Abstract
Influenza A (FLUAV) and influenza B (FLUBV) viruses are human and/or animal pathogens widely studied due to their importance to public health and animal production. Both FLUAV and FLUBV possess a genome composed of eight viral gene segments. For reverse genetics of influenza viruses, transcription of the mRNA for the viral proteins is typically done from a plasmid encoding an RNA polymerase II (pol II) promoter element upstream of cloned viral cDNA and expressed like host mRNA. On the other side, the synthesis of the negative-sense, single-stranded, uncapped vRNAs can be accomplished by the host’s RNA polymerase I (pol I). The reverse genetics for influenza has allowed the manipulation of influenza genomes incorporating heterogeneous sequences into different segments of the influenza genome, such as reporter genes. In this chapter, we outline the protocol from the generation of reverse genetic plasmid that can be applied for the cloning of any of the segments of FLUAV or FLUBV. Furthermore, we describe a protocol for generating FLUAV or FLUBV recombinant viruses carrying Nanoluciferase (NLuc) in the PB1 gene using reverse genetics. Finally, we delineate a microneutralization protocol using FLUAV-NLuc or FLUBV-NLuc viruses optimized for the use of antibodies from different sources (mice, ferrets, avian, etc.), which provides a more sensitive, reliable, and avidity-independent method to assess the presence of neutralizing antibodies against FLUAV or FLUBV.
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Acknowledgments
DRP’s research is funded by a subcontract 75N93021C00014 from the Centers for Influenza Research and Response (CEIRR) from the National Institute of Allergy and Infectious Diseases (NIAID). Additional funds were obtained by DRP under GRANT12901999, Proposal 2019-05890, Accession Number 1022658 from the National Institute of Food and Agriculture (NIFA), US Department of Agriculture. DRP receives additional support from the Georgia Research Alliance and the Caswell S. Eidson endowment funds from the University of Georgia.
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Caceres, C.J., Gay, L.C., Faccin, F.C., Pérez, D.R. (2024). Use of Reverse Genetics for the Generation of Recombinant Influenza Viruses Carrying Nanoluciferase. In: Pérez, D.R. (eds) Reverse Genetics of RNA Viruses. Methods in Molecular Biology, vol 2733. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3533-9_4
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DOI: https://doi.org/10.1007/978-1-0716-3533-9_4
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