title: Selection and characterization of brivudin resistant bovine herpesvirus type 5.
abstract: Bovine herpesvirus type 5 (BoHV-5) is the agent of meningoencephalitis, an important disease of cattle in South America. The neuropathogenesis of BoHV-5 infection is poorly understood and most previous research focused on the role of envelope glicoproteins in neurovirulence. Thymidine kinase (TK) is a viral enzyme necessary for virus replication in neurons and, therefore, represents a potential target for virus attenuation. The selection and characterization of BoHV-5 variants resistant to the nucleoside analog brivudin (BVDU), which selects TK-defective viruses is here described. Several BVDU-resistant clones were obtained after multiple passages in tissue culture in the presence of BVDU and one clone (BoHV-5/R-27) was further characterized. The selected clone replicated to similar titers and produced plaques with similar size and morphology to those of wild-type virus (SV507/99). The genetic stability of the resistant virus was demonstrated after ten passages in cell culture in the absence of the drug. Moreover, the drug-resistant virus showed reduced virulence in a rabbit model: virus inoculation in four rabbits did not result in disease, in contrast with 75% morbidity (3/4) and 50% mortality (2/2) among rabbits inoculated with the parental virus. These results demonstrate that BoHV-5 is sensitive to BVDU and that drug-resistant mutants can be readily selected upon BVDU treatment. BVDU-resistant mutants, likely defective in TK, retained their ability to replicate in tissue culture yet were attenuated for rabbits. This strategy to obtain TK-defective BoHV-5 may be useful to study the role of TK in BoHV-5 neuropathogenesis and for vaccine development.
authors.affiliation: Programa de Pós-Graduação em Medicina Veterinária, Centro de Ciências Rurais, Universidade Federal de Santa Maria , Santa Maria, RS , Brasil.
authors.forename: Mário Celso Sperotto
authors.initials: MC
authors.lastname: Brum
authors.forename: Cyndia Mara Bezerra
authors.initials: CM
authors.lastname: Dos Santos
authors.forename: Rudi
authors.initials: R
authors.lastname: Weiblen
authors.forename: Eduardo Furtado
authors.initials: EF
authors.lastname: Flores
keywords: BoHV-5; brivudin; drug resistant; thymidine kinase
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