Rapid and efficient construction of recombinant bovine herpesvirus 1 genomes.
Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle. Recombinant bovine herpesvirus 1 viruses (rBoHV) have been studied extensively as potential vaccines for BoHV-1 associated diseases. A method is described which advances protocols used currently for constructing rBoHV by producing recombinant viruses free of parent virus. The method, restriction endonuclease mediated recombination (REMR), utilises a unique NsiI site in the BoHV-1 genome. Following NsiI digestion the two genomic fragments are prevented from recombining by dephosphorylation. However, when the genomic fragments are co-transfected into a susceptible cell-line with a third DNA fragment (DNA bridge), which encodes DNA homologous to the digested viral termini, the three DNA molecules are able to undergo homologous recombination and produce infectious BoHV-1. During the recombination process foreign DNA within the DNA bridge is incorporated into the BoHV-1 genome, producing rBoHV. In the absence of the DNA bridge virus reconstitution does not occur thus eliminating contamination by the nonrecombinant parent virus. As REMR used an NsiI site occurring naturally in the BoHV-1 genome it can be used for the insertion of foreign DNA into the genome without any prior modifications. REMR could also be applied to any herpesvirus for which the genome sequence is known.
Mahony
Timothy J
TJ
Queensland Agricultural Biotechnology Centre, Agency for Food and Fibre Sciences, Gehrmann Laboratories, Research Road, 4072, Queensland, Brisbane, Australia. timothy.mahony@dpi.qld.gov.au
McCarthy
Fiona M
FM
Gravel
Jennifer L
JL
Young
Peter L
PL
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D016679:Genome, Viral; D007242:Herpesvirus 1, Bovine / Q000235:genetics* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013997:Time Factors; D014162:Transfection
D004279:DNA, Viral; C048503:ATGCAT-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific