Molecular characterization of Brazilian isolates of orf virus

Veterinary Microbiology 73 (2000) 253±259

Molecular characterization of Brazilian isolates of orf virus C. Mazura,*, I.I. Ferreirab, F.B. Rangel Filhoa, R. Gallerb a

Instituto de VeterinaÂria, Universidade Federal Rural do Rio de Janeiro, Km 47 Rodovia Rio-S.Paulo, 23890-000 SeropeÂdica, RJ, Brazil b Departamento de BioquõÂmica e Biologia Molecular, Fundac,aÄo Oswaldo Cruz. Av.Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil Received 17 July 1998; received in revised form 26 May 1999; accepted 22 September 1999

Abstract Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modi®ed methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed ®eld gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was con®rmed by these hybridizations. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Orf virus; Parapoxvirus; Poxvirus

1. Introduction Orf virus (contagious pustular dermatitis or scabby mouth virus) is a parapoxvirus that causes an exanthematic dermatitis mainly in goats and sheep. The orf virus can also infect man and is considered an occupational zoonosis (Robinson and Ballassu, 1981). The lesions are characterized initially by macules which progress through papules, vesicles *

Corresponding author. Tel.: ‡55-21-6821109; fax: ‡55-21-6821711. E-mail address: mazur@dbbm.®ocruz.br (C. Mazur) 0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 1 5 1 - 0

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and pustules, until scab formation, during 3±4 weeks. These scabs, rich in viral particles, drop after lesion healing and are considered important in viral transmission, keeping infectivity for long periods, under favorable conditions. The lesions are generally mild and commonly occur in young animals, mainly on the lips (Mazur and Machado, 1990). Nevertheless, some outbreaks may be marked by exacerbated virulence, causing signi®cant mortality and morbidity in suckling animals (Mazur and Machado, 1989). Orf virus, together with bovine papular stomatitis and milker's nodule virus (paravaccinia), is classi®ed in the Parapoxvirus genus (Fenner, 1976). Although viral isolates may be readily grouped as parapoxvirus, the species classi®cation remains problematic (Gassman et al., 1985). Molecular analysis of parapoxvirus isolates based on DNA restriction patterns, revealed genetic heterogeneity among isolates of the same viral species (Wittek et al., 1980). Therefore, such analysis may be useful to characterize molecularly parapoxvirus members and allow precise identi®cation and classi®cation of viral isolates (Gassman et al., 1985). The classi®cation of the three viruses above in the parapoxvirus genus was determined by biological assays (Huck, 1966), by serology (Rosenbusch and Reed, 1983), by DNA hybridization and DNA restriction pattern analysis (Gassman et al., 1985). Under these conditions, any new isolate may be tentatively related with one of the described members of the genus (orf, bovine pustular stomatitis and paravaccinia viruses). A number of outbreaks of a disease suggesting parapox infection in sheep and goats has been observed in Brazil. For a positive identi®cation of the etiologic agent, DNA restriction pattern analysis may be useful (Ra®i and Burger, 1985; Robinson et al., 1987). For this purpose, viral DNA is usually obtained from viral growth in cellular monolayers and puri®ed by ultracentrifugation (Esposito et al., 1978; Robinson et al., 1982, 1987). In this regard, we have developed a simpli®ed method for DNA extraction directly from scabs, which in addition to reducing time and cost (Mazur et al., 1991), allows molecular analysis of ®eld isolates unaltered by adaptation to growth in cell cultures. Here, we describe the heterogeneity among Brazilian viral isolates detected by restriction enzyme analyses and hybridization of viral DNA genomic blots to the cloned HindIII-G fragment. 2. Material and methods 2.1. Viruses The viruses used in this study were obtained from three distinct geographic regions of Brazil. Orf-A virus was isolated from goats in Conselheiro Lafaiete (20.48S; 43.58W), State of Minas Gerais (Mazur and Machado, 1990) and represents the southeast region of Brazil. Orf-R virus was isolated from an outbreak in sheep in Santa Maria (29.48S; 53.58W), State of Rio Grande do Sul (southern region), in 1992. Orf-NE1 and NE2 viruses were isolated from an outbreak in goats near the city of Recife (8.08S; 34.58W), in the State of Pernambuco (northeastern region), 1993. Viral isolates orf-A, NE1 and NE2 were inoculated in young goats and orf-R in sheep to obtain viral mass, each virus in one animal. For this purpose, animals were inoculated by scarifying the area of the inner thighs with scab suspensions (10% in PBS, pH 7.2). The animals were kept under

