title: Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells.
abstract: The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.
authors.affiliation: Division of Molecular Biology, Institute for Animal Health, Pirbright Laboratory, Ash Road, Surrey GU24 ONF, Woking, UK.
authors.forename: S C
authors.initials: SC
authors.lastname: Das
authors.forename: M D
authors.initials: MD
authors.lastname: Baron
authors.forename: M A
authors.initials: MA
authors.lastname: Skinner
authors.forename: T
authors.initials: T
authors.lastname: Barrett
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D005587:Fowlpox virus / Q000235:genetics* / Q000254:growth & development; D006041:Goats; D018107:Peste-des-petits-ruminants virus / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012302:Rinderpest virus / Q000254:growth & development; D012756:Sheep; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics* / Q000254:growth & development; D014709:Vero Cells; D014764:Viral Proteins; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases