Construction of a viable BHV1 mutant lacking most of the short unique region

The 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies entirely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presence of nonessential genes by a simple, rapid method of insertional mutagenesis. Our analysis indicates that all the genes present in the K-fragment, except the glycoprotein D gene, are nonessential for replication of BHV1 in tissue culture. After inserting a copy of the glycoprotein D gene into the thymidine kinase locus of BHV1 it was possible to delete the entire HindIII K-fragment and the contiguous 0.36 kb O-fragment in one step. The deletion mutant, which contains 7 kb less BHV1 DNA than wt virus and lacks ORF1, US2, the genes for protein kinase, glycoprotein G, glycoprotein I, and most of glycoprotein E was still replication-competent.