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Reverse Genetics of RNA Viruses pp 251–273Cite as

Plasmid-Based Reverse Genetics of Influenza A Virus

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1602))

Abstract

Influenza A viruses have broad host range with a recognized natural reservoir in wild aquatic birds. From this reservoir, novel strains occasionally emerge with the potential to establish stable lineages in other avian and mammalian species, including humans. Understanding the molecular changes that allow influenza A viruses to change host range is essential to better assess their animal and public health risks. Reverse genetics systems have transformed the ability to manipulate and study negative strand RNA viruses. In the particular case of influenza A viruses, plasmid-based reverse genetics approaches have allowed for a better understanding of, among others, virulence, transmission, mechanisms of antiviral resistance, and the development of alternative vaccines and vaccination strategies. In this chapter we describe the cloning of cDNA copies of viral RNA segments derived from a type A influenza virus into reverse genetics plasmid vectors and the experimental procedures for the successful generation of recombinant influenza A viruses.

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References

  1. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (2012) Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego

    Google Scholar 

  2. Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, Recuenco S, Ellison JA, Davis CT, York IA, Turmelle AS, Moran D, Rogers S, Shi M, Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X, Frace M, Lindblade KA, Cox NJ, Anderson LJ, Rupprecht CE, Donis RO (2012) A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci U S A 109(11):4269–4274. doi:10.1073/pnas.1116200109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen X, Recuenco S, Gomez J, Chen LM, Johnson A, Tao Y, Dreyfus C, Yu W, McBride R, Carney PJ, Gilbert AT, Chang J, Guo Z, Davis CT, Paulson JC, Stevens J, Rupprecht CE, Holmes EC, Wilson IA, Donis RO (2013) New world bats harbor diverse influenza A viruses. PLoS Pathog 9(10):e1003657. doi:10.1371/journal.ppat.1003657

    Article  PubMed  PubMed Central  Google Scholar 

  4. Brunotte L, Beer M, Horie M, Schwemmle M (2016) Chiropteran influenza viruses: flu from bats or a relic from the past? Curr Opin Virol 16:114–119. doi:10.1016/j.coviro.2016.02.003

    Article  PubMed  Google Scholar 

  5. Webster RG, Yakhno M, Hinshaw VS, Bean WJ, Murti KG (1978) Intestinal influenza: replication and characterization of influenza viruses in ducks. Virology 84(2):268–278

    Article  CAS  PubMed  Google Scholar 

  6. Webster RG, Hinshaw VS, Bean WJ, Sriram G (1980) Influenza viruses: transmission between species. Philos Trans R Soc Lond Ser B Biol Sci 288(1029):439–447

    Article  CAS  Google Scholar 

  7. Hinshaw VS (1998) Influenza in other Species (Seal, Whale, and Mink). In: Nicholson KG, Webster RG, Hay AJ (eds) Textbook of Influenza. Blackwell Science Ltd., Oxford, pp 163–167

    Google Scholar 

  8. Reid AH, Taubenberger JK, Fanning TG (2004) Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol 2(11):909–914. doi:10.1038/nrmicro1027

    Article  CAS  PubMed  Google Scholar 

  9. Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, Ma SK, Cheung CL, Raghwani J, Bhatt S, Peiris JS, Guan Y, Rambaut A (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459(7250):1122–1125. doi:10.1038/nature08182

    Article  CAS  PubMed  Google Scholar 

  10. Sorrell EM, Wan H, Araya Y, Song H, Perez DR (2009) Minimal molecular constraints for respiratory droplet transmission of an avian-human H9N2 influenza A virus. Proc Natl Acad Sci U S A 106(18):7565–7570. doi:10.1073/pnas.0900877106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus AD, Fouchier RA (2012) Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336(6088):1534–1541. doi:10.1126/science.1213362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sutton TC, Finch C, Shao H, Angel M, Chen H, Capua I, Cattoli G, Monne I, Perez DR (2014) Airborne transmission of highly pathogenic H7N1 influenza virus in ferrets. J Virol 88(12):6623–6635. doi:10.1128/JVI.02765-13

    Article  PubMed  PubMed Central  Google Scholar 

  13. Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y (2012) Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486(7403):420–428. doi:10.1038/nature10831

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Stobart CC, Moore ML (2014) RNA virus reverse genetics and vaccine design. Viruses 6(7):2531–2550. doi:10.3390/v6072531

    Article  PubMed  PubMed Central  Google Scholar 

  15. Engelhardt OG (2013) Many ways to make an influenza virus--review of influenza virus reverse genetics methods. Influenza Other Respir Viruses 7(3):249–256. doi:10.1111/j.1750-2659.2012.00392.x

    Article  CAS  PubMed  Google Scholar 

  16. de Wit E, Spronken MI, Vervaet G, Rimmelzwaan GF, Osterhaus AD, Fouchier RA (2007) A reverse-genetics system for Influenza A virus using T7 RNA polymerase. J Gen Virol 88(Pt 4):1281–1287. doi:10.1099/vir.0.82452-0

    Article  PubMed  Google Scholar 

  17. 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(16):9345–9350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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(11):9679–9682

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Hoffmann E, Neumann G, Hobom G, Webster RG, Kawaoka Y (2000) “Ambisense” approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template. Virology 267(2):310–317

    Article  CAS  PubMed  Google Scholar 

  20. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG (2000) A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97(11):6108–6113. doi:10.1073/pnas.100133697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

M.A. is funded by the Intramural Research Program of the NIH, NIAID, A. S. G.-R., J. S., A. O. and D. R. P.’s research is funded by the University of Georgia, NIH contract HHSN272201400008C, and Scientific Cooperative Agreements with ARS-USDA. L.M-S is funded by the University of Rochester and the NH contract HHSN272201400005C.

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Authors and Affiliations

  1. Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA

    Matthew Angel & Ana Silvia Gonzalez-Reiche

  2. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, Rochester, NY, 14642, USA

    Luis Martinez-Sobrido

  3. Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA

    Daniel R. Perez, Jefferson Santos & Adebimpe Obadan

Authors
  1. Daniel R. Perez
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  2. Matthew Angel
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  3. Ana Silvia Gonzalez-Reiche
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  4. Jefferson Santos
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  5. Adebimpe Obadan
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  6. Luis Martinez-Sobrido
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Corresponding author

Correspondence to Daniel R. Perez .

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Editors and Affiliations

  1. Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

    Daniel R. Perez

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Perez, D.R., Angel, M., Gonzalez-Reiche, A.S., Santos, J., Obadan, A., Martinez-Sobrido, L. (2017). Plasmid-Based Reverse Genetics of Influenza A Virus. In: Perez, D. (eds) Reverse Genetics of RNA Viruses. Methods in Molecular Biology, vol 1602. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6964-7_16

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  • DOI: https://doi.org/10.1007/978-1-4939-6964-7_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6962-3

  • Online ISBN: 978-1-4939-6964-7

  • eBook Packages: Springer Protocols

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