Abstract
Intercontinental spread of highly pathogenic avian influenza A(H5N1) viruses poses significant pandemic risks and necessitates strong protective countermeasures. We evaluated the therapeutic efficacy of the neuraminidase inhibitor oseltamivir, the polymerase inhibitors baloxavir and favipiravir, and an ion-channel blocker amantadine, against severe influenza A(H5N1) virus infection in female BALB/c mice. Baloxavir (≥10 mg/kg, 1 dose) fully protected mice from death, significantly reduced virus respiratory replication, and prevented neuroinvasion. Oseltamivir (≥100 mg/kg/day for 5 days) provided limited survival benefits, reduced lung titers but failed to prevent viral neuroinvasion. Favipiravir (≥100 mg/kg/day for 5 days) provided partial protection, although did not reduce viral titers in lungs and brain. Amantadine provided no benefits. Although all drugs inhibited A(H5N1) viruses in vitro, in vivo correlations did not extend beyond baloxavir. Our results indicate that baloxavir is the most reliable treatment to address both respiratory replication and systemic spread of contemporary A(H5N1) viruses in mice and should be considered in pandemic planning.
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Data availability
Data generated in this study are provided in the text of the manuscript, figures, tables, supplemental figures, and in source data files. NGS sequence data are available on the National Center for Biotechnology Information Sequence Read Archive under PRJNA1416403 BioProject Accession (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1416403). Plaque assay images are available on Figshare at https://doi.org/10.6084/m9.figshare.30918779. Source data files are provided with this paper. Source data are provided with this paper.
References
Nair, H. et al. Global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis. Lancet 378, 1917–1930 (2011).
Swayne, D. E. et al. Strategic challenges in the global control of high pathogenicity avian influenza. Rev. Sci. Tech. Special Edition, 89–102 (2024).
Garg, S. et al. Highly pathogenic avian influenza A(H5N1) virus infections in humans. N. Engl. J. Med. 392, 843–854 (2025).
Xu, X., Subbarao, Cox, N. J. & Guo, Y. Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261, 15–19 (1999).
Pardo-Roa, C. et al. Cross-species and mammal-to-mammal transmission of clade 2.3.4.4b highly pathogenic avian influenza A/H5N1 with PB2 adaptations. Nat. Commun. 16, 2232 (2025).
Rolfes, M. A. et al. Human infections with highly pathogenic avian influenza A(H5N1) viruses in the United States from March 2024 to May 2025. Nat. Med. 31, 3889–3898 (2025).
Bruno, A. et al. First case of human infection with highly pathogenic H5 avian Influenza A virus in South America: a new zoonotic pandemic threat for 2023? J. Travel Med. 30, taad032 (2023).
Castillo, A. et al. The first case of human infection with H5N1 avian Influenza A virus in Chile. J. Travel Med. 30, taad083 (2023).
Kandeil, A. et al. Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America. Nat. Commun. 14, 3082 (2023).
Song, J. H. et al. Intranasally administered whole virion inactivated vaccine against clade 2.3.4.4b H5N1 influenza virus with optimized antigen and increased cross-protection. Virol. J. 22, 131 (2025).
Patel, N. et al. Single-dose avian influenza A(H5N1) Clade 2.3.4.4b hemagglutinin-Matrix-M((R)) nanoparticle vaccine induces neutralizing responses in nonhuman primates. Nat. Commun. 16, 6625 (2025).
Taaffe, J. et al. An overview of influenza H5 vaccines. Lancet Respir. Med. 13, e20–e21 (2025).
Fabrizio, T. P. et al. Genotype B3.13 influenza A(H5N1) viruses isolated from dairy cattle demonstrate high virulence in laboratory models, but retain avian virus-like properties. Nat. Commun. 16, 6771 (2025).
Jones, J. C. et al. Baloxavir improves disease outcomes in mice after intranasal or ocular infection with Influenza A virus H5N1-contaminated cow’s milk. Nat. Microbiol. 10, 836–840 (2025).
Andreev, K. et al. Genotypic and phenotypic susceptibility of emerging avian influenza A viruses to neuraminidase and cap-dependent endonuclease inhibitors. Antivir. Res. 229, 105959 (2024).
Andreev, K. et al. Antiviral susceptibility of highly pathogenic avian influenza A(H5N1) Viruses Circulating Globally in 2022-2023. J. Infect. Dis. 229, 1830–1835 (2024).
Bai, A. D., Srivastava, S., Al Baluki, T., Razak, F. & Verma, A. A. Oseltamivir treatment vs supportive care for seasonal influenza requiring hospitalization. JAMA Netw. Open 8, e2514508–e2514508 (2025).
Hayden, F. G. et al. Baloxavir marboxil for uncomplicated influenza in adults and adolescents. N. Engl. J. Med. 379, 913–923 (2018).
Furuta, Y., Komeno, T. & Nakamura, T. Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase. Proc. Jpn Acad. Ser. B Phys. Biol. Sci. 93, 449–463 (2017).
Shiraki, K. & Daikoku, T. Favipiravir, an anti-influenza drug against life-threatening RNA virus infections. Pharmacol. Ther. 209, 107512 (2020).
Bright, R. A. et al. Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet 366, 1175–1181 (2005).
Dong, G. et al. Adamantane-Resistant Influenza A Viruses in the World (1902–2013): frequency and Distribution of M2 Gene Mutations. PLOS ONE 10, e0119115 (2015).
Sleeman, K. et al. In vitro antiviral activity of favipiravir (T-705) against drug-resistant influenza and 2009 A(H1N1) viruses. Antimicrob. Agents Chemother. 54, 2517–2524 (2010).
Gubareva, L. V. et al. Assessing baloxavir susceptibility of influenza viruses circulating in the United States during the 2016/17 and 2017/18 seasons. Eurosurveillance 24, 13–17 (2019).
Subbarao, E. K., London, W. & Murphy, B. R. A single amino-acid in the Pb2-gene of influenza-A virus is a determinant of host range. J. Virol. 67, 1761–1764 (1993).
Liu, S. et al. Substitution of D701N in the PB2 protein could enhance the viral replication and pathogenicity of Eurasian avian-like H1N1 swine influenza viruses. Emerg. Microbes Infect. 7, 75 (2018).
Nieto, A. et al. Identification of Rare PB2-D701N mutation from a patient with severe influenza: contribution of the PB2-D701N mutation to the pathogenicity of human influenza. Front. Microbiol. 8, 575 (2017).
Sediri, H., Schwalm, F., Gabriel, G. & Klenk, H.-D. Adaptive mutation PB2 D701N promotes nuclear import of influenza vRNPs in mammalian cells. Eur. J. Cell Biol. 94, 368–374 (2015).
Zhu, W. et al. Dual E627K and D701N mutations in the PB2 protein of A(H7N9) influenza virus increased its virulence in mammalian models. Sci. Rep. 5, 14170 (2015).
Rodriguez, A., Pérez-González, A. & Nieto, A. Cellular human CLE/C14orf166 protein interacts with influenza virus polymerase and is required for viral replication. J. Virol. 85, 12062–12066 (2011).
Tipih, T. et al. Highly pathogenic avian influenza H5N1 clade 2.3.4.4b genotype B3.13 is highly virulent for mice, rapidly causing acute pulmonary and neurologic disease. Nat. Commun. 16, 5738 (2025).
Aoki FY. 44 - Antiviral Drugs for Influenza and Other Respiratory Virus Infections. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (Eighth Edition) (eds Bennett, J. E., Dolin, R. & Blaser, M. J.) (W.B. Saunders, 2015).
Aoki, F. Y. & Sitar, D. S. Clinical pharmacokinetics of amantadine hydrochloride. Clin. Pharmacokinet. 14, 35–51 (1988).
Rayner, C. R. et al. Pharmacokinetic-pharmacodynamic determinants of oseltamivir efficacy using data from phase 2 inoculation studies. Antimicrob. Agents Chemother. 57, 3478–3487 (2013).
Ando, Y. et al. Pharmacokinetic and pharmacodynamic analysis of baloxavir marboxil, a novel cap-dependent endonuclease inhibitor, in a murine model of influenza virus infection. J. Antimicrob. Chemother. 76, 189–198 (2021).
