Thymidine kinase (TK) negative mutants of herpesviruses are generally less virulent than wild type strains. In hopes of developing a recombinant permanently attenuated strain of channel catfish virus (CCV), we identified a unique CCV encoded thymidine kinase and mapped the CCV TK gene on the viral genome. The channel catfish virus (CCV) encodes a thymidine kinase (TK) which is biochemically distinguishable from the TK of the host cell line, channel catfish ovary (CCO), and other herpesvirus TK's. A TK deficient CCO cell line (CCOBr) resistant to 5-bromo2-deoxyuridine was isolated. CCV infection of the TK deficient cells produced high TK activity and this activity was compared to CCO-TK activity using nucleoside substrate competition, feedback inhibition and phosphate donor specificity assays. CCV-TK was more competitively inhibited by deoxy-purines than CCO-TK and showed less dTTP mediated feedback inhibition. CCV-TK was unique among herpesvirus TK's in its inability to utilize CTP as a phosphate donor. CCV genomic DNA libraries were constructed into plasmid pUC 19 and cosmic pHC 79. A 1-B-D ((Ara-T)) resistant mutant of CCV ((CCVAr)) was isolated and found to be TK negative. Cationic liposome mediated co-transfection of cloned wild type CCV DNA fragments with CCVAr whole DNA in marker rescue assays, mapped the TK mutation within a 3.0 Kb fragment on the right portion of the Eco RI D and E fragments as defined by Chousterman et al. (1979 J. Virol. 31:63-85). This location within the direct repeat ends of the genome indicates divergence from previously identified herpesvirus gene arrangements and in conjunction with unique TK substrate specificity intensifies the interest in sequencing this region for evolutionary comparisons. The CCV TK gene will be deleted from the genome and the virulence of the resultant mutant analyzed.