title: Entirely plasmid-based reverse genetics system for rotaviruses.
abstract: Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein-tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Kanai
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192 Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Komoto
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.|Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kawagishi
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.|Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.
authors.forename: Ryotaro
authors.initials: R
authors.lastname: Nouda
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Nagasawa
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.
authors.forename: Misa
authors.initials: M
authors.lastname: Onishi
authors.affiliation: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Matsuura
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192 Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Taniguchi
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan; tkobayashi@biken.osaka-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000821:virology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D018165:Orthoreovirus / Q000235:genetics* / Q000378:metabolism; D030102:Orthoreovirus, Mammalian / Q000235:genetics* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D059386:Reverse Genetics / Q000379:methods*; D014161:Transduction, Genetic; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D009097:Multienzyme Complexes; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C104282:nsp1 protein, Rotavirus; D049452:Green Fluorescent Proteins; D008156:Luciferases; D008780:Methyltransferases; D009713:Nucleotidyltransferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus
keywords: reporter virus; reverse genetics; rotavirus; vaccine