Summary
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 the most powerful genetic tool in modern virology. The generation of influenza A viruses by reverse genetics has lagged mainly due to the inherent technical difficulties of providing a sufficient amount of all eight viral RNAs from cloned cDNA. A breakthrough was made in 1999 by utilizing the cellular enzyme RNA polymerase I for the synthesis of influenza viral RNAs. Although slightly different methods are being used in different laboratories for the 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 live-attenuated vaccines, and the use of influenza virus as vaccine and gene delivery vectors.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
1. Boyer, J.-C. and Haenni, A.-L. (1994) Infectious transcripts and cDNA clones of RNA viruses. Virology 198, 415–426.
2. Palese, P., Zheng, H., Engelhardt, O. G., Pleschka, S., and Garcia-Sastre, A. (1996) Negative-strand RNA viruses: Genetic engineering and applications. Proc. Natl. Acad. Sci. USA 93, 11354–11358.
3. Lamb, R. A. and Krug, R. M. (2001) Orthomyxoviridae: The viruses and their replication, in Fields Virology (D. Knipe and P. M. Howle, eds.), pp. 1487–1532. Philadelphia: Lippincott Williams & Wilkins.
4. Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G., and Kawaoka, Y. (1999) Generation of influenza A viruses entirely from cloned cDNAs. Proc. Natl. Acad. Sci. USA 96, 9345–9350.
5. Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G., and Garcia-Sastre, A. (1999) Rescue of influenza A virus from recombinant DNA. J. Virol. 73, 9679–9682.
6. Neumann, G. and Kawaoka, Y. (2002) Synthesis of influenza virus: New impetus from an old enzyme, RNA polymerase I. Virus Res. 82, 153–158.
7. Hoffmann, E., Nuemann, G., Hobom, G., and Webster, R. G. (2000) “Ambisense” approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template. Virology 267, 310–317.
8. Neumann, G., Fujii, K., Kino, Y., and Kawaoka, Y. (2005) An improved reverse genetics system for influenza A virus generation and its implications for vaccine production. Proc. Natl. Acad. Sci. USA 102, 16825–16829.
9. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Plainview, NY: Cold Spring Harbor Laboratory Press.
10. Hoffmann, E., Krauss, S., Perez, D., Webby, R., and Webster, R. G. (2002) Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine 20, 3165–3170.
11. Lee, C. W., Senne, D. A., and Suarez, D. L. (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, 3175–3181.
12. Lee, C. W., Lee, Y. J., Senne, D. A., and Suarez, D. L. (2006) Pathogenic potential of North American H7N2 avian influenza virus: A mutagenesis study using reverse genetics. Virology 353, 388–395.
13. Lee, C. W., Senne, D. A., and Suarez, D. L. (2006) Production of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens. Clin. Vac. Immunol. 13, 395–402.
14. Massin, P., Rodrigues, P., Marasescu, M., van der Werf, S., and 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, 13811–13816.
15. Tang, Y., Lee, C. W., Zhang, Y., Senne, D. A., Dearth, R., Byrum, B., Perez, D., Suarez, D. L., and Saif, Y. M. (2005) Isolation and characterization of H3N2 influenza A virus from turkeys. Avian Dis. 49, 207–213.
Acknowledgments
The authors would like to thank Dr. Gerd Hobom (Institut fur Mikro- und Molekularbiologie, Giessen, Germany) for kindly providing the pHH21 vector and Dr. Yoshihiro Kawaoka (University of Wisconsin) for providing 293T cells and plasmids for A/WSN/33 (H1N1) reverse genetics.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lee, CW., Suarez, D. (2008). Reverse Genetics of the Avian Influenza Virus. In: Spackman, E. (eds) Avian Influenza Virus. Methods in Molecular Biology™, vol 436. Humana Press. https://doi.org/10.1007/978-1-59745-279-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-279-3_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-939-0
Online ISBN: 978-1-59745-279-3
eBook Packages: Springer Protocols