Development of a novel single-step reverse genetics system for the generation of classical swine fever virus

We describe an alternative reverse genetics system for generating classical swine fever virus (CSFV) based on swine RNA polymerase I promoter (pSPI)-mediated vRNA transcription. The recombinant plasmid pSPTI/SM harboring a full-length CSFV Shimen strain cDNA, flanked by a swine RNA polymerase I (pol I) promoter sequence at the 5' end and a murine pol I terminator sequence at the 3' end, was constructed. When the plasmid pSPTI/SM was introduced into PK-15 cells by transfection, an infectious CSFV with termini identical to those of the parental virus was generated directly. CSFV rescued from this reverse genetics system exhibited similar growth kinetics and plaque formation compared with the parental CSFV. When the novel reverse genetics system was used to generate the CSFV vaccine C-strain, infectious virus was detected in the supernatant of PK-15 cells transfected with the recombinant plasmid pSPTI/C. This novel reverse genetics system is a simple and efficient tool for the investigation of the structure and function of the viral genome, for molecular pathogenicity studies, and for the development of genetically engineered vaccines for CSFV.