2018 in Northern and Central Italy, and in 2011 in Austria, Germany and Spain. These wild hares were found dead for causes other than EBHS or were healthy hares shot during the hunting season. Forty-three out of 322 hare samples from Italy and 14 out of 299 samples from Austria and Germany were positive for HaCV-GII.2 by RT-PCR using universal primers for lagoviruses and primers specific for HaCV. Sequence analysis of the full capsid protein gene conducted on 12 strains representative of different years and locations indicated that these viruses belong to the same, single cluster as the prototype strain initially identified at the hares' farm (HaCV_Bs12_1). The relatively high level of genetic variation (88% nt identity) within this cluster suggests HaCVs may have been circulating widely in Europe for some time., (© 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. A recombinant Myxoma virus (strains MYXV-Tol and ha-MYXV) was rapidly identified, harbouring a 2.8 kbp insertion containing evolved duplicates of M060L, M061L, M064L, and M065L genes from myxoma virus (MYXV) or other Poxviruses. Since 2017, 1616 rabbits and 125 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ha-MYXV strains. A subset of the positive samples (20%) from both species was tested for the insert with MYXV being detected in rabbits and the recombinant MYXV in hares. Recently, three wild rabbits were found dead South of mainland Portugal, showing skin oedema and pulmonary lesions that tested positive for the 2.8 kbp insert. Sequencing analysis showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs and eyelids of rabbits by electron microscopy, and isolation in RK13 cells attested virus infectivity. Despite that the analysis of complete genomes may predict the recombinant MYXV strains' ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, raising serious concerns for the future of the Iberian wild leporids while emphasises the need for the continuous monitoring of MYXV and recombinant MYXV in both species.

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.)

In recent decades, cases of autochthonous hepatitis E (HE) have sharply increased in European countries where foodborne transmission is considered the main route of HE virus (HEV) transmission. Although rabbits are considered the main reservoir of the zoonotic HEV-3ra subtype, information on the role of wild lagomorphs in the epidemiology of HEV remains scarce. The aim of this study therefore was to assess the circulation of HEV in European wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis), the most important lagomorph species in Spanish Mediterranean ecosystems. Liver samples from 372 wild rabbits and 78 Iberian hares were analysed using a broad-spectrum RT-PCR that detects HEV genotypes 1-8. None of the 450 lagomorphs tested were positive for HEV infection. To the best of our knowledge, this is the first study to assess HEV circulation in wild rabbits in Spain and the first to evaluate HEV infection in Iberian hares. Our results indicate absence of HEV circulation in wild rabbits and Iberian hares in southern Spain during the study period, which suggests that the risk of transmission of HEV from wild lagomorphs to other species, including humans, is low., (© 2020 Blackwell Verlag GmbH.)

Background: Rabbit hemorrhagic disease virus (RHDV, Lagovirus europeus GI.1) induces a contagious and highly lethal hemorrhagic disease in rabbits. In 2010 a new genotype of lagovirus (GI.2), emerged in Europe, infecting wild and domestic population of rabbits and hares., Case Presentation: We describe the infection with a GI.2 strain, "Bremerhaven-17", in captive mountain hares (Lepus timidus) in a zoo facility in Germany. Postmortem examination revealed RHD-like lesions including necrotizing hepatitis. RT-qPCR and AG-ELISA confirmed presence of GI.2. Recombination and phylogenetic analysis grouped the identified strain with other GI.2 strains, sharing nucleotide identity of 91-99%., Conclusion: Our findings confirm that mountain hares are susceptible to GI.2 infection, due to a past recombination event facilitating virus spillover from sympatric rabbits.

