First Detection and Circulation of RHDV2 in New Zealand.

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.

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.

A Putative Novel Hepatitis E Virus Genotype 3 Subtype Identified in Rabbit, Germany 2016.

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.

A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies.

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.

Outbreak of rabbit hemorrhagic disease virus 2 in the southwestern United States: first detections in southern California.

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).

Detection of Myxoma Virus DNA in Ticks from Lagomorph Species in Spain Suggests Their Possible Role as Competent Vector in Viral Transmission.

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.

Widespread occurrence of the non-pathogenic hare calicivirus (HaCV Lagovirus GII.2) in captive-reared and free-living wild hares in Europe.

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.

Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus).

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.

Lagovirus europeus GI.2 (rabbit hemorrhagic disease virus 2) infection in captive mountain hares (Lepus timidus) in Germany.

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.

First description of a herpesvirus infection in genus Lepus.

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.

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.

Coinfections of Novel Polyomavirus, Anelloviruses and a Recombinant Strain of Myxoma Virus-MYXV-Tol Identified in Iberian Hares.

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.

Absence of Hepatitis E virus circulation in wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) in Mediterranean ecosystems in Spain.

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.

Myxoma virus jumps species to the Iberian hare.

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.

First outbreak of myxomatosis in Iberian hares (Lepus granatensis).

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.

Genetic Characterization of a Recombinant Myxoma Virus in the Iberian Hare (Lepus granatensis).

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.

Molecular methods in detection and epidemiologic studies of rabbit and hare viruses: a review.

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.

Carrion flies as sentinels for monitoring lagovirus activity in Australia.

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.

Hepatitis E Virus Infection in European Brown Hares, Germany, 2007-2014.

Rabbit-associated hepatitis E viruses (HEVs) cause zoonotic infections. We investigated 2,389 hares in Germany during 2007-2014. Complete genome characterization of a hare-associated HEV strain revealed close genomic relatedness to rabbit-associated HEV strains. Although hare-specific HEV seroprevalence was low, at 2.6%, hares represent a potential source of sporadic HEV infections.