An over-lapping bacterial artificial chromosome system that generates vectorless progeny for Channel catfish herpesvirus

College of Veterinary Medicine, Mississippi State University Institute for Digital Biology, Mississippi Agriculture and Forestry Experiment Station, MSU Life Sciences and Biotechnology Institute, Mississippi State University, Mississippi State, Mississippi 39762, USA

^* To whom correspondence should be addressed. Email: dkunec{at}cvm.msstate.edu.

	Abstract

Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild type (wt) virus are extremely valuable tools for elucidating the role of specific genes in virus pathophysiology as well as for making vaccines. The Ictalurid herpesvirus 1 (Channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here we describe cloning the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BAC). These clones allowed us to regenerate vectorless wild type CCVs with an indistinguishable phenotype from the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full length CCV BAC by replacing the CCV ORF5 with the BAC cassette and co-transfecting CCO cells. The viral progeny we used to transform Escherichia coli and the resulting BAC had only one of the 18 kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of generating an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants.