The European wild rabbit ( Oryctolagus cuniculus) is a key prey species on the Iberian Peninsula, and several predator species that are at risk of extinction are dependent on them as prey. A new rabbit hemorrhagic disease (RHD) virus genotype (GI.2/RHDV2/b) emerged in 2010 and posed a threat to wild rabbit populations. During a survey aimed at investigating RHD epidemiology in wild rabbits, GI.2/RHDV2/b was detected by duplex real-time PCR in carcasses of one Mediterranean pine vole ( Microtus duodecimcostatus) and two white-toothed shrews ( Crocidura russula). Laboratory New Zealand white rabbits that were challenged with inocula obtained from the liver of the small mammals died showing RHD lesions, confirming the infectiousness of the isolates. Phylogenetic analysis of the VP60 gene nucleotide sequences showed complete homology between the isolates from the two small mammal species and a high degree of similarity, but not complete homology, to GI.2/RHDV2/b sequences from wild rabbits. The GI.2/RHDV2/b genotype has not been reported in species outside the order Lagomorpha

Calvete, C.

Calvo, A.J.

Calvo, J.H.

Jiménez de Bagüés, M.P.

Luján, L.

Mendoza, M.

Molín, J.

Monroy, F.

Sarto, M.P.

English

Repositorio Universidad de Zaragoza

DOI: 10.7589/2018-05-124 Journal of Wildlife Diseases, 55(2), 2019, pp. 000–000 Wildlife Disease Association 2019Detection of Rabbit Hemorrhagic Disease Virus GI.2/RHDV2/B in theMediterranean Pine Vole (Microtus duodecimcostatus) and White-Toothed Shrew (Crocidura russula)Carlos Calvete,1,5 Manuel Mendoza,1 Marı´a P. Sarto,1 Marı´a P. Jime´nez de Bagu¨e´s,1 Lluis Luja´n,2 Je´ssicaMolı´n,2 Antonio J. Calvo,3 Fernando Monroy,3 and Jorge H. Calvo1,4 1Animal Production and HealthDepartment, Agrifood Research and Technology Centre of Aragon (CITA), Agrifood Institute of Aragon–IA2 (CITA–Zaragoza University), Avda, Montan˜ana 930, 50059 Zaragoza, Spain; 2Animal Pathology Department, University ofZaragoza, Agrifood Institute of Aragon–IA2 (CITA–Zaragoza University), 50013 Zaragoza, Spain; 3Animal HealthManagement, TRAGSATEC, C/Julia´n Camarillo 6 A, 28037 Madrid, Spain; 4Aragonese Foundation for Research &Development (ARAID), 50018 Zaragoza, Spain; 5Corresponding author (email: ccalvete@aragon.es)ABSTRACT: The European wild rabbit (Oryctola-gus cuniculus) is a key prey species on the IberianPeninsula, and several predator species that are atrisk of extinction are dependent on them as prey.A new rabbit hemorrhagic disease (RHD) virusgenotype (GI.2/RHDV2/b) emerged in 2010 andposed a threat to wild rabbit populations. Duringa survey aimed at investigating RHD epidemiol-ogy in wild rabbits, GI.2/RHDV2/b was detectedby duplex real-time PCR in carcasses of oneMediterranean pine vole (Microtus duodecimcos-tatus) and two white-toothed shrews (Crocidurarussula). Laboratory New Zealand white rabbitsthat were challenged with inocula obtained fromthe liver of the small mammals died showingRHD lesions, confirming the infectiousness of theisolates. Phylogenetic analysis of the VP60 genenucleotide sequences showed complete homologybetween the isolates from the two small mammalspecies and a high degree of similarity, but notcomplete homology, to GI.2/RHDV2/b sequencesfrom wild rabbits. The GI.2/RHDV2/b genotypehas not been reported in species outside the orderLagomorpha.Key words: Crocidura russula, GI.2/RHDV2/b, lagovirus, Microtus duodecimcostatus, Orycto-lagus cuniculus, rabbit hemorrhagic disease, smallmammals.Rabbit hemorrhagic disease (RHD) is afatal, highly infectious disease that affectsEuropean rabbits (Oryctolagus cuniculus).According to nomenclature proposed by LePendu et al. (2017), RHD is caused by thecalicivirus species Lagovirus europaeus (GI.1genotype). In 2010, a new RHD virusgenotype, GI.2/RHDV2/b, was identified.Since then, it has spread throughout WesternEuropean and is able to infect several speciesof hares, suggesting a wider spectrum of hoststhan other RHD viruses (Le Pendu et al.2017). We detected GI.2/RHDV2/b in thelivers of two species of small wild mammals,identifying infections by this virus in speciesoutside the order Lagomorpha.We obtained samples during a project tostudy