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isolation, housed and treated individually and observed daily for lesion evolution. Care was taken to avoid cross contamination during animal handling. Scabs formed weeks later were collected and stored at ÿ208C. 2.2. Nucleic acid isolation The DNA isolation was as described in Mazur et al. (1991). The only modi®cation was that the phenol±chloroform extraction was carried out using a rocking platform. This was essential to allow the extraction of high molecular weight DNA used for genomic restriction pattern analysis by pulsed ®eld electrophoresis. The best phenol±chloroform extraction required slow rocking twice for 20 min each time. Supernatants resulting from phase separation after centrifugation at 1600  g were transferred only with cut tips, to avoid damage by shearing. DNA was precipitated with ethanol and NaCl, resupended in TE buffer and kept at 48C. 2.3. Agarose gel pulsed ®eld electrophoresis Approximately 200 ng of viral DNA were digested with endonucleases HindIII, EcoRI or ClaI and fractionated on 1.2% TRIS borate-EDTA (TBE), pH 8.0, agarose gels using pulsed ®eld electrophoresis. Electrophoretic conditions used were 150 V, 90 mA, 10 s ®xed pulse, during a total of 12±24 h. After ethidium bromide staining, the fractionated DNA was blotted onto nylon membranes. 2.4. Molecular cloning and DNA hybridization For DNA cloning purposes, extracted viral DNA from orf-A was digested with HindIII endonuclease and resolved by agarose gel electrophoresis. An agarose slice containing the HindIII-G viral genome fragment was cut out and the DNA puri®ed using Gelase (Epicentre Technology, Inc.). This HindIII-G DNA was ligated into plasmid pGEM1-2 (PROMEGA) and used to transform E.coli MC1061. A plasmid containing inserted fragment was selected, named pB38, and used as probe in hybridization experiments, after labeling with 32P by nick translation (Sambrook et al., 1989). 3. Results Electron microscopy was performed to morphologically characterize viral particles present in scabs of orf-A virus isolate. The observed viral particles, consistent with their classi®cation within the parapoxvirus genus, were oval-shaped, measuring approximately 300ÿ350 nm 200ÿ250 nm (Mazur and Machado, 1989). The morphology of orf-NE and R viruses was also con®rmed by electron microscopy (data not shown). To establish the DNA restriction pro®le of the Brazilian viral isolates, high molecular weight DNA samples isolated directly from scabs using our method (Mazur et al., 1991) were digested with ClaI, EcoRI and HindIII and the resulting DNA fragments were resolved by pulsed ®eld agarose gel electrophoresis. These analyses are shown in Fig. 1 and include the hybridization pro®les, when plasmid pB38 was used as probe. Table 1

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Fig. 1. Pulsed ®eld electrophoretic and hybridization analysis of Brazilian isolates viral DNA. (a) agarose gel (150 v., 90 mA, pulse 10 s, 19 h.) showing orf-A DNA digestion; (c) agarose gel, (150 v., 90 mA, pulse 10 s, 20 h), showing orf-NE1 DNA digestion; (e) agarose gel (150 v., 90 mA, pulse 10 s, 22 h), showing orf-R DNA digestion; (b), (d), (f): autoradiograms obtained by molecular hybridization with labeled pB38 of viral DNA fragments separated on gels shown in panels (a), (c) and (e), respectively. *The arrows indicate the HindIII G fragment in panel a and the bands which correspond to this cloned fragment present in the DNA of the other viral isolates in panels (c) and (f).

