title: Development of a novel single step reverse genetics system for feline calicivirus.
abstract: The reverse genetics system is a useful tool to generate infectious virus. Feline calicivirus (FCV), a member of the genus Vesivirus in the family Caliciviridae, has a positive sense, single-stranded RNA genome. Two reverse genetics systems have been established for FCV; however, these methods need multi-steps to produce progeny infectious virus. In this study, a novel plasmid-based single step reverse genetics system for FCV has been developed. The plasmid carries FCV F4 strain genomic sequence with an introduced silent mutation. In addition, at the 5'- and 3'-end, a human elongation factor-1α promoter and a cis-acting hepatitis delta virus ribozyme following poly-A, were added, respectively. When the plasmid was transfected into Crandell-Rees feline kidney cells, progeny FCV was generated. The reverse genetics system-derived FCV (rFCV) showed similar growth kinetics and antigenic characteristics and had identical genomic terminals to those of the original FCV F4 strain. The presence of the introduced silent mutation in the rFCV genomic cDNA supported that the progeny virus was originated from the plasmid. This novel FCV reverse genetics system is simple and can be used to evaluate the functions of the viral genome, proteins, and phenotypic characterization of FCV strains in the future.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan; Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, 44691 OH, USA. Electronic address: oka-t@nih.go.jp.
authors.forename: Tomoichiro
authors.initials: T
authors.lastname: Oka
authors.affiliation: Division of Biosafety Control and Research, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Takagi
authors.affiliation: Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi 252-0880, Kanagawa, Japan.
authors.forename: Yukinobu
authors.initials: Y
authors.lastname: Tohya
mesh_terms: D000818:Animals; D017927:Calicivirus, Feline / Q000235:genetics* / Q000502:physiology; D002415:Cats; D002460:Cell Line; D005822:Genetic Vectors; D003698:Hepatitis Delta Virus / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000235:genetics; D059386:Reverse Genetics / Q000379:methods*; D014779:Virus Replication
chemical_list: D020648:Peptide Elongation Factor 1; D016337:RNA, Catalytic
keywords: Elongation factor-1 alpha promoter; Positive sense, single-stranded RNA virus; Reverse genetics