During the necropsies of Iberian hares obtained in 2018/2019, along with signs of the nodular form of myxomatosis, other unexpected external lesions were also observed. Histopathology revealed nuclear inclusion bodies in stromal cells suggesting the additional presence of a nuclear replicating virus. Transmission electron microscopy further demonstrated the presence of herpesvirus particles in the tissues of affected hares. We confirmed the presence of herpesvirus in 13 MYXV-positive hares by PCR and sequencing analysis. Herpesvirus-DNA was also detected in seven healthy hares, suggesting its asymptomatic circulation. Phylogenetic analysis based on concatenated partial sequences of DNA polymerase gene and glycoprotein B gene enabled greater resolution than analysing the sequences individually. The hare' virus was classified close to herpesviruses from rodents within the Rhadinovirus genus of the gammaherpesvirus subfamily. We propose to name this new virus Leporid gammaherpesvirus 5 (LeHV-5), according to the International Committee on Taxonomy of Viruses standards. The impact of herpesvirus infection on the reproduction and mortality of the Iberian hare is yet unknown but may aggravate the decline of wild populations caused by the recently emerged natural recombinant myxoma virus., Competing Interests: No competing interests.

Viruses are ubiquitous in nature; however, very few have been identified in the Leporid species. In the fall of 2018, an outbreak of myxomatosis in Iberian hares ( Lepus granatensis ) was reported in Spain and a novel recombinant myxoma virus strain (MYXV-Tol) was identified. To investigate variability within the recombinant region of the MYXV-Tol and identify any potential viral coinfections, samples (ear, eyelid or vaginal) of Iberian hares were collected from Spain and analyzed. The presence of the recombinant region of the MYXV-Tol was confirmed in six out of eleven samples analyzed. Additionally, a polyomavirus (family Polyomaviridae ), representing a putative new species, and anelloviruses (family Anelloviridae ) belonging to two putative species were identified, some as coinfection with the recombinant MYXV-Tol. The two polyomavirus genomes were identified in two hares and share >99% genome-wide identity. Based on the analysis of their large T-antigen, the new polyomavirus clusters in a distant clade from other mammals sharing <64% amino acid identity. A total of 14 anelloviruses were identified, which share 63-99% genome-wide identity. Overall, our results show a coinfection of different DNA viruses in the studied samples and raise awareness regarding the extensive unsampled diversity of viruses in hares.

Myxomatosis is an infectious disease caused by myxoma virus (MYXV; genus Leporipoxvirus), which affects the European wild rabbit (Oryctolagus cuniculus) and sporadically brown hares (Lepus europaeus). Here, we describe the first outbreak of myxomatosis in Iberian hares (Lepus granatensis). Between mid-July and the end of September 2018, around 530 dead animals were detected in Iberian hare populations in southern Spain. The apparent mean mortality rate was 56.7%, and the estimated mean case fatality rate was 69.2%. Histopathological and molecular results confirmed MYXV infections in all hares analysed. To the authors' knowledge, this is the first myxomatosis outbreak causing a high mortality in hares and the first detailed characterization of a myxomatosis outbreak in the Iberian hare. The absence of cases in sympatric wild rabbits suggests differences in the susceptibility between both lagomorph species to the virus strain implicated in the outbreak. After the first case, the number of affected areas increased sharply affecting most of the Iberian Peninsula where the Iberian hare is present. Further studies are required to elucidate the origin of the implicated MYXV strain as well as to assess the impact of this outbreak on the Iberian hare populations., (© 2019 Blackwell Verlag GmbH.)

The study of myxoma virus (MYXV) infections in the European rabbit (Oryctolagus cuniculus) has produced one of the most accepted host-pathogen evolutionary models. To date, myxomatosis has been limited to the European rabbit with sporadic reports in hares. However, reports of widespread mortalities in the Iberian hare (Lepus granatensis) with myxomatosis-like clinical signs indicate a potential species jump has occurred. The presence of MYXV DNA was confirmed by PCR in 244 samples received from regional veterinary services, animal health laboratories, hunters or rangers over a 5-month period. PCR analysis of 4 MYXV positive hare samples revealed a 2.8 kb insertion located within the M009 gene with respect to MYXV. The presence of this insertion was subsequently confirmed in 20 samples from 18 Spanish provinces. Sanger sequencing and subsequent analysis show that the insert contained 4 ORFs which are phylogenetically related to MYXV genes M060, M061, M064 and M065. The complete MYXV genome from hare tissue was sequenced using Ion torrent next-generation technology and a summary of the data presented here. With the exception of the inserted region, the virus genome had no large scale modifications and 110 mutations with respect to the MYXV reference strain Lausanne were observed. The next phase in the evolution of MYXV has taken place as a host species jump from the European rabbit to the Iberian hare an occurrence which could have important effects on this naïve population., (© 2019 Blackwell Verlag GmbH.)

Lagoviruses are an essential tool for managing wild rabbit populations in Australia. Our understanding of lagovirus epidemiology in Australia currently depends on members of the public submitting liver samples from dead lagomorphs (i.e. rabbits and hares) through a monitoring program called Rabbitscan. However, many wild lagomorphs die in inaccessible locations or are scavenged before sampling can occur, leading to considerable sampling bias. In this study, we screened field-caught carrion flies for the presence of lagoviruses to monitor virus circulation patterns in the landscape, with an aim to establish a less biased epidemiological surveillance tool. Carrion flies were collected from two study sites over a 22-month period and these samples were used to optimize and validate molecular testing methods in this sample type for the currently circulating lagovirus variants. Virus was clearly detectable in field-caught carrion flies using optimized SYBR-green RT-qPCR and RT-PCR assays. However, variant identification was frequently hindered by the low virus loads present in carrion fly samples and spurious RT-PCR amplification. This was overcome by frequent sampling, which effectively acts as replicate sampling to verify inconclusive results. There was generally good correlation between virus detections and variant identification in carrion flies and in samples recovered from wild lagomorphs. The methods reported here provide an additional surveillance tool to monitor lagovirus spread and circulation at a landscape scale, which in turn can help to guide more effective rabbit management programs., (© 2019 Blackwell Verlag GmbH.)

Various PCR-based assays for rabbit viruses have gradually replaced traditional virologic assays, such as virus isolation, because they offer high-throughput analysis, better test sensitivity and specificity, and allow vaccine and wild-type virus strains to be fully typed and differentiated. In addition, PCR is irreplaceable in the detection of uncultivable or fastidious rabbit pathogens or those occurring in low quantity in a tested sample. We provide herein an overview of the current state of the art in the molecular detection of lagomorph viral pathogens along with details of their targeted gene or nucleic acid sequence and recommendations for their application. Apart from the nucleic acids-based methods used for identification and comprehensive typing of rabbit viruses, novel methods such as microarray, next-generation sequencing, and mass spectrometry (MALDI-TOF MS) could also be employed given that they offer greater throughput in sample screening for viral pathogens. Molecular methods should be provided with an appropriate set of controls, including an internal amplification control, to confirm the validity of the results obtained.

Myxomatosis is a lethal disease in wild European and domestic rabbits ( Oryctolagus cuniculus ), which is caused by a Myxoma virus (MYXV) infection-a leporipoxvirus that is found naturally in some Sylvilagus rabbit species in South America and California. The introduction of MYXV into feral European rabbit populations of Australia and Europe, in the early 1950s, demonstrated the best-documented field example of host-virus coevolution, following a cross-species transmission. Recently, a new cross-species jump of MYXV has been suggested in both Great Britain and Spain, where European brown hares ( Lepus europaeus ) and Iberian hares ( Lepus granatensis ) were found dead with lesions consistent with those observed in myxomatosis. To investigate the possibility of a new cross-species transmission event by MYXV, tissue samples collected from a wild Iberian hare found dead in Spain (Toledo region) were analyzed and deep sequenced. Our results reported a new MYXV isolate (MYXV Toledo) in the tissues of this species. The genome of this new virus was found to encode three disruptive genes ( M009L , M036L , and M152R ) and a novel ~2.8 kb recombinant region, which resulted from an insertion of four novel poxviral genes towards the 3' end of the negative strand of its genome. From the open reading frames inserted into the MYXV Toledo virus, a new orthologue of a poxvirus host range gene family member was identified, which was related to the MYXV gene M064R . Overall, we confirmed the identity of a new MYXV isolate in Iberian hares, which, we hypothesized, was able to more effectively counteract the host defenses in hares and start an infectious process in this new host.

The San Juan Mountains of southern Colorado provide subalpine habitat for a suite of mammalian species including Canada lynx (Lynx canadensis), moose (Alces alces) and snowshoe hare (Lepus americanus). In the winter field season of 2016 five faecal samples from lynx, and one each from moose and snowshoe hare were collected to identify small single-stranded DNA viruses associated with these three prominent species. Thirty-two novel viruses were identified and classified as members of two well established ssDNA families Genomoviridae (n = 22) and Microviridae (n = 10) and one recently proposed new family, Smacoviridae (n = 1). In addition one highly novel circular ssDNA virus was identified which at present does not group with any known family. A high level of genomovirus diversity was identified from faeces collected between and across the three mammal species, with full genome-wide pairwise comparisons showing 57%-97% identity. Twenty genomoviruses can be assigned to the genus Gemycircularvirus and represent 11 species, and two into a distinct species in the genus Gemykolovirus. The single smacovirus identified from moose also represents a distinct smacovirus species. Ten microviruses, seven from moose, one from snowshoe hare and two from lynx, all are part of the Gokushovirinae subfamily. The two from lynx are highly similar to a microvirus previously detected in domestic cat (sharing 88%-90% genome-wide identity), indicating this may be a common felid gut microbiome associated virus. Our findings highlight the broad range of diverse ssDNA viruses present in three mammals inhabiting the San Juan Mountains., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Tick-borne encephalitis (TBE) is one tick-transmitted disease where the human incidence has increased in some European regions during the last two decades. We aim to find the most important factors causing the increasing incidence of human TBE in Sweden. Based on a review of published data we presume that certain temperature-related variables and the population densities of transmission hosts, i.e. small mammals, and of primary tick maintenance hosts, i.e. cervids and lagomorphs, of the TBE virus vector Ixodes ricinus, are among the potentially most important factors affecting the TBE incidence. Therefore, we compare hunting data of the major tick maintenance hosts and two of their important predators, and four climatic variables with the annual numbers of human cases of neuroinvasive TBE. Data for six Swedish regions where human TBE incidence is high or has recently increased are examined by a time-series analysis. Results from the six regions are combined using a meta-analytical method., Results: With a one-year time lag, the roe deer (Capreolus capreolus), red deer (Cervus elaphus), mountain hare (Lepus timidus) and European hare (Lepus europaeus) showed positive covariance; the Eurasian elk (moose, Alces alces) and fallow deer (Dama dama) negative covariance; whereas the wild boar (Sus scrofa), lynx (Lynx lynx), red fox (Vulpes vulpes) and the four climate parameters showed no significant covariance with TBE incidence. All game species combined showed positive covariance., Conclusions: The epidemiology of TBE varies with time and geography and depends on numerous factors, i.a. climate, virus genotypes, and densities of vectors, tick maintenance hosts and transmission hosts. This study suggests that the increased availability of deer to I. ricinus over large areas of potential tick habitats in southern Sweden increased the density and range of I. ricinus and created new TBEV foci, which resulted in increased incidence of human TBE. New foci may be established by TBE virus-infected birds, or by birds or migrating mammals infested with TBEV-infected ticks. Generally, persistence of TBE virus foci appears to require presence of transmission-competent small mammals, especially mice (Apodemus spp.) or bank voles (Myodes glareolus).

Rabbit haemorrhagic disease virus (RHDV, or GI.1) is a calicivirus in the genus Lagovirus that has been widely utilized in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI.1a-Aus, previously called RHDVa-Aus, is a GI.1a virus detected in January 2014, and the novel lagovirus GI.2 (previously known as RHDV2). Furthermore, an additional GI.1a strain, GI.1a-K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain-specific multiplex RT-PCR assay was developed, which allows fast, cost-effective and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal RT-qPCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissue samples, which contained the non-structural genes of GI.1a-Aus and the structural genes of GI.2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI.2, requires further studies., (© 2017 Blackwell Verlag GmbH.)

