Transfection of bovine cell culture with bovine herpesvirus 4 DNA obtainedby cell nuclear extraction

Bovine herpes virus 4 (BHV-4) is a gamma-herpesvirus not associated with cl early defined clinical entities in cattle. The BHV-4 genome consists of a l inear dsDNA of approximately 145 Kbp which is only partially characterized and sequenced. We set up a rapid and practical method to isolate BHV-4 DNA from infected cell culture. Microfuged infected cells after exposure to hig h salt concentration and detergent allowed viral DNA to be purified. Electr ophoretic analysis of the digested DNA showed a complete digestion, corresp onding to a classical EcoRI banding pattern of strains Movar 33/63, LVR and DN 599. Moreover the biological integrity of viral DNA here obtained, was demonstrated by transfection experiments. BHV-4 DNA was capable of forming CPE after transfection into BAE-7372 cells. Transfected cells specifically reacted with a BHV-4 infected cow serum demonstrating the presence of viral particles. The possibility of obtaining infectious viral DNA using this me thod may facilitate the construction of recombinant viruses. Specifically, through the use of cotransfection experiments with deleted or mutated viral DNA sequences, the infectious clones isolated could provide the basis for an increased understanding of BHV-4 viral gene expression, replication and pathogenesis.