Abstract
New World fruit bats were recently found to harbor two distinct and previously unknown influenza A viruses (IAVs) of the subtypes H17N10 and H18N11. Although viral genome sequences were detected in the liver, intestine, lung, and kidney of infected bats and the complete genome sequences have been isolated from their rectal swab samples, all attempts to isolate an infectious virus from bats in nature have failed. The lack of an infectious bat IAV isolate was overcome by reverse genetic approaches that led to the generation of an infectious virus in vitro. Using such synthetic bat IAVs enabled the identification of their unconventional cell entry via major histocompatibility complex II (MCH-II) molecules and their ability to replicate in mice, ferrets, and bats. Importantly, we also showed that these synthetic recombinant bat IAVs are not able to reassort with conventional IAVs, preventing the acquisition of enhanced transmission properties in non-bat species by reassortment with conventional IAVs. As authentic viruses are key for understanding the molecular biology of bat IAVs, in this chapter, we describe our recently established reverse genetics protocol for generating H17N10 and H18N11 in vitro. This step-by-step protocol starts with cloning of cDNA copies of the viral RNA segments into reverse genetics plasmids, followed by the generation of a highly concentrated stock and finally a method to determine viral titers.
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Acknowledgments
Figures were created with Biorender.com. This work was supported by grants from the European Research Council (ERC) to M.S. (NUMBER 882631—Bat Flu). This work was also partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), an NIAID-funded Center of Excellence for Influenza Research and Response (CEIRR, contract number 75N93021C00014) to A.G.-S.
Disclosures
The A.G.-S. laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7 Hills Pharma, PharmaMar, ImmunityBio, Accurius, nanoComposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories, and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, PharmaMar, Paratus, CureLab Oncology, CureLab Veterinary, Synairgen, and Pfizer, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, and AstraZeneca. A.G.-S. is the inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work.
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Kessler, S., García-Sastre, A., Schwemmle, M., Ciminski, K. (2024). Reverse Genetics of Bat Influenza A Viruses. 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_5
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DOI: https://doi.org/10.1007/978-1-0716-3533-9_5
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