European brown hare syndrome virus (EBHSV) was detected in a faecal swab collected from a wolf carcass in Northern Italy. The full-length genome of the EBHSV WOLF/17/2016/ITA strain was determined. In the VP60 capsid gene, the wolf strain displayed the highest genetic identity (99.2-99.1% nucleotide and 99.6-99.7% amino acid) with two EBHSV strains recently found in the intestinal content of a red fox and in the spleen and liver of a hare in Northern Italy. This finding poses interrogatives on the potential role of carnivores as EBHSV passive carriers, favoring the introduction and spread of the virus among different hare populations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Recently, a change of hepatitis E from being a typical travel-associated disease to an autochthonous zoonosis in Germany was observed. An increasing number of autochthonous infections with the hepatitis E Virus (HEV) have been recognized in developed countries. Venison from wild boar is already known to be a potential source of infection, if not prepared properly by the consumer. In Germany, certain wild animals are known to be a reservoir for HEV. However, current information is missing about European brown hares (Lepus europaeus) and wild rabbits (Oryctolagus cuniculus). Thus, a total of 833 hunting-harvested animals (European brown hares n = 669; wild rabbits n = 164) were tested for the occurrence of HEV RNA and HEV antibodies. For this, liver and blood specimens were taken after hunts in six German federal states. HEV antibodies were found by ELISA in 2.2% (624/14) of European brown hares, but no HEV RNA was detectable by nested real-time RT-PCR. In contrast, a seroprevalence of 37.3% (126/47) was observed for wild rabbits, and 17.1% (164/28) of the samples were HEV RNA positive. Genomic analysis revealed that these partial sequences clustered within the rabbit clade of HEV-3 genotype. In addition, one rabbit sequence segregated into subtype 3g of HEV-3. Highest seroprevalences for hares and rabbits were detected in the federal states of Bavaria and of Schleswig-Holstein, respectively. Comparing urban, rural and insular areas, the highest seroprevalence was shown for wild rabbits in rural areas and for European brown hares on the northern island Fehmarn. This study provides evidence that European brown hares and wild rabbits from Germany can be infected with HEV. The different prevalences indicate that wild rabbits are a potential reservoir for HEV in Germany, whereas European brown hares seem to be only of minor importance for the epidemiology of HEV., (© 2017 Blackwell Verlag GmbH.)

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2., (© 2016 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

Ruminant pestiviruses cause important economic losses in livestock and the epidemiological role of free-ranging sympatric wildlife is of special interest for the implementation of pestivirus eradication plans. Moreover, the emergence of high mortality outbreaks of pestivirus in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) since 2001 in the border between Spain and France has increased the value of knowing the hosts that role pestivirus infection. In the present study, pestivirus infection was assessed in 94 sera from wild hunted European hares (Lepus europaeus) collected in two different areas: Pyrenees (alpine and subalpine ecosystems) versus Non Pyrenees (non alpine and subalpine ecosystems). The presence of antibodies against Border Disease Virus (BDV) and Bovine Viral Diarrhea Virus (BVDV) was evaluated by means of the Virus Neutralization Test and the presence of viral RNA in sera samples was assessed by Reverse transcription-polymerase chain reaction (RT-PCR). A total of 34 out of 94 (36.2%; CI95 0.26-0.46) sera presented neutralizing antibodies against ruminant pestiviruses, and significant differences between BDV4 and BVDV1 titres were found in 7 hares. In the Pyrenean area not statistically significant seroprevalence was observed when comparing with the Non Pyrenean area. RT-PCR analysis of sera samples resulted all negative. The results of the present study indicate that the European hare is susceptible to pestivirus infection and that could be involved in the epidemiology of ruminant pestiviruses. To the authors' knowledge, this is the third wild non-artiodactyl with reported antibodies against ruminant pestivirus after the rabbit and Bennet's wallaby., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, belongs to the Flaviviridæ family and contains a single-strand positive-sense RNA genome, which upon virus entry and uncoating, functions as mRNAs and thus can be directly translated into proteins by host cell machinery. To date the HCV origin remains unclear and HCV life cycle and pathogenesis are not enlightened processes due to the absence of HCV efficient cell cultures systems or animals models. Here we show that rabbit and hare HCV-like viruses, RHCV and HHCV respectively, are formed after the inoculation of genomic DNA in Madin-Darby bovine kidney cell line cultures. RHCV is closely related to the HCV-1a/HCV-1b genotypes and HHCV is more closely related to the HCV-1b genotype. These findings could contribute to the understanding of HCV origin as well as clarify the virus life cycle, pathogenesis, evolution and diversity.

Meat products from HEV-infected reservoir animal species are capable of transmitting HEV to humans and represent a public health concern. Human HEV cases have been linked to the consumption of raw or undercooked pig liver sausages, pork, and game meats, such as wild boars and deer worldwide. Direct exposure to swine or wild game species might also represent a source of HEV transmission especially for veterinarians, hunters, or butchers. A limited amount of data is available on HEV prevalence in wild boars in Italy and no data are available for other wild game species intended for human consumption. In this study, the circulation of HEV in four different animal species hunted in north-western Italy was evaluated to gain insight into the infection levels and the genetic diversity of the virus in such animal populations. Liver samples of 372 wild boars, 30 roe deer, 47 European hares and 38 coypus were analyzed for HEV RNA by real-time RT-PCR; positive samples were then sequenced and submitted to phylogenetic analysis. HEV RNA was detected in the livers of 7/372 (1.9%) wild boars tested, while no sample was positive for roe deer, European hare, and coypu. Phylogenetic analysis showed that wild boar HEV sequences belonged to HEV subtypes 3e, 3c, and 3f. Our results indicate that HEV is circulating only in wild boar among the considered game species in north-western Italy and suggest a potential zoonotic risk related to handling and/or consumption of raw or undercooked meat and products made of the liver from this species.

We examined sera from snowshoe hares (Lepus americanus) livetrapped in the northern Greater Yellowstone Area (GYA), US, for antibodies to Brucella abortus, Francisella tularensis, and snowshoe hare virus (SSHV). Zero of 90, 0 of 67, and 40 of 100 samples were antibody positive for B. abortus, F. tularensis, and SSHV, respectively. Hares were trapped from 2009 to 2012, and of the six animals that were captured twice with at least 1 yr between captures, four developed antibody to SSHV, indicating active exposure to the agent. These findings suggest snowshoe hares in the GYA do not play a significant role as a reservoir of B. abortus, but do maintain the zoonotic, encephalitic SSHV in the population.

Rabbit haemorrhagic disease virus (RHDV), a member of the genus Lagovirus, causes rabbit haemorrhagic disease (RHD), a fatal hepatitis of rabbits, not previously reported in hares. Recently, a new RHDV-related virus emerged, called RHDV2. This lagovirus can cause RHD in rabbits and disease and mortality in Lepus capensis (Cape hare). Here we describe a case of RHDV2 infection in another hare species, Lepus corsicanus, during a concurrent RHD outbreak in a group of wild rabbits. The same RHDV2 strain infected rabbits and a hare, also causing a RHD-like syndrome in the latter. Our findings confirmed the capability of RHDV2 to infect hosts other than rabbits and improve the knowledge about the epidemiology and the host range of this new lagovirus., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.)

One of the most severe European rabbit (Oryctolagus cuniculus) pathogens is myxoma virus (MYXV), a rabbit-specific leporipoxvirus that causes the highly lethal disease myxomatosis. Other leporid genera, Sylvilagus and Lepus, encompass species with variable susceptibilities to MYXV, but these do not develop the lethal form of the disease. The protective role of the retinoic acid-inducible gene-I (RIG-I/DDX58) in sensing MYXV in nonpermissive human myeloid cells prompted the study of the RIG-I-like receptor (RLR) family evolution in the three leporid genera. This viral-sensor family also includes the melanoma differentiation-associated factor 5 (MDA5/IFIH1), and the laboratory of genetics and physiology 2 (LGP2/DHX58). Considering specifically the MYXV susceptible host (European rabbit) and one of the virus natural long-term hosts (Sylvilagus bachmani, brush rabbit), the amino acid differences of positively selected sites in RIG-I between the two species were located in the protein region responsible for viral RNA recognition and binding, the repressor domain. Such differences might play a determinant role in how MYXV is sensed. When looking for episodic selection on MDA5 and LGP2 of the eastern cottontail (Sylvilagus floridanus), we also uncovered evidence of selective pressures that might be exerted by a species-specific leporipoxvirus, the Shope fibroma virus. Finally, a putative alternative splicing case was identified in Oryctolagus and Lepus MDA5 isoforms, corresponding to the deletion of one specific exon. This study provided the first insights into the evolution of the leporid RLR gene family that helps illuminate the origins of the species-specific innate responses to pathogens and more specifically to MYXV.

