Use of PCR and immunofluorescence to detect bovine herpesvirus 1 recombinants.
Homologous recombination occurs frequently between strains of the same alphaherpesvirus species. Studies of this phenomenon require techniques that can 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 between 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 polyclonal virus preparations, a PCR protocol alone does not differentiate between samples containing recombinant viruses (gC+/gE+) and those containing a mixture of both single deleted parental strains (gC-/gE+ and gC+/gE-), and false positives resulting from recombination could occur. To reduce this possibility, double-label immunofluorescence staining of isolated plaques was developed, which coupled with PCR, allows straightforward discrimination between parental strains and progeny recombinant viruses. This assay will be useful for further studies of recombination, especially those evaluating the potential emergence of recombinants between BHV-1 marker vaccine and wildtype strains.
Schynts
F
F
Department of Infectious and Parasitic Diseases, Laboratory of Virology and Immunology, Faculty of Veterinary Medicine, University of Liège, B43bis, B-4000 Sart Tilman Liège, Belgium.
Vanderplasschen
A
A
Hanon
E
E
Rijsewijk
F A
FA
van Oirschot
J T
JT
Thiry
E
E
D000818:Animals; D002417:Cattle; D005455:Fluorescent Antibody Technique / Q000662:veterinary; D006023:Glycoproteins / Q000235:genetics; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000302:isolation & purification*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D011995:Recombination, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics
D006023:Glycoproteins; D014759:Viral Envelope Proteins