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Table 1 Restriction enzyme pattern of DNA orf virus isolates orf-A

orf-NEa

orf-R

HindIII fragments A B C D E F G H Total

59.5 20.9 17.9 13.4 11.1 8.3 5.4b ± 136.8

38.1 24.0 22.3 19.3 15.0 6.3 6.2 5.9c 137.1

33.5 24.5 22.3 15.4 14.2 13.6 8.3c 5.4 137.2

EcoRI fragments A B C D E F Total

63.5c 32.5 18.4 12.0 6.0 4.8 137.2

76.9 28.6c 16.0 5.7 5.2 4.1 136.5

70.0 44.0c 16.4 3.4 3.0 2.0 138.8

ClaI fragments A B C D Total

91.7 21.7c 17.4 6.9 137.7

91.0 20.4c 19.7 6.5 137.6

85.0 32.2c 20.2 ± 137.4

a

Molecular weights are expressed in kilobases, the term orf-NE was used to refer to orf-NE1 and orf-NE2. Fragment cloned in pGEM-2 plasmid, construction referred to here as pB38. c Fragments that hybridized with pB38, used as molecular probe. b

shows the molecular weight estimates for DNA restriction fragments from Brazilian viral isolates. As expected orf-NE1 and NE2 showed the same restriction pro®le (data not shown), since they were isolated from animals of the same species and originated from the same geographical origin. An extra ClaI site was observed in viral DNA from caprines as compared to ovine viral DNA samples (Fig. 1, panels A lane 3; C, lane 1; E lane 3). Viral isolates of caprines from different geographic areas, as orf-A and NE, also differed in their DNA restriction pro®les by comparing the molecular weights of DNA fragments resulting from digestion of both DNAs with HindIII and EcoRI (Table 1). The hybridizations with plasmid pB38 indicates similarity among the pro®les of Brazilian viral isolates when the respective viral DNAs were digested with ClaI. With HindIII digestion, the hybridization pro®le was similar for viruses isolated from caprines, in contrast to the isolate from ovines (see Table 1and Fig. 1, panels (b), (d), (f), lanes 2). Hybridization of the cloned probe to EcoRI digested DNA fragments is an example of variability between isolates derived from the same host (caprines) by identifying a band of approximately 63.5 Kb for orf-A and 16.0 Kb for orf-NE (Fig. 1, panel (b), lane 4; panel (f), lane 4, respectively).

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4. Discussion Orf virus is endemic all over the world, with variable virulence for a range of hosts that includes man, domestic and wild sheep, goats and dogs (Robinson and Lyttle, 1992). In vaccinated or naturally infected animals, reinfection may occur months later. Mechanisms of protection, or its apparent absence in some cases, after parapoxvirus infection, are not satisfactorily understood. In this regard a more precise characterization of the viral isolates might provide some evidence for the relevance of genetic variation in pathogenesis and immune response. Caprine and ovine herds are bred in different areas of Brazil and in all of them outbreaks of disease which resemble parapox virus infections can be observed with variable frequency. In epidemiological investigations, the restriction enzyme digestion pattern of viral DNA could indicate the geographic origin and movement of a viral strain, what may cause outbreaks in different areas. Orf viral isolates have been characterized in many countries, but few studies are available on Brazilian parapox virus isolates. We have used direct extraction of viral DNA from scabs and its analysis with a de®ned set of restriction enzymes and pulsed-®eld electrophoresis to allow us to characterize Brazilian viral isolates. The genome size of Brazilian viral isolates, as deduced by adding up bands from the same gel lane, was found to vary from 136.5 to 138.7 Kb, a value which is in the same order of magnitude as observed for other parapoxviruses (Gassman et al., 1985). When typing viral DNA with HindIII, a major variable was the G fragment of orf-A virus genome which, as shown by hybridization, appeared to correspond to bands of different sizes in the DNA of the other Brazilian viral isolates. This is a variable area of the genome as seen for NZ strains (Robinson et al., 1987) and it is not yet known whether this variability is simply due to changes in restriction sites or rearrangements involving DNA regions. Brazilian and New Zealand (NZ) viral isolates (Robinson et al., 1987) are readily distinguishable observing the HindIII pro®les as the latter show a conserved 76 kb HindIII-A which is not observed in the DNA of Brazilian viral isolates. Apparently, an extra HindIII restriction site in the genomic region corresponding to the conserved 76 kb fragment of NZ strains (HindIII-A fragment) is present in the DNA of the Brazilian isolates. We were able to distinguish viral isolates derived from different animal hosts as well as viral isolates from the same host species that originated from different geographical locations. Having set the pro®les, we are undertaking a larger surveillance on Brazilian territory to characterize the parapoxviruses that appear among goat and ovine herds. This data should be relevant to detecting viral spread and its relation to new outbreaks. Acknowledgements This work was supported by the International Foundation for Science, Sweden, and National Council of Research (CNPq). We thank Dr. Rudi Weiblen for sending the orf-R virus from Santa Maria, Rio Grande do Sul State and Marcos Gustavo da Silva Ribeiro, LaboratoÂrio de AnaÂlise e Produc,aÄo de Imagens±FIOCRUZ±for illustration.

