title: Replication-incompetent influenza A viruses that stably express a foreign gene.
abstract: A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.|Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.
authors: Makoto
authors: M
authors: Ozawa
authors: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.
authors: Sylvia T
authors: ST
authors: Victor
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.
authors: Andrew S
authors: AS
authors: Taft
authors: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.
authors: Shinya
authors: S
authors: Yamada
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.
authors: Chengjun
authors: C
authors: Li
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.
authors: Masato
authors: M
authors: Hatta
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.
authors: Subash C
authors: SC
authors: Das
authors: Influenza Virus Research Center, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan.
authors: Emi
authors: E
authors: Takashita
authors: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.
authors: Satoshi
authors: S
authors: Kakugawa
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.
authors: Eileen A
authors: EA
authors: Maher
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.
authors: Gabriele
authors: G
authors: Neumann
authors: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA.|Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.|ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan.|Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.
authors: Yoshihiro
authors: Y
authors: Kawaoka
mesh_terms: D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005814:Genes, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
chemical_list: D006388:Hemagglutinins; C502893:PB2 protein, influenza virus; D012367:RNA, Viral; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D009439:Neuraminidase