Exploiting persistent infection for selection of bovine herpesvirus 4 recombinants.
Bovine herpesvirus 4 (BoHV-4) is a gamma-herpesvirus with no clear disease association, and due to its biological characteristics, has been suggested as a gene delivery vector. It was demonstrated previously that recombinant BoHV-4 carrying a neomycin-resistance gene was able to infect a human rhabdomyosarcoma cell line (RD-4), resulting in no detectable cytopathic effect (CPE) and allowing selection of G418-resistant persistently-infected cells containing circular episomal viral DNA [Donofrio, G., Cavirani, S., van Santen, V.L., 2000a. Establishment of a cell line persistently infected with recombinant BoHV-4. J. Gen. Virol. 81, 1807-1814.]. Those cells produce infectious virus and infection is predominantly non-permissive and non-cytopathic. Starting from these results, the ability of RD-4 cells to sustain persistent infection was combined with positive selection activity conferred by the neomycin-expression cassette insert, as an easier way to select recombinants of BoHV-4 following homologous recombination in permissive cells. A tool for selecting BoHV-4 recombinants was developed by drug positive selection.
Donofrio
G
G
Dipartimento di Salute Animale, Sezione di Malattie Infettive degli Animali, Facoltà di Medicina Veterinaria, Università di Parma, via del Taglio 8, 43100 Parma, Italy. gaetano.donofrio@unipr.ir
Martignani
E
E
Cavirani
S
S
Flammini
C F
CF
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D018274:Electroporation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026021:Herpesvirus 4, Bovine / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity*; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014773:Virology / Q000379:methods
D004279:DNA, Viral; D049452:Green Fluorescent Proteins; D009355:Neomycin