Channel Catfish Herpesvirus (CCV) Encodes a Functional Thymidine Kinase Gene: Elucidation of a Point Mutation That Confers Resistance to Ara-T

Abstract

The channel catfish herpesvirus (CCV) thymidine kinase (TK) gene was mapped on the CCV genome by marker rescue analysis using a TK-deficient channel catfish ovary cell line (CCO), a TK-negative CCV mutant, and a panel of cloned CCV genomic DNA fragments. The TK-deficient cell line (CCOBr) was isolated after repeated propagation of CCO cells in increasing concentrations of 5-bromo-2′-deoxyuridine. Infection of CCOBr cells with CCV produced high levels of TK activity. The TK- virus (CCVAr) was isolated after repeated propagation in the presence of the TK-activated antiherpetic agent, 1-β-d-arabinofuranosylthymine (Ara-T). A CCV genomic DNA library was constructed into cosmid pHC 79. Marker rescue analysis mapped the mutation within a 3.1-kb fragment located internal to the 18-kb repeat ends of the CCV genome. These genomic coordinates contained a putative TK gene identified by homology to other herpesvirus TK and cellular deoxycytidine kinase genes. DNA sequencing of the mapped coordinates identified the presence of a single mutation in the CCVAr mutant virus which resulted in a stop codon at amino acid position 97. These results functionally confirm that ORF 5 identified by Davison (Virology 186, 9-14, 1992) is the TK gene and show that CCV is amenable to marker rescue and marker transfer genetic analyses extensively used for investigations of the molecular biology of other herpesviruses.