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References Esposito, J.J., Obueski, J.F., Nakano, J.H., 1978. Orthopoxvirus DNA: strain differentiation by eletrophoresis of restriction endonuclease fragmented virion DNA. Virology 89, 53±66. Fenner, F., 1976. Classi®cation and nomenclature of viruses. Second Report of the International Committee on Taxonomy on Viruses. Intervirol. 7, 28. Gassman, U., Wyler, R., Wittek, R., 1985. Analysis of parapoxvirus genomes. Arch. Virol. 83, 17±31. Huck, R.A., 1966. A paravaccinia virus isolated from cows' teats. Vet. Rec. 78, 503±505. Mazur, C., Machado, R.D., 1989. Detection of contagious pustular dermatitis virus of goats in a severe outbreak. Vet. Rec. 125, 419±420. Mazur, C., Machado, R.D., 1990. The isolation and identi®cation of the contagious ecthyma virus of caprines in cell cultures. Rev. Microbiol. SaÄo Paulo 21, 127±130. Mazur, C.R., Filho, F.B., Galler, R., 1991. Molecular analysis of contagious pustular dermatitis virus: a simpli®ed method for viral DNA extraction from scab material. J. Virol. Methods 35, 265±272. Ra®i, F., Burger, D., 1985. Comparison of contagious ecthyma virus genomes by restriction endonucleases. Arch. Virol. 84, 283±289. Robinson, A.J., Ballassu, T.C., 1981. Contagious pustular dermatitis orf. Vet. Bull. 51, 771±782. Robinson, A.J., Barns, G., Fraser, K., Carpenter, E., Mercer, A.A., 1987. Conservation and variation in orf virus genome. Virology 157, 13±23. Robinson, A.J., Ellis, G., Ballassu, T.C., 1982. The genome of orf virus: Restriction endonucleases analysis of viral DNA isolated from lesions in sheep. Arch Virol. 71, 43±55. Robinson, A.J., Lyttle, D.J., 1992. Parapoxviruses: Their Biology and Potential as Recombinant Vaccines. Recombinant Poxviruses, Chapter 9, CRC Press, Boca Raton, pp. 285±327. Rosenbusch, R.F., Reed, D.E., 1983. Reaction of convalescent bovine antisera with strain-speci®c antigens of parapoxviruses. Am. J. Vet. Res. 44, 875±890. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: a Laboratory Manual. 2nd ed. Laboratory Press, Cold Spring Harbor, pp. 10.2±10.12. Wittek, R., Herlyn, M., Schumperli, D., Bachmann, P.A., Mayr, A., Wyler, R., 1980. Genetic and antigenic heterogeneity of different parapoxvirus strains. Intervirol. 13, 33±41.