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.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a typical tick-borne pathogen that causes an increasing number of severe infections in many parts of Africa, Asia, the Middle East, and the Balkans, as well as in some other parts of Europe. The virus is transmitted primarily by Hyalomma spp., and the spectrum of natural hosts for CCHFV is broad, including wild and domestic animals. Although, the presence of CCHFV was hypothesized in Hungary, no significant research activity has been carried out in the past 30 years. In the present study, we provide serological evidence of CCHFV infection in Lepus europeus using newly developed antibody detection assays. Of 198 samples, 12 (6%) were positive for immunoglobulin G antibody against CCHFV, with 2 independent detection assays. This observation indicates a need for a large-scale surveillance to estimate the potential public health risk of CCHFV in Hungary.

Endogenous retroviruses, non-retroviral RNA viruses and DNA viruses have been found in the mammalian genomes. The origin of Hepatitis C virus (HCV), the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans, remains unclear since its discovery. Here we show that fragments homologous to HCV structural and non-structural (NS) proteins present in the European rabbit (Oryctolagus cuniculus) and hare (Lepus europaeus) genomes replicate in bovine cell cultures. The HCV genomic homolog fragments were demonstrated by RT-PCR, PCR, mass spectrometry, and replication in bovine cell cultures by immunofluorescence assay (IFA) and immunogold electron microscopy (IEM) using specific MAbs for HCV NS3, NS4A, and NS5 proteins. These findings may lead to novel research approaches on the HCV origin, genesis, evolution and diversity.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

TRIM5α proteins recruit and restrict incoming cytoplasmic retroviruses. Primate TRIM5α sequence diversity underlies species-specific restriction and is likely caused by selective pressure from ancient pathogenic infections. Here we show that TRIM5α from the European brown hare restricts diverse retroviruses. Furthermore, it differs significantly in sequence from TRIM5α from the closely related rabbit, suggesting evolutionary changes in the last 12 million years since these species diverged. We propose that, like primates, lagomorphs have been subject to selective pressure from TRIM5-sensitive viruses, possibly related to the endogenous lentivirus RELIK found in both rabbits and hares.

Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in Albania. Ticks collected from cattle grazing in the endemic areas of Albania were tested for presence of CCHFV RNA, while serum samples collected from goats, cattle, hares, and birds were tested for the presence of specific IgG antibodies to CCHFV. One of the 31 pools prepared, consisting of four female Hyalomma spp. ticks, was found to carry CCHFV RNA with 99.2-100% homology to sequences detected in patients from the same region. Antibodies were not detected in cattle, hares, and birds, but 2/10 goats presented high titers of IgG antibodies. The shepherd of that flock was a member of a family affected by CCHF 10 days before the collection of goats' sera, and he presented a mild form of the disease.

The retroviral genus Lentivirus comprises retroviruses characterised from five mammalian orders. Lentiviruses typically undergo rapid rates of evolution, a feature that has allowed recent evolutionary relationships to be elucidated, but has also obscured their distant evolutionary past. However, the slowdown in the rate of evolution associated with genome invasion, as has occurred in the European rabbit, enables longer-term lentiviral evolutionary history to be inferred. Here we report the identification of orthologous RELIK proviruses in the European hare, demonstrating a minimum age of 12 million years for the lagomorph lentiviruses. This finding indicates an association between lentiviruses and their hosts covering much of the evolutionary history of the lagomorphs, and taking place within species with a worldwide distribution.

