title: Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein.
abstract: We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.
  
  
  OBJECTIVE
  AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan Department of Infection Control and Disease Prevention, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Takenaka-Uema
authors.affiliation: Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Murata
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan Department of Infection Control and Disease Prevention, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Gen
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Ishihara-Saeki
authors.affiliation: Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Uchida
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Murakami
authors.affiliation: Department of Infection Control and Disease Prevention, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Haga
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan ahorimo@mail.ecc.u-tokyo.ac.jp akashi@mail.ecc.u-tokyo.ac.jp.
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Akashi
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan ahorimo@mail.ecc.u-tokyo.ac.jp akashi@mail.ecc.u-tokyo.ac.jp.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Horimoto
mesh_terms: D000818:Animals; D002044:Bunyaviridae Infections / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism / Q000473:pathology / Q000821:virology; D006224:Cricetinae; D015870:Gene Expression; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008526:Medulla Oblongata / Q000378:metabolism / Q000473:pathology / Q000821:virology; D051379:Mice; D030781:Organisms, Genetically Modified; D016851:Orthobunyavirus / Q000235:genetics / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins