Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant.
OBJECTIVE
To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1).


METHODS
The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue 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, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests.


CONCLUSIONS
The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf 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.
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan 66506, USA.
S I
SI
Chowdhury
C S
CS
Ross
B J
BJ
Lee
V
V
Hall
H J
HJ
Chu
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004279:DNA, Viral / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006566:Herpesviridae Infections / Q000517:prevention & control / Q000662:veterinary; D007242:Herpesvirus 1, Bovine / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D007106:Immune Sera / Q000096:biosynthesis; D007700:Kinetics; D009500:Neutralization Tests / Q000662:veterinary; D010957:Plasmids / Q000737:chemistry; D011817:Rabbits; D011897:Random Allocation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014614:Vaccines, Synthetic / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics*; D014764:Viral Proteins; D017201:Virus Shedding
D004279:DNA, Viral; D007106:Immune Sera; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins