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Molecular Identification of a New Pestivirus Associated With View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Diposit Digital de Documents de la UAB Journal of Wildlife Diseases, 40(4), 2004, pp. 796±800 q Wildlife Disease Association 2004 Molecular Identi®cation of a New Pestivirus Associated with Increased Mortality in the Pyrenean Chamois (Rupicapra pyrenaica pyrenaica) in Spain Ana Hurtado,1,3 Gorka Aduriz,1 Nieves Go mez,1 Beatriz Oporto,1 Ramo n A. Juste,1 Santiago Lavin,2 Jorge R. Lopez-Olvera,2 and Ignasi Marco21Department of Animal Health, Instituto Vasco de InvestigacioÂn y De- sarrollo Agrario Neiker, Berreaga 1, 48160 Derio, Bizkaia, Spain; 2 Servei d'Ecopatologia de Fauna Salvatge, Facultat de Veterinaria, Universitat Autonoma de Barcelona, 08193-Bellaterra, Spain; 3 Corresponding author (email: [email protected]) ABSTRACT: Pestivirus infection was identi®ed stock can cause a variety of economically in 16 of 17 chamois during an outbreak of a important syndromes (Houe, 1999), in- previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica pyrenaica)in cluding reproductive failure, poor devel- northeastern Spain in 2001±02. By analysis of opment, enteric disease, and a hemorrhag- the 59 noncoding regions of the virus, we as- ic syndrome. Fetal infection can lead to signed it to the border disease virus cluster with birth of persistently infected (PI) animals pairwise similarity values ranging from 82.1% to 88.1%. It will be important to investigate the that become chronic shedders of the virus. association of this pestivirus with disease in In goats, natural infections with pestivirus- Pyrenean chamois. es do not appear to be a problem because Key words: Border disease virus, chamois, ®eld cases of clinical disease are rare. In- pestivirus, reverse transcriptase polymerase fection of pregnant goats may produce se- chain reaction, Rupicapra pyrenaica. vere placentitis and high death rates among fetuses; few PI kids are born alive The genus Pestivirus belongs to the after maternal infection (Depner et al., family Flaviviridae and four main geno- 1991; Lonken et al., 1991; Pratelli et al., types are recognized by sequence analysis: bovine viral diarrhea virus types 1 and 2 1999). (BVDV-1 and BVDV-2), border disease vi- An outbreak of a previously unreported rus (BDV) of sheep, and classical swine fe- disease was detected in Pyrenean chamois ver virus (CSFV). These genotypes can be (Rupicapra pyrenaica pyrenaica) in Cata- further subdivided into subtypes (Becher lonia (northeastern Spain) between Feb- et al., 1999; Paton et al., 2000; Vilcek et ruary 2001 and May 2002. Animals were al., 2001; Beer et al., 2002; Hurtado et al., found within 11,000 ha in the National 8 9 2003); several pestivirus strains have been Hunting Reserve of Alt Pallars (42 35 N, 8 9 described in wildlife as separate groups 1 20 E). Mortality was dif®cult to evaluate (Fischer et al., 1998; Becher et al., 1999). because of landscape characteristics, but a The genome of pestiviruses consists of a decrease in the population was observed single, positive stranded RNA molecule during the annual census. In 2000, before that comprises a long, single open reading the disease was detected, the population frame and two noncoding regions (NCRs) was estimated to be 3,880 chamois and at at the 59 and 39 ends. The 59NCR consists the end of 2002, the population dropped of highly conserved regions useful for to 2,678. The aim of this study was to iden- polymerase chain reaction (PCR)-based tify and characterize a pestivirus found in diagnosis of pestiviruses from a wide range diseased chamois. of hosts, intercalated by three variable re- Seventeen Pyrenean chamois were cap- gions (Deng and Brock, 1993) where nu- tured by hand, culled, or found dead. Ages cleotide substitutions accounting for dif- ranged from 1 yr to 11 yr of age (three ferences among pestivirus types are locat- females and 14 males). Blood, serum, ed. spleen, or kidney samples, or a combina- Pestivirus infections in domestic live- tion of these, were collected and analyzed. 796 Downloaded From: https://bioone.org/journals/Journal-of-Wildlife-Diseases on 18 Nov 2020 Terms of Use: https://bioone.org/terms-of-use SHORT COMMUNICATIONS 797 TABLE 1. Results of tests for pestivirus antigens and antibodies in sera and tissues of Pyrenean chamois from Spain during 2001±02. Age Abb Agc PCRd Chamois no. Sexa (yr) (serum) (sample) (sample) RP-1/01 M 3 Neg Pos (ser) ND RP-2/01 M 7 ND Pos (spl) Neg (spl) RP-3/01 F 4 Neg Pos (ser) ND RP-4/01 M 10 Neg Pos (ser) ND RP-5/01 F 9 Neg Pos (ser) ND RP-7/01 M 2 ND Pos (spl) Pos (spl) RP-8/01 M 1 Neg Pos (ser) ND RP-1/02 M 8 Neg Pos (ser) Neg (kid) RP-3/02e M 9 Neg Pos (ser) Pos (spl) RP-4/02 M 8 ND Neg (spl) Neg (spl) RP-5/02f M 11 Neg Pos (ser) Pos (spl) RP-6/02 M Adult ND Pos (spl) Neg (spl) RP-8/02e M 5 Neg Pos (ser) Pos (spl) RP-9/02 M 7 Neg Pos (ser) Pos (spl, bl) RP-10/02 M 5 Neg Pos (ser) Posg (bl) RP-11/02 M 8 Neg Pos (ser) Pos (spl, bl) RP-12/02e,f,h F 4 Neg Pos (ser) Posg (bl) a M 5 male; F 5 female. b Tests for serum antibody by blocking enzyme-linked immunosorbent assay (ELISA). Neg 5 no antibody detected; ND 5 not done. c Tests for pestivirus antigen by sandwich ELISA. Pos 5 samples positive for pestivirus antigen; Neg 5 negative for pestivirus antigen; ser 5 serum; spl 5 spleen. d PCR 5 polymerase chain reaction. ND 5 not done; Neg 5 negative; Pos 5 positive; spl 5 spleen; kid 5 kidney; bl 5 blood. e PCR positive for Anaplasma phagocytophilum. f PCR positive to piroplasm. g 59 noncoding region of the pestivirus sequenced. h Results were consistent at ®ve time points (see text). One animal (RP12/02) was kept alive in 48-kDa glycoprotein of BVDV (Haines et captivity for more than 1 mo and blood and al., 1992). Ampli®cation of viral RNA by serum samples were taken ®ve times dur- PCR with panpestivirus primers 324/326 ing this period (Table 1). Tests were con- was conducted as previously described ducted to detect piroplasms by PCR ac- (Hurtado et al., 2003). The ampli®ed DNA cording to Georges et al. (2001) and for fragments from two animals were puri®ed Anaplasma by following the methods of and both strands were sequenced (285 base Pusterla et al. (2000). Serologic analyses pairs [bp]) as described by Hurtado et al. were performed on 13 sera by a blocking (2003) to identify the pestivirus genotype. enzyme-linked immunosorbent assay Serum samples also were tested for Brucella (ELISA, Synbiotics, Lyon, France) that antibodies by the rose bengal agglutination speci®cally detects antibodies directed and complement ®xation tests and maedi/ against a protein (p80/125) common to all visna antibodies by agar gel immunodiffu- strains of BVDV and BDV. Sera or tissue sion (AGID) (Of®ce International des Epi- homogenates also were tested for pestivirus zooties, 2000). The GenBank database antigen by the sandwich ELISA antigen (www.ncbi.nlm.nih.gov/Genbank/index. test (Synbiotics). Immunohistochemistry html) was searched for related sequences (IHC) on formalin-®xed, paraf®n-embed- and multiple sequence alignments were ded spleen and kidney samples was con- performed using the program AlignX (vec- ducted by using the monoclonal anti-pes- tor NTI8.0 Suite, InforMax, North Be- tivirus antibody 15C5 directed against the thesda, Maryland, USA) with an engine Downloaded From: https://bioone.org/journals/Journal-of-Wildlife-Diseases on 18 Nov 2020 Terms of Use: https://bioone.org/terms-of-use 798 JOURNAL OF WILDLIFE DISEASES, VOL. 40, NO. 4, OCTOBER 2004 based on the Clustal W algorithm The presence of pestivirus was demon- (Thompson et al., 1994). Alignments were strated by antigen-capture ELISA, PCR visually checked to take into account sec- ampli®cation, IHC, or a combination of ondary structures in the 59NCR and the these in 16 chamois. Lack of PCR ampli- aligned sequence data were used to con- ®cation and positive IHC staining in a few struct a phylogenetic tree. Similarity ma- samples probably was due to sample deg- trices were constructed from aligned se- radation. Absence of antibodies in all se- quence data (242 positions compared) by rum samples could have been due to re- single distance by using the Jukes and cent infections; however, absence of anti- Cantor correction for multiple base chang- bodies in the animal kept alive longer than es (Jukes and Cantor, 1969). The phylo- 1 mo might mean that this was a PI ani- genetic tree was constructed by the neigh- mal. However, it is not known if chamois bor-joining method (Saitou and Nei, 1987) can become persistently infected with pes- as implemented in MEGA version 2.1 tiviruses and how long a PI chamois would (Kumar et al., 2001). live. Affected chamois had nonspeci®c signs, Two hypotheses should be considered. such as dif®culty moving and absence of One hypothesis is that the pestivirus infec- ¯ight reaction due to human presence. tion appeared several years previously, but They were cachectic and had pallid mu- remained undetected until it spread cous membranes. Eleven chamois had throughout a large part of the population. varying degrees of alopecia, mainly of the This hypothesis is supported by evidence trunk and neck, with skin hyperpigmen- of persistent viremia in RP-12/02. Alter- tation. All chamois had lower red blood natively, the disease could have been the cell counts, hematocrits, and hemoglobin result of introduction of a virulent patho- values, compared to reference values (un- gen with an ef®cient transmission rate.
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