the epidemiology of RHD in anexperimental European rabbit population infour 1 ha enclosures (P1–P4). The experimen-tal population was located on property ownedby the Agrifood Research and TechnologyCentre (CITA) in Zaragoza Province, Spain(41851055 00N, 0839024 00W), where natural wildrabbit populations were absent. The enclo-sures, which were 2 km apart, were enclosedby 2.5-m-high wire mesh to prevent thepassage of rabbits and their kits, as well asterrestrial predators, but did not preventaccess by small mammals or birds. Rabbitslived in natural warrens inside the enclosures.From 2013 to 2016, the population wasmonitored at least twice per week to collectrabbits that had died from RHD, and from2014 to 2016, the carcasses of sympatric smallmammals were also collected. In all, 37rabbits that had died from acute RHD werefound, and four small mammal carcasses werecollected: two Mediterranean pine voles(Microtus duodecimcostatus) were found inSeptember 2014 (Md-Sep14-P3) and Novem-ber 2014 (Md-Nov14-P3), both in enclosureP3, and two white-toothed shrews (Crocidurarussula) were detected in February 2015 in P1(Cr-Feb15-P1) and in June 2015 in P4 (Cr-Jun15-P4).Both of the pine voles were adults. CarcassMd-Sep14-P3 was found partially consumed,without a digestive tract or kidneys (sex1undetermined), whereas Md-Nov14-P3 was afemale in a state of advanced pregnancy. Onewhite-toothed shrew, Cr-Feb15-P1, was anadult male, while the other (Cr-Jun15-P4) wasa young individual of undetermined sex. Atnecropsy, no sign of injury or damage causedby predators, scavengers, or insects wasevident in any of the last three carcasses. Anintense, generalized congestion of the mainorgans was apparent in the pregnant pine volespecimen (Md-Nov14-P3); however, no signof any disease was detected in the remainingthree carcasses.Liver samples from the 37 rabbits and thefour small mammals were analyzed usingduplex real-time PCR (qPCR) to detectRNA from GI.1b/RHDV and GI.2/RHDV2/blagoviruses in a single analysis (Calvete et al.2018). Each sample was amplified in duplicatein the same run, and the quantification cycle(Cq) value was calculated as an average ofboth amplifications. To demonstrate theinfectiousness of the virus from the qPCR-positive, small-mammal samples, experimen-tal rabbit infections were performed undernegative pressure in the BSL2 facilities ofCITA. Eight 12-wk-old, laboratory-raisedNew Zealand white rabbits (Oryctolaguscuniculus) were inoculated intramuscularlywith 0.3 mL of supernatant (0.22 lm pore-size filtered) derived from 0.2 g of small-mammal livers homogenized in 1 mL of sterilephosphate-buffered saline. Two rabbits,reared in separate cages, were injectedsimultaneously with the inoculum obtainedfrom each small-mammal liver, even if theliver was qPCR negative. Infections werecarried out in separate experiments per-formed across several months.The rabbits were monitored for 3 wkpostinoculation. Deceased animals were nec-ropsied; surviving animals were also necrop-sied after being humanely euthanized.Samples from the liver, spleen, kidneys,thymus, and duodenum were collected todetect viral RNA by qPCR. A subset of thesame tissue samples was fixed in 10% bufferedformalin and processed using standard proce-dures for histological examination. The entireVP60 gene of the virus was sequenced(Calvete et al. 2018), from the liver samplesof 37 wild rabbits, as well as from thelaboratory rabbits that died from RHD.Therefore, the full capsid VP60 sequencewas used to infer phylogenetic relationships inthis study. Phylogenetic trees were estimatedusing the maximum likelihood (ML) methodavailable in MEGA7 (Kumar et al. 2016)under the best-fit nucleotide substitutionmodel determined by the same software.The Tamura 3-parameter model (Tamura1992) with a discrete gamma distributionwas used to model evolutionary rate differ-ences among sites (five categories; Gammaparameter G¼0.1917). Bootstrap resamplingwas performed on 1,000 replicates of the MLtrees. The antibody status of the survivinginoculated rabbits was tested on days 0 and 21after inoculation using a commercial indirectELISA test (Ingezim Rabbit, INGENASALab, Madrid, Spain). The animal