Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells

The suitability of recombinant T7 polymerase produced using either the high ly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two m ammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine a nd caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste d es 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 polyme rase, was found to be non-cytopathic in a variety of primary and establishe d cell lines of mammalian origin and was suitable for use in virus rescue e xperiments. MDBK cells and primary cells, unlike Vero cells, could not be e fficiently transfected and so were unsuitable for virus rescue. Optimal con ditions 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 vi ruses which grow in Vero cells. (C) 2000 Elsevier Science B.V. All rights r eserved.