title: Generation of influenza A viruses entirely from cloned cDNAs.
abstract: We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 x 10(3) plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x 10(4)-5 x 10(7) pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, 2015 Linden Drive West, Madison, WI 53706, USA.
authors.forename: G
authors.initials: G
authors.lastname: Neumann
authors.forename: T
authors.initials: T
authors.lastname: Watanabe
authors.forename: H
authors.initials: H
authors.lastname: Ito
authors.forename: S
authors.initials: S
authors.lastname: Watanabe
authors.forename: H
authors.initials: H
authors.lastname: Goto
authors.forename: P
authors.initials: P
authors.lastname: Gao
authors.forename: M
authors.initials: M
authors.lastname: Hughes
authors.forename: D R
authors.initials: DR
authors.lastname: Perez
authors.forename: R
authors.initials: R
authors.lastname: Donis
authors.forename: E
authors.initials: E
authors.lastname: Hoffmann
authors.forename: G
authors.initials: G
authors.lastname: Hobom
authors.forename: Y
authors.initials: Y
authors.lastname: Kawaoka
mesh_terms: D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D018076:DNA, Complementary; D004279:DNA, Viral / Q000235:genetics*; D004285:Dogs; D015707:HN Protein / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology; D007668:Kidney; D051379:Mice; D010957:Plasmids; D012318:RNA Polymerase I / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; D015707:HN Protein; D012367:RNA, Viral; D015678:Viral Structural Proteins; D012318:RNA Polymerase I