Mutagenesis Of A Bovine Herpesvirus Type 1 Genome Cloned As An Infectious Bacterial Artificial Chromosome: Analysis Of Glycoprotein N And/or E Genes Deletion Mutants In Vitro

Bovine Herpesvirus type 1 (BHV1) can cause a variety of clinical diseases, such as infectious bovine rhinotracheitis(IBR), infectious pustular vulvovaginitis(IPV) and abortion. In addition to some South American countries, BHV1 infection widespread in the world currently, countries in the world have isolated the virus, the spread of some Chinese provinces and cities also. BHV1 infection on milk yield of cows, bull fertility, as well as causative force cattle have a greater influence to the world's cattle industry and have caused tremendous economic lossesFor a long time,the main method studying on the functional genes of herpesvirus was to construct genetic-deleting mutants.However,it was tedious to produce recombinant mutants using homologous recombinant in infected cells. In 1997,Messerle et al.,German researehers,firstly constructed infectious clone of MCMV based on baeterial artificial chxomosomes(BACs).This technique actualized that the genomes of infectious virus could be stored,propagated or modificated by BAC-plasmid pattern in E.coli.and it was convenient and rapid to manipulate the technique. This technique lead to advancement in studying on gene functions of herpesvirus,and provided new ways to develop high-performance herpesviral vetor system.In this study,using two-step Red-mediated recombination technology in Escherichia coli,the glycoprotein(g)E gene, gN gene, the double of gE and gN genes were respectively deleted from pBHV1-ZJ, an bacterial artificial chromosome(BAC) clone carrying whole genome of bovine herpesvirus type 1. The resulting BAC clones, pBHV1-ΔgE or pBHV1-ΔgN, were transfected into bovine kidney cells (MDBK) and rBHV1-ΔgE or rBHV1-ΔgN were recovered, but the double deletion mutant was not recovered after transfection. The growth kinetics and plaque tests indicated that rBHV1-ΔgN exhibit markedly lowered virus titres than the parental virus rBHV1 in MDBK and rBHV1-ΔgE did not, plaque sizes of rBHV1-ΔgE and rBHV1-ΔgN were respectively reduced by 37% and 82% compared to rBHV1. The BHV1 viruses deleted gE or/and gN gene will help to research the cooperation of gE and gN in the viral replication and to develop new marked vaccines.