Generation of Influenza A Viruses Entirely from Cloned cDNAs

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 96; no. 16; pp. 9345 - 9350
• Main Authors: Neumann, Gabriele, Watanabe, Tokiko, Ito, Hiroshi, Watanabe, Shinji, Goto, Hideo, Gao, Peng, Hughes, Mark, Perez, Daniel R., Donis, Ruben, Hoffmann, Erich, Hobom, Gerd, Kawaoka, Yoshihiro
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 03-08-1999; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Animals; Biological Sciences; Cell Line; Chick Embryo; Cloning; Complementary DNA; Deoxyribonucleic acid; DNA; DNA, Complementary; DNA, Viral - genetics; Dogs; Helper viruses; HN Protein - genetics; Humans; Infections; Influenza; Influenza A virus; Influenza A virus - genetics; Influenza A virus - physiology; Influenza virus; Kidney; Mice; Orthomyxoviridae; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA Polymerase I - metabolism; RNA, Viral - genetics; Transfection; Viral RNA; Viral Structural Proteins - genetics; Virus Replication; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.16.9345

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 1 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 $>1\times 10^{3}$ 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× 104-5× 107 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.