Elsevier

Veterinary Microbiology

Volume 73, Issue 4, 11 May 2000, Pages 253-259
Veterinary Microbiology

Molecular characterization of Brazilian isolates of orf virus

https://doi.org/10.1016/S0378-1135(99)00151-0Get rights and content

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 modified 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 field 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 confirmed by these hybridizations.

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 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 significant mortality and morbidity in suckling animals (Mazur and Machado, 1989).

Orf virus, together with bovine papular stomatitis and milker’s nodule virus (paravaccinia), is classified in the Parapoxvirus genus (Fenner, 1976). Although viral isolates may be readily grouped as parapoxvirus, the species classification 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 identification and classification of viral isolates (Gassman et al., 1985). The classification 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 identification of the etiologic agent, DNA restriction pattern analysis may be useful (Rafii and Burger, 1985, Robinson et al., 1987). For this purpose, viral DNA is usually obtained from viral growth in cellular monolayers and purified by ultracentrifugation (Esposito et al., 1978, Robinson et al., 1982, Robinson et al., 1987). In this regard, we have developed a simplified method for DNA extraction directly from scabs, which in addition to reducing time and cost (Mazur et al., 1991), allows molecular analysis of field 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.

Section snippets

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 (20.4°S; 43.5°W), 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 (29.4°S; 53.5°W), 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

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 classification within the parapoxvirus genus, were oval-shaped, measuring approximately 300−350 nm× 200−250 nm (Mazur and Machado, 1989). The morphology of orf-NE and R viruses was also confirmed by electron microscopy (data not shown).

To establish the DNA restriction profile of the Brazilian viral isolates, high molecular

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

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, Laboratório de Análise e Produção de Imagens–FIOCRUZ–for illustration.

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