Abstract
Reverse genetics is the process of generating an RNA virus from a cDNA copy. Reverse genetics systems have truly transformed our ability to manipulate and study negative-strand RNA viruses. Plasmid-based reverse genetics approaches for influenza viruses provide a better understanding of virulence, transmission, mechanisms of antiviral resistance, and the development of alternative vaccines and vaccination strategies. Studying the molecular changes that allow influenza A viruses (IAVs) to transmit among animal species is important to better understand their animal health and public health risks. In this chapter, the cloning of cDNA copies of IAV’s RNA segments into a reverse genetics plasmid vector, the experimental procedures for studying viral polymerase activity, and the successful generation of recombinant IAVs are described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (2012) Ninth report of the international committee on taxonomy of viruses. Elsevier Academic Press, San Diego
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. https://doi.org/10.1073/pnas.1116200109
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. https://doi.org/10.1371/journal.ppat.1003657
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. https://doi.org/10.1073/pnas.0900877106
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. https://doi.org/10.1126/science.1213362
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. https://doi.org/10.1128/JVI.02765-13
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. https://doi.org/10.1038/nature10831
Finch C, Li W, Perez DR (2015) Design of alternative live attenuated influenza virus vaccines. Curr Top Microbiol Immunol 386:205–235. https://doi.org/10.1007/82_2014_404
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
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. https://doi.org/10.1111/j.1750-2659.2012.00392.x
Perez DR, Angel M, Gonzalez-Reiche AS, Santos J, Obadan A, Martinez-Sobrido L (2017) Plasmid-based reverse genetics of influenza A virus. Methods Mol Biol 1602:251–273. https://doi.org/10.1007/978-1-4939-6964-7_16
Nogales A, Perez DR, Santos J, Finch C, Martinez-Sobrido L (2017) Reverse genetics of influenza B viruses. Methods Mol Biol 1602:205–238. https://doi.org/10.1007/978-1-4939-6964-7_14
Te Velthuis AJW, Long JS, Barclay WS (2018) Assays to measure the activity of influenza virus polymerase. Methods Mol Biol 1836:343–374. https://doi.org/10.1007/978-1-4939-8678-1_17
Pena L, Vincent AL, Ye J, Ciacci-Zanella JR, Angel M, Lorusso A, Gauger PC, Janke BH, Loving CL, Perez DR (2011) Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses. J Virol 85(1):456–469. https://doi.org/10.1128/JVI.01503-10
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. https://doi.org/10.1073/pnas.100133697
Acknowledgments
This work was funded by the University of Georgia, NIH contract HHSN272201400008C, and Scientific Cooperative Agreements with ARS-USDA.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.
About this protocol
Cite this protocol
Perez, D.R., Seibert, B., Ferreri, L., Lee, CW., Rajao, D. (2020). Plasmid-Based Reverse Genetics of Influenza A Virus. In: Spackman, E. (eds) Animal Influenza Virus. Methods in Molecular Biology, vol 2123. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0346-8_4
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0346-8_4
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0345-1
Online ISBN: 978-1-0716-0346-8
eBook Packages: Springer Protocols