Intra-patient neuraminidase mutations in avian H5N1 influenza virus reduce sialidase activity to complement weaker hemagglutinin binding and facilitate human infection

PLoS Pathog. 2026 Jan 23;22(1):e1013863. doi: 10.1371/journal.ppat.1013863.

Yohei Watanabe ¹ ², Madiha S Ibrahim ³, Yasuha Arai ², Daisuke Kuroda ⁴, Emad M Elgendy ³, Shin-Ichi Nakakita ⁵, Yohei Takeda ⁶ ⁷, Vuong Nghia Bui ⁸, Takao Ono ⁹, Shota Ushiba ¹⁰, Tomo Daidoji ² ¹¹, Nongluk Sriwilaijaroen ¹² ¹³, Haruko Ogawa ⁶, Kazuhiko Matsumoto ¹⁴, Yasuo Suzuki ¹³, Takaaki Nakaya ²

Affiliations

1 Department of Virology, The JIKEI University School of Medicine, Tokyo, Japan.
2 Department of Infectious Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
3 Department of Virology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt.
4 Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan.
5 Division of Functional Glycomics, Kagawa University, Kagawa, Japan.
6 Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
7 Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
8 Virology Department, National Institute of Veterinary Research, Hanoi, Vietnam.
9 Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, The University of Osaka, Osaka, Japan.
10 Murata Manufacturing Co., Ltd, Kyoto, Japan.
11 Department of Pathobiology, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan.
12 Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand.
13 Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
14 SANKEN, The University of Osaka, Osaka, Japan.

PMID: 41575943 DOI: 10.1371/journal.ppat.1013863

Abstract

Clade 2.2 H5N1 influenza viruses have caused an unusually high number of human infections, providing a unique opportunity to investigate early molecular steps associated with host adaptation. Although most work has focused on hemagglutinin (HA), the contribution of neuraminidase (NA) to these early adaptive events has remained unclear. By analyzing publicly available sequences from clade 2.2-infected patients, we identified 20 NA mutations and compared their phenotypes to 20 mutations acquired during diversification in primary human airway cells under drug-free conditions. Most patient-derived NA mutations resulted in modest reductions in sialidase activity, keeping activity within a functional range that supported improved replication in α2,6 sialylglycan (α2,6 Sia)-dominant environments, whereas excessive reduction impaired fitness. Notably, the phenotypes of culture-selected and patient-derived mutations were highly concordant, suggesting that these NA changes arose through natural selection rather than Antiviral pressure. Re-analysis of patient sequences further revealed that many adaptive NA mutations co-occur with HA mutations that confer only weak, partial α2,6 Sia binding. Using reverse genetics, we found that such naturally occurring HA/NA mutation pairs acted cooperatively in a receptor-context-dependent manner to support α2,6-associated replication relative to HA-only mutants, placing these variants within a constrained "early-adaptation space" characterized by limited α2,6 engagement and moderately reduced NA activity. Together, these findings indicate that early human adaptation of clade 2.2 H5N1 involves not only HA and PB2, but also incremental, cooperative tuning of NA function. Monitoring coordinated HA-NA evolution may therefore improve risk assessment frameworks for zoonotic influenza viruses poised at early stages of human host adaptation.

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HY-14818

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99.97%, Influenza Virus Inhibitor

Influenza Virus

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