CONSTRUCTION OF A VIABLE BHV1 MUTANT LACKING MOST OF THE SHORT UNIQUEREGION

The 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies enti rely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presen ce of nonessential genes by a simple, rapid method of insertional muta genesis. Our analysis indicates that all the genes present in the K-fr agment, except the glycoprotein D gene, are nonessential for replicati on of BHV1 in tissue culture. After inserting a copy of the glycoprote in D gene into the thymidine kinase locus of BHV1 it was possible to d elete the entire HindIII K-fragment and the contiguous 0.36kb O-fragme nt 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, glyc oprotein G, glycoprotein I, and most of glycoprotien E was still repli cation-competent.