Use of PCR and immunofluorescence to detect bovine herpesvirus 1 recombinants

Homologous recombination occurs frequently between strains of the same alph aherpesvirus species. Studies of this phenomenon require techniques that ca n differentiate parental strains from putative recombinant progeny viruses. Usually, progeny viruses generated by co-infection of two distinguishable parental strains are first cloned by selection of a single plaque and then characterised by PCR. An assay designed to investigate recombination betwee n two bovine herpesvirus 1 (BHV-1) strains lacking either the glycoprotein gC or gE ORF is described. A PCR assay was developed in which a single step co-amplifies both BHV-1 glycoprotein-encoding sequences. Because the usual procedure for virus isolation, viral plaque picking, can lead to polyclona l virus preparations, a PCR protocol alone does not differentiate between s amples containing recombinant viruses (gC + /gE +) and those containing a m ixture of both single deleted parental strains (gC - /gE + and gC + /gE -), and false positives resulting from recombination could occur. To reduce th is possibility, double-label immunofluorescence staining of isolated plaque s was developed, which coupled with PCR, allows straightforward discriminat ion between parental strains and progeny recombinant viruses. This assay wi ll be useful for further studies of recombination, especially those evaluat ing the potential emergence of recombinants between BHV-1 marker vaccine an d wildtype strains. (C) 2001 Elsevier Science B.V. All rights reserved.