Ison, M. G. & Scheetz, M. H. Understanding the pharmacokinetics of Favipiravir: implications for treatment of influenza and COVID-19. EBioMedicine 63, 103204 (2021).
Abdel-Ghafar, A. N. et al. Update on Avian Influenza A (H5N1) virus infection in humans. N. Engl. J. Med. 358, 261–273 (2008).
Bassetti, M., Sepulcri, C., Giacobbe, D. R. & Fusco, L. Treating influenza with neuraminidase inhibitors: an update of the literature. Expert Opin. Pharmacother. 25, 1163–1174 (2024).
In: Antivirals for Pandemic Influenza: Guidance on Developing a Distribution and Dispensing Program (National Academies Press, 2008).
Ward, P., Small, I., Smith, J., Suter, P. & Dutkowski, R. Oseltamivir (Tamiflu) and its potential for use in the event of an influenza pandemic. J. Antimicrob. Chemother. 55, i5–i21 (2005).
Loregian, A., Mercorelli, B., Nannetti, G., Compagnin, C. & Palu, G. Antiviral strategies against influenza virus: towards new therapeutic approaches. Cell Mol. Life Sci. 71, 3659–3683 (2014).
Gu, C. Y. et al. A human isolate of bovine H5N1 is transmissible and lethal in animal models. Nature 636, 711–718 (2024).
Kiso, M., Uraki, R., Yamayoshi, S. & Kawaoka, Y. Efficacy of baloxavir marboxil against bovine H5N1 virus in mice. Nat Commun 16, 5356 (2025).
Govorkova, E. A. et al. Susceptibility of highly pathogenic H5N1 influenza viruses to the neuraminidase inhibitor oseltamivir differs in vitro and in a mouse model. Antimicrob. Agents Chemother. 53, 3088–3096 (2009).
Schünemann, H. J. et al. WHO Rapid Advice Guidelines for pharmacological management of sporadic human infection with avian influenza A (H5N1) virus. Lancet Infect. Dis. 7, 21–31 (2007).
Guan, W. et al. Baloxavir marboxil use for critical human infection of avian influenza A H5N6 virus. Med 5, 32–41.e35 (2024).
Salomon, R. et al. The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04. J. Exp. Med. 203, 689–697 (2006).
Fang, Q. & Wang, D. Advanced researches on the inhibition of influenza virus by Favipiravir and Baloxavir. Biosaf. Health 2, 64–70 (2020).
Furuta, Y. et al. Mechanism of action of T-705 against influenza virus. Antimicrob. Agents Chemother. 49, 981–986 (2005).
Pascua, P. N. Q. et al. Antiviral Susceptibility of Influenza A(H5N1) Clade 2.3.2.1c and 2.3.4.4b Viruses from Humans, 2023-2024. Emerg. Infect. Dis. 31, 751–760 (2025).
Spector, R. Transport of amantadine and rimantadine through the blood-brain barrier. J. Pharmacol. Exp. Ther. 244, 516–519 (1988).
Ilyushina, N. A., Hoffmann, E., Salomon, R., Webster, R. G. & Govorkova, E. A. Amantadine-oseltamivir combination therapy for H5N1 influenza virus infection in mice. Antivir. Ther. 12, 363–370 (2007).
Smee, D. F., Hurst, B. L., Wong, M. H., Bailey, K. W. & Morrey, J. D. Effects of double combinations of amantadine, oseltamivir, and ribavirin on influenza A (H5N1) virus infections in cell culture and in mice. Antimicrob. Agents Chemother. 53, 2120–2128 (2009).
Bauer, L., Benavides, F. F. W., Veldhuis Kroeze, E. J. B., de Wit, E. & van Riel, D. The neuropathogenesis of highly pathogenic avian influenza H5Nx viruses in mammalian species including humans. Trends Neurosci. 46, 953–970 (2023).
Leung, B. W., Chen, H. & Brownlee, G. G. Correlation between polymerase activity and pathogenicity in two duck H5N1 influenza viruses suggests that the polymerase contributes to pathogenicity. Virology 401, 96–106 (2010).
Ison, M. G. et al. Early treatment with baloxavir marboxil in high-risk adolescent and adult outpatients with uncomplicated influenza (CAPSTONE-2): a randomised, placebo-controlled, phase 3 trial. Lancet Infect. Dis. 20, 1204–1214 (2020).
Kuo, Y. C., Lai, C. C., Wang, Y. H., Chen, C. H. & Wang, C. Y. Clinical efficacy and safety of baloxavir marboxil in the treatment of influenza: a systematic review and meta-analysis of randomized controlled trials. J. Microbiol. Immunol. Infect. 54, 865–875 (2021).
Shah, S. et al. Clinical outcomes of baloxavir versus oseltamivir in patients hospitalized with influenza A. J. Timicrob. Chemother. 75, 3015–3022 (2020).
Ishiguro, N. et al. Clinical and virologic outcomes of baloxavir compared with oseltamivir in pediatric patients with influenza in Japan. Infect. Dis. Ther. 14, 833–846 (2025).
Aoki, F. Y. et al. Early administration of oral oseltamivir increases the benefits of influenza treatment. J. Antimicrob. Chemother. 51, 123–129 (2003).
Treanor, J. J. et al. Efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. US Oral Neuraminidase Study Group. JAMA 283, 1016–1024 (2000).
Leang, S. K. & Hurt, A. C. Fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza viruses to the neuraminidase inhibitor class of antivirals. J. Vis. Exp. 122, 55570 (2017).
Patel, M. C. et al. An optimized cell-based assay to assess influenza virus replication by measuring neuraminidase activity and its applications for virological surveillance. Antivir. Res. 208, 105457 (2022).
Kaplan, E. L. & Meier, P. Nonparametric-estimation from incomplete observations. J. Am. Stat. Assoc. 53, 457–481 (1958).
Reed, L. J. & Muench, H. A simple method of estimating fifty percent endpoints. Am. J. Epidemiol. 27, 493–497 (1938).
Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98 (1999).
Goldhill, D. H. et al. The mechanism of resistance to favipiravir in influenza. Proc. Natl. Acad. Sci. USA 115, 11613–11618 (2018).
Acknowledgements
The authors thank Keith A. Laycock, PhD, ELS, for scientific editing of the manuscript, David Carey, Heather Weinberg, and Chelsi Stultz for animal husbandry, the St. Jude Hartwell Center for Bioinformatics and Biotechnology for next-generation sequencing, Patrick Seiler, Jeri-Carol Crumpton for experimental assistance, Rebecca Poulson, College of Veterinary Medicine, University of Georgia for providing A/red-shouldered hawk/North Carolina/W22-121/2022 (H5N1), A/lesser scaup/Georgia/W22-145E/2022 (H5N1) and A/Texas/37/2024 (H5N1) influenza viruses, and the field staff of Wildlife Rescue Center, Bangladesh, for sample collection of A/duck/Bangladesh/61785/2024 (H5N1), A/duck/Bangladesh/49780/2021 (H5N1), A/duck/Bangladesh/61790/2024 (H5N1), A/duck/Bangladesh/59132/2023 (H5N1), A/duck/Bangladesh/59016/2023 (H5N1). Graphical Fig. S5 was created using BioRender. This project was funded by the U.S. NIAID, U.S. NIH, and U.S. DHHS under contract 75N93021C00016. This content is the responsibility of the authors and does not necessarily represent official views of the U.S. NIH.
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K.A., R.J.W., and E.A.G. conceptualized the research project. K.A., J.C.J., A.K., and P.V. acquired and/or analyzed the data. K.A. and E.A.G. wrote original drafts, and all authors reviewed and edited subsequent drafts. Funding was acquired by R.J.W.
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Andreev, K., Jones, J.C., Kandeil, A. et al. Baloxavir outperforms oseltamivir, favipiravir, and amantadine in treating lethal influenza A(H5N1) HA clade 2.3.4.4b infection in mice. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69721-5
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DOI: https://doi.org/10.1038/s41467-026-69721-5