The complete nucleotide consequence of snowshoe hare (Lepus americanus) virus (SSHV) genome was first determined. Phylogenetic analysis indicated the identity of the study strain in the complete S segment and an L-segment with a length of 145 amino acid residues with SSHV sequences from the database GenBank (J02390, AF393325). The homology of an M segment of the study segment was 99.7% with the consequences of the same from the database GenBank (K02539). La Crosse virus is most genetically close to SHV among the representatives of the California encephalitis serocomplex.

Solid-phase enzyme immunoassay, neutralization test, and the hemagglutination-inhibition test were used to study the sera from human beings (152 samples), agricultural animals (n = 77), hares (n = 3), and wild birds (n = 69), collected in 2006-2007 in the Kuban River estuary (Temryuk District, Krasnodar Territory). There were specific antibodies against viruses of West Nile (WH), tick-borne encephalitis (TBE) (Flaviviridae, Flavivirus), Sindbis (Togaviridae, Alphavirus), the antigenic complex of California, Batai (Bunyaviridae, Orthobunyavirus), Dhori (Orthomyxoviridae, Thogotovirus). The findings suggest the presence of arboviruses from 6 transmitting mosquitoes and ticks in the study area and human infection by the viruses of the antigenic complex of California (20-47%), Batai (3-15%), West Nile (3-12%), Dhori (2%). The index agricultural animals (horses, cattle) were observed to have specific antibodies to the viruses of WN (8-15%), TBE (0-2%), Sindbis (2-9%), the antigenic complex of California (27-54%). Out of the representatives of the wild fauna, virus-neutralizing antibodies to Sindbis virus were found in European hares (Lepus europaeus), California complex virus in gulls (Larus argentatus) and terns (Sterna hirundo), WN and Sindbis viruses in herons (Ardea purpurea), and WN and California complex viruses in bald-coots (Fulica atra).

Investigations regarding European brown hare syndrome virus (EBHSV) in European brown hares (Lepus europaeus) in Slovakia were undertaken in order to detect the possible presence of EBHSV and to evaluate its phylogenetic position. Liver and/or serum samples were obtained from 135 European brown hares shot by hunters in eight regional hunting areas. From 36 animals corresponding liver and serum samples were available; from the remaining 49 and 50 animals only liver or serum samples were examined, respectively. Samples were tested for antibodies against EBHSV and for viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and RT-PCR products were subsequently sequenced. Additionally, matrilinear hare haplotypes were analyzed in order to detect potential familial susceptibility to EBHSV. Sixty-three of 86 sera contained antibodies against EBHSV, whereas 15 of 85 liver samples were PCR positive. Of the latter, 14 were sequenced, revealing three new strains of EBHSV. Fifteen different matrilinear haplotypes were identified, but no correlation was found between haplotype and susceptibility to EBHSV infection. Our findings confirmed the existence of EBHSV in Slovakia and reinforce the need for determining EBHSV status when importing hares for restocking.

Genetic diversity between French European brown hare syndrome (EBHS) viruses since the disease appeared has been evaluated. Nucleotide sequencing of the partial capsid protein genes of 169 EBHS viruses collected from various parts of France between 1989 and 2003, three reference strains, and a Greek EBHSV collected in 2002 revealed a maximum nucleotide divergence of 11.7%, indicating a high level of conservation between viruses. Two major groups were identified. The first group contained EBHS viruses collected since 1989 from different parts of France, the reference strains, and all of the viruses located in the far north of France. In this group, three genogroups were clearly identified as mainly related to their geographic origin. The distribution of the viruses suggests that the early viruses have not disappeared and have slowly evolved in their area of origin. The second group, supported by a significant bootstrap value, contained the Greek EBHSV with the French EBHS viruses collected between 1999 and 2003 from regions of southern France. It constitutes a newly identified genogroup. Our results demonstrate strong differences in genetic evolution between EBHSV and rabbit haemorrhagic disease virus, with persistence of the earlier EBHS viruses and interaction between the geographical and temporal distributions.