experimentswere done in compliance with the provisionsof Spanish national and European laws andapproved by the CITA Ethical Committee forAnimal Experimentation (protocol 2014-19).Duplex qPCR was positive for GI.2/RHDV2/b in one pine vole (Md-Nov14-P3)and in both white-toothed shrew samples (Cr-Feb15-P1 and Cr-Jun15-P4), with Cq valuesof 36.5, 32.5, and 38, respectively. All of the37 wild rabbits were also qPCR-positive forthe same virus at mean (SD) Cq values of 18.7(2.04). This value is in agreement with Calveteet al. (2018), who reported Cq values rangingfrom 18.03 to 18.30 using the same qPCRprocedures in rabbits that had died from acuteRHD caused by the same virus. AlthoughqPCR was not developed to quantify viralRNA copy numbers (Calvete et al. 2018), thehigh Cq values found in the small mammalssuggested a lower quantity of viral particlesthan in rabbits killed by acute disease.Death by acute RHD occurred in bothlaboratory rabbits infected with the inoculumobtained from Md-Nov14-P3 and in onerabbit infected with the inoculum from Cr-Jun15-P4. Major histopathological lesionswere found in the primary target tissues ofthe RHD virus, such as the liver, spleen, lung,and kidneys. Most of the severe lesions were2 JOURNAL OF WILDLIFE DISEASES, VOL. 55, NO. 2, APRIL 2019found in the liver, displaying acute necrotizinghepatitis. Changes in the spleen includeddiffuse red pulp hyaline necrosis, whereas inthe lungs, multifocal acute alveolar edema andhemorrhages were seen resulting from mas-sive disseminated intravascular coagulation.Multifocal acute tubular necrosis and fibrinthrombi were also observed in kidneys. Thepresence of GI.2/RHDV2/b in the liver tissuewas confirmed by qPCR with Cq valuesranging from 16.4 to 20.No histopathological changes were ob-served in the rabbits that survived challenge.The results of the qPCR analysis werenegative for all tissue samples except for theliver and the spleen of the surviving rabbitchallenged with the Cr-Jun15-P4 inoculum.In this instance, GI.2/RHDV2/b RNA wasfound with high Cq values in the spleen andliver (40.5 and 42, respectively), which were,nevertheless, out of the lowest limit ofdetection (Cq¼38.7) established for qPCR(Calvete et al. 2018). Seroconversion wasdetected in two surviving rabbits: one chal-lenged with Cr-Feb15-P1 inoculum, and thesurvivor from the Cr-Jun15-P4 inoculumchallenge. The results are summarized inTable 1.Phylogenetic patterns of the viral samplesfrom the 37 wild rabbits, whose sequenceswere deposited in GenBank under the acces-sion numbers KY498543–52 and KY498555–81 (Fig. 1 and Table 2), showed evidence ofhigh viral diversification, with just eight pairsof identical sequences. Clustering was mainlyrelated to year of isolation, and a completehomology of nucleotide sequences occurredonly between samples obtained from rabbitsthat died in the same enclosure, in the sameor in a contiguous month (seven pairs), withthe exception of one pair of samples (Zar2015-5 and Zar2015-7) collected in differentenclosures but in the same month.The complete VP60 sequences obtainedfrom the dead rabbits inoculated with the pinevole (GenBank accession number KY498553)or the white-toothed shrew inoculum (Gen-Bank accession number KY498554) had 100%nucleotide homology, even though the white-toothed shrew was found in a differentenclosure 7 mo after the pine vole. Nocomplete homology with any wild rabbitsample was found, although the small-mam-mal isolate sequences were more similar tothose collected during 2015–16. The pine voleisolate, collected in November 2014, clusteredamong the 2015 wild rabbit samples. It alsoTABLE 1. Results of qPCR to detect GI.2/RHDV2/b virus RNA in the liver of small mammals found dead inenclosures (P1–P4) housing European rabbits (Oryctolagus cuniculus). Two, laboratory-raised, New Zealandwhite rabbits were inoculated simultaneously with the inoculum obtained from each small mammal liver. Resultsof rabbit experimental infection, including rabbit liver qPCR and seroconversion in surviving rabbits, aresummarized. Identification of small mammals (ID): Md-Sep14-P3 and Md-Nov14-P3, Mediterranean pine voles(Microtus duodecimcostatus) found dead in September and November 2014, respectively, both in enclosure P3;Cr-Feb15-P1 and Cr-Jun15-P4, white-toothed shrews (Crocidura russula) found dead in February and June2015 in P1 and P4 enclosures, respectively.Small mammals Results of rabbit challengesID Liver Cq Rabbit no. RHD death Liver Cq SeroconversionMd-Sep14-P3 Negative 1 No Negative No2 No Negative NoMd-Nov14-P3 36.5 3 Yes 16.4 NAa4 Yes 19.4 NACr-Feb15-P1 32.5 5 No Negative Yes6 No Negative NoCr-Jun15-P4 38.0 7 Yes 20.0 NA8 No 40.5 Yesa NA¼Not applicable.CALVETE ET AL.