GENERATION OF CYTOPATHOGENIC SUBGENOMIC RNA OF CLASSICAL SWINE FEVER VIRUS IN PERSISTENTLY INFECTED PORCINE CELL-LINES

Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a c DNA clone of Alfort/187, were used to establish persistently infected cultures of the swine kidney cell lines SK-6 and PK-41. It was found t hat 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supern atants collected upon passaging of the cells-continuously contained hi gh titers of infectious virus. In six separate cultures persistently i nfected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be p assaged repeatedly, suggesting the generation of a mutant virus. Analy sis of RNA from such cultures revealed the presence of a subgenomic vi ral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicat ed and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral pr otein NS3 thought to be involved in pestivirus cytopathogenicity. (C) 1997 Elsevier Science B.V.