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Reverse Genetics of Influenza Virus

  • Chang-Won LeeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1161)

Abstract

Reverse genetics is the creation of a virus from a full-length cDNA copy of the viral genome, referred to as an “infectious clone,” and is one of the most powerful genetic tools in modern virology. Since its development in 1999, plasmid-based reverse genetics has been effectively applied to numerous aspects of influenza studies which include revolutionizing the production of seasonal and pandemic influenza vaccine seed strains. Although continual improvement in reverse genetics system is being made in different laboratories for the efficient rescue of the influenza virus, the basic concept of synthesizing viral RNA using RNA polymerase I remains the same. Coupled with in vitro mutagenesis, reverse genetics can be applied widely to accelerate progress in understanding the influenza virus life cycle, the generation of customized vaccine seed strains, development of live-attenuated vaccines, and the use of influenza virus as vaccine and gene delivery vectors.

Key words

Influenza Reverse genetics Infectious clone Mutagenesis Cloning 

Notes

Acknowledgments

The authors would like to thank Dr. Gerd Hobom (Institut fur Mikro- und Molekularbiologie, Giessen, Germany) for kindly providing pHH21 vector and Dr. Yoshihiro Kawaoka (University of Wisconsin) for providing plasmids for A/WSN/33 (H1N1) reverse genetics.

References

  1. 1.
    Engelhardt OG (2013) Many ways to make an influenza virus: review of influenza virus reverse genetics methods. Influenza Other Respir Viruses 7:249–256PubMedCrossRefGoogle Scholar
  2. 2.
    Boyer J-C, Haenni A-L (1994) Infectious transcripts and cDNA clones of RNA viruses. Virology 198:415–426PubMedCrossRefGoogle Scholar
  3. 3.
    Palese P, Zheng H, Engelhardt OG, Pleschka S, Garcia-Sastre A (1996) Negative-strand RNA viruses: genetic engineering and applications. Proc Natl Acad Sci U S A 93:11354–11358PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Lamb RA, Krug RM (2001) Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howle PM (eds) Fields virology. Lippincott Williams & Wilkins, Philadelphia, PA, pp 1487–1532Google Scholar
  5. 5.
    Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, Gao P, Hughes M, Perez DR, Donis R, Hoffmann E, Hobom G, Kawaoka Y (1999) Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A 96:9345–9350PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Fodor E, Devenish L, Engelhardt OG, Palese P, Brownlee GG, Garcia-Sastre A (1999) Rescue of influenza A virus from recombinant DNA. J Virol 73:9679–9682PubMedCentralPubMedGoogle Scholar
  7. 7.
    Neumann G, Kawaoka Y (2002) Synthesis of influenza virus: new impetus from an old enzyme, RNA polymerase I. Virus Res 82:153–158PubMedCrossRefGoogle Scholar
  8. 8.
    Hoffmann E, Nuemann G, Hobom G, Webster RG (2000) “Ambisense” approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template. Virology 267:310–317PubMedCrossRefGoogle Scholar
  9. 9.
    Neumann G, Fujii K, Kino Y, Kawaoka Y (2005) An improved reverse genetics system for influenza A virus generation and its implications for vaccine production. Proc Natl Acad Sci U S A 102:16825–16829PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Chen H, Ye J, Xu K, Angel M, Shao H, Ferrero A, Sutton T, Perez DR (2012) Partial and full PCR-based reverse genetics strategy for influenza viruses. PLoS One 7:e46378PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Plainview, NYGoogle Scholar
  12. 12.
    Hoffmann E, Krauss S, Perez D, Webby R, Webster RG (2002) Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine 20:3165–3170PubMedCrossRefGoogle Scholar
  13. 13.
    Wang L, Lee CW (2009) Sequencing and mutational analysis of the non-coding regions of influenza A virus. Vet Microbiol 135:239–247PubMedCrossRefGoogle Scholar
  14. 14.
    Lee CW, Senne DA, Suarez DL (2004) Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza. Vaccine 22:3175–3181PubMedCrossRefGoogle Scholar
  15. 15.
    Lee CW, Lee YJ, Senne DA, Suarez DL (2006) Pathogenic potential of North American H7N2 avian influenza virus: a mutagenesis study using reverse genetics. Virology 353:388–395PubMedCrossRefGoogle Scholar
  16. 16.
    Lee CW, Senne DA, Suarez DL (2006) Production of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens. Clin Vaccine Immunol 13:395–402PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Czudai-Matwich V, Schnare M, Pinkenburg O (2013) A simple and fast system for cloning influenza A virus gene segments into pHW2000- and pCAGGS-based vectors. Arch Virol 158:2049–2058PubMedCrossRefGoogle Scholar
  18. 18.
    Mostafa A, Kanrai P, Ziebuhr J, Pleschka S (2013) Improved dual promotor-driven reverse genetics system for influenza viruses. J Virol Methods 193:603–610PubMedCrossRefGoogle Scholar
  19. 19.
    Massin P, Rodrigues P, Marasescu M, van der Werf S, Naffakh N (2005) Cloning of the chicken RNA polymerase I promoter and use for reverse genetics of influenza A viruses in avian cells. J Virol 79:13811–13816PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Wang Z, Duke GM (2007) Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells. Virol J 4:102PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Song MS, Baek YH, Pascua PN, Kwon HI, Park SJ, Kim EH, Lim GJ, Choi YK (2013) Establishment of Vero cell RNA polymerase I-driven reverse genetics for Influenza A virus and its application for pandemic (H1N1) 2009 influenza virus vaccine production. J Gen Virol 94:1230–1235PubMedCrossRefGoogle Scholar
  22. 22.
    Tang Y, Lee CW, Zhang Y, Senne DA, Dearth R, Byrum B, Perez D, Suarez DL, Saif YM (2005) Isolation and characterization of H3N2 influenza A virus from Turkeys. Avian Dis 49:207–213PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Food Animal Health Research Program, Ohio Agricultural Research and Development CenterThe Ohio State UniversityWoosterUSA

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