title: Development of a novel single-step reverse genetics system for the generation of classical swine fever virus.
abstract: 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.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Huining
authors.initials: H
authors.lastname: Pang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Yiluo
authors.initials: Y
authors.lastname: Tan
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China. zspan@whu.edu.cn.
authors.forename: Zishu
authors.initials: Z
authors.lastname: Pan
mesh_terms: D000818:Animals; D002460:Cell Line; D006691:Classical Swine Fever / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D059386:Reverse Genetics / Q000379:methods*; D013552:Swine; D014162:Transfection
chemical_list: D012367:RNA, Viral