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 drugresistant 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.

A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model

“…The ability of the recombinant BoHV-5tk∆ to replicate in cell culture was not adversely affected ( Fig. 1), as previously described in bovine and rabbit cells (Brum et al 2010b, Silva et al 2010. Replication of BoHV-5tk∆ in the nasal mucosa of lambs, however, was significantly reduced (Table 1, Fig.2), confirming previous observations in rabbits (Silva et al 2010) and calves (Santos et al 2011) and extending previous observations with tk-deleted PRV and BoHV-1 strains in pigs and calves, respectively (Kaashoek et al 1996, Ferrari et al 1998.…”

Toll-like receptor expression in the nervous system of bovine alpha-herpesvirus-infected calves

“…The neuropathogenesis of BoHV-5 is not completely understood. However, multiple factors, either host, viral or environmental, likely influence the development of clinical signs and neuropathology (Brum et al, 2010). Previous studies were primarily focused on the role of envelope glycoproteins in neurovirulence (Chowdhury et al, 2000(Chowdhury et al, , 2002Kaashoek et al, 1998).…”

A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection

“…In a previous studies, a Brazilian BoHV-5 TK negative strain (BoHV-5TKΔ) was shown to be attenuated in a rabbit model (Silva et al 2010) and a BoHV-5 brivudin-resistant strainprobably defective in TK activity-was also attenuated in rabbits (Brum et al 2009). However, in none of these studies the ability of the virus to spread into the CNS following IN inoculation was addressed.…”