Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant

Objective-To construct and characterize a recombinant glycoprotein (g)E gen e-deleted bovine herpesvirus (BHV) type 1 (BHV-1). Procedure-The BHV-1 gE gene-coding region and the flanking upstream and dow nstream sequences were cloned. The aforementioned cloned DNA was digested w ith suitable enzymes to release the amino terminal two thirds of that regio n, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), B HV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (b lue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus , gE Delta 3.1IBR, was characterized in vitro and in vivo. The ability of t he recombinant virus to induce BHV-1 neutralizing antibodies in infected ca lves was investigated by plaque-reduction tests. Results and Conclusions-The gE Delta 3.1IBR virus contained a deletion in t he Viral gE gene-coding sequences where a stable chimeric reporter (beta-ga l) gene was inserted. One-step growth kinetics and virus yield of the recom binant and parent Viruses were similar, but early after infection, the reco mbinant virus yield was comparatively less. After intranasal inoculation, t he recombinant gE Delta 3.1IBR virus replicated in the upper respiratory tr act of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo cal f experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.