—SHORT COMMUNICATIONS 3exhibited a high similarity in nucleotidesequences and a complete homology at theamino acid level with a rabbit samplecollected from the same enclosure (P3) inMarch 2016 (17 mo later).Both pine voles and white-toothed shrewsare species that are sympatric to the Europeanwild rabbit. The findings of our survey may bethe consequence of passive viral transmissionto small mammals (with no effective virusreplication), due to their scavenging habits, orby the ingestion of dead, infected rabbit tissueor feces. Supporting this idea, Shien and Lee(2000) detected viral RNA in the liver ofpiglets up to 14 d after a subcutaneousinoculation of GI.1, although there was noevidence of viral replication in the porcinetissues. Moreover, Zheng et al. (2003) dem-onstrated the presence of GI.1 viral RNA inthe lymph nodes and livers of domestic catsexperimentally fed RHD virus-infected rabbitlivers. The RNA was detected 45–52 d afterfeeding, although viral replication was also notobserved.Genetic variability of RHD virus is high andmainly driven by time, with exact homologiesoccurring between isolates obtained in tem-poral proximity (Muller et al. 2009). There-fore, the complete homology that we foundbetween the small-mammal isolate sequenceswas unique. Given the long-term survival ofRHD viruses in animal tissues—at least 91 d(Henning et al. 2005)—the complete homol-ogy of the VP60 sequence could be aconsequence of the fact that carcasses ofRHD-infected rabbits may have been acces-sible to small mammals for several monthsfollowing the last RHD outbreak, thus allow-ing for the ingestion of the same or relatedviruses. Under this hypothesis, the role ofsmall mammals in RHD epidemiology may benegligible or restricted to viral disseminationby feces at a local scale.The virus that was found in small mammals,however, was capable of infecting laboratoryrabbits and causing RHD. If passive trans-mission to small mammals occurred, then thedetected viral RNA would be expected to haveoriginated from immunocomplexes of GI.2/TABLE 2. Sequences of the entire VP60 gene of rabbit hemorrhagic disease virus GI.2/RHDV2/b strain used forphylogenetic analysis. The Zar2013-1 to Zar2016-4 strains were detected in a Mediterranean pine vole (Microtusduodecimcostatus) and two white-toothed shrews (Crocidura russula) associated with European wild rabbits(Oryctolagus cuniculus) in Zaragoza Province, Spain. The names of the strains conform to those shown in Figure1.Sequences GenBank accession numbersZar2013-1 to Zar2016-4 KY498543–81Group France and Italy FR819781, HE819400, HE800529–32, HE819400, HE800529–32, KC345611–13,JX106023, KT737803, KC907712Italy-Co12 KT737802Italy-Sr12-2 KC741409Group Malta KJ957809–10Group Portugal I KF442963–4, KF442961–2, KM115697–8, KM115680–2, KM115711, KM115689,KM115683Group Portugal II KT000290–91, KT000306, KT000313, KT000329, KT000337, KT000339, KT000342,KM115714–16, KM115712–13Portugal-CBVal16 KM979445Spain-CBAnd1 KP090976Spain-N11 KM878681Spain-Rij06-12 KP129395Spain-Seg08-12 KP129396Spain-Tar06-12 KP129397Spain-Zar11-11 KP129398Spain-Zar06-12 KP1293994 JOURNAL OF WILDLIFE DISEASES, VOL. 55, NO. 2, APRIL 2019RHDV2/b particles and antibodies. That is,the existence of an infective virus would notbe expected unless both small mammals diedbefore they could mount an immune responseagainst the viral antigens. Therefore, thefinding of infective virus might point to thepossibility of cross-species infection. Thispossibility has already been suggested byMercha´n et al. (2011), who reported thedetection of RHD viral RNA belonging tothe GI.1 genotype in two wild mouse speciesin Spain, and by Rocha et al. (2017), whoreported the experimental transmission ofGI.1 virus from rabbits to these same mousespecies. Given that these small-mammalspecies are sympatric to European leporidsand that the origin of RHD etiological agentsis not well understood (Abrantes et al. 2012),there is a need for further studies to betterunderstand the role of small mammals inRHD epidemiology.The study was funded by a Research andDevelopment collaboration project betweenAgrifood Research and Technology Centre ofAragon (CITA) and the Animal HealthDepartment of TRAGSATEC (INMUNIZA-DOS 1316) as well as by the E-RTA2014-00009-00-00 project from the National Insti-tute for Agricultural and Food Research andTechnology (INIA).LITERATURE CITEDAbrantes J, van der Loo W, Le Pendu J, Esteves PJ. 2012.Rabbit haemorrhagic disease (RHD) and rabbithaemorrhagic disease virus (RHDV): A review. VetRes 4:12.Calvete C, Mendoza M, Alcaraz A, Sarto MP, Jime´nez deBagu¨e´s MP, Calvo AJ, Monroy F, Calvo JH. 2018.Rabbit haemorrhagic disease: Cross-protection andcomparative pathogenicity of GI.2/RHDV2/b andGI.1b/RHDV lagoviruses in a challenge trial. VetMicrobiol 219:87–95.Henning J, Meers J, Davies PR, Morris RS. 2005. Survivalof rabbit haemorrhagic disease virus (RHDV) in theenvironment. Epidemiol Infec 133:719–730.Kumar S, Stecher G, Tamura K. 2016. MEGA7:Molecular Evolutionary Genetics Analysis version7.0 for bigger datasets. Mol Biol Evol 33:1870–1874.Le Pendu J, Abrantes J, Bertagnoli S, Guitton JS, Le Gall-Recule´ G, Lopes AM, Marchandeau S, Alda F,Almeida T, Ce´lio AP, et al. 2017. Proposal for aunified classification system and nomenclature oflagoviruses. J Gen Virol 98:1658–1666.FIGURE 1. Maximum likelihood tree of the VP60gene of of rabbit hemorrhagic disease virus, GI.2/RHDV2/b. Bootstrap values of the nodes are shownnext to them. Scale bar indicates the nucleotidesubstitutions per site. To facilitate visualization somegroups were collapsed. Zar2013-1 to Zar2016-4 aresequences obtained from a Mediterranean pine vole(Microtus duodecimcostatus) and two white-toothedshrews (Crocidura russula) associated with Europeanwild rabbits (Oryctolagus cuniculus) in ZaragozaProvince, Spain. Collecting date and rabbit populationfor them are in parentheses. GenBank accessionnumbers of the sequences used in this study are inTable 2. Isolates from M. duodecimcostatus (M.duo)and C. russula (C.rus) in bold.CALVETE ET AL.—SHORT COMMUNICATIONS 5Mercha´n T, Rocha G, Alda F, Silva E, Thompson G, deTrucios SH, Page´s A. 2011. Detection of rabbithaemorrhagic disease virus (RHDV) in nonspecificvertebrate hosts sympatric to the European wildrabbit (Oryctolagus cuniculus). Infect Genet Evol 11:1469–1474.Muller A, Freitas J, Silva E, Le Gall-Recule G,Zwingelstein F, Abrantes J, Esteves PJ, Alves PC,van der Loo, W, Kolodziejek J, et al. 2009. Evolutionof rabbit haemorrhagic disease virus (RHDV) in theEuropean rabbit (Oryctolagus cuniculus) from theIberian Peninsula. Vet Microbiol 135:368–373.Rocha G, Alda F, Page´s A, Mercha´n T. 2017. Experi-mental transmission of rabbit haemorrhagic diseasevirus (RHDV) from rabbit to wild mice (Mus spretusand Apodemus sylvaticus) under laboratory condi-tions. Infect Gen Evol 47:94–98.Shien JH, Lee LH. 2000. Susceptibility of piglets to rabbithemorrhagic disease virus following experimentalinfection. Can J Vet Res 64:134–137.Tamura K. 1992. Estimation of the number of nucleotidesubstitutions when there are strong transition-trans-version and G þ C-content biases. Mol Biol Evol 9:678–687.Zheng T, Lu G, Napier AM, Lockyer SJ. 2003. No virusreplication in domestic cats fed with RHDV-infectedrabbit livers. Vet Microbiol 95:61–73.Submitted for publication 11 May 2018.Accepted 20 July 2018.6 JOURNAL OF WILDLIFE DISEASES, VOL. 55, NO. 2, APRIL 2019

Detection of Rabbit Hemorrhagic Disease Virus GI.2/RHDV2/b in the Mediterranean Pine Vole ( Microtus duodecimcostatus) and White-Toothed Shrew ( Crocidura russula)

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Detection of Rabbit Hemorrhagic Disease Virus GI.2/RHDV2/b in the Mediterranean Pine Vole ( Microtus duodecimcostatus) and White-Toothed Shrew ( Crocidura russula)

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