Date of Award

1990

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Ronald L. Thune

Abstract

The channel catfish herpesvirus (CCV) encodes a thymidine kinase (Tk) which is biochemically distinguishable from it's host, the channel catfish ovary (CCO), and other herpesvirus Tk's. Tk deficient CCO cells (CCOBr) were developed by propagating CCO cells in increasing concentrations of 5-bromo-2$\sp\prime$-deoxyuridine (BUdR). CCV infection produced high Tk activity in these Tk deficient cells. This activity was compared to CCO-Tk in ATP and CTP phosphate donor assays, nucleoside substrate-competition and dTTP feedback inhibition assays. The viral Tk was more competitively inhibited by deoxypurines than cellular Tk and showed less dTTP mediated feedback inhibition. CCO-Tk utilized lower concentrations of ATP more effectively than CCV-Tk. Neither CCV-Tk nor CCO-Tk utilized CTP as a phosphate donor. This is in contrast to other herpesvirus Tk's and indicates a divergence from mammalian herpesviruses. To map the CCV-Tk gene within the CCV genome, a Tk-negative mutant of CCV was isolated by passing the virus in the presence of BUdR and then selecting an isolate (CCVAr) that was resistant to 1 mM 1-$\beta$-D-arabinofuranosylthymine (Ara-T). A CCV incomplete genomic library was constructed by cloning restriction endonuclease EcoRI digested viral DNA into plasmid pUC-19. A complete library was constructed into cosmid pHC-79 by cloning a partial EcoRI digest of viral DNA. Four cosmid clones were isolated that collectively encompassed 98% of the genome. The cosmid CCV-DNA clones and the pUC-19 subfragments were used in cationic-liposome mediated co-transfections with whole CCVAr-DNA in marker-rescue assays. CCVAr rescue, scored by $\sp{14}$CdT mediated plaque autoradiography on CCOBr cells, mapped the mutation within the direct repeat ends of the genome.

Pages

100

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