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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 \u03b12,6 sialylglycan (\u03b12,6 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 \u03b12,6 Sia binding. Using reverse genetics, we found that such naturally occurring HA\/NA mutation pairs acted cooperatively in a receptor\u2013context-dependent manner to support \u03b12,6-associated replication relative to HA-only mutants, placing these variants within a constrained \u201cearly-adaptation space\u201d characterized by limited \u03b12,6 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\u2013NA evolution may therefore improve risk assessment frameworks for zoonotic influenza viruses poised at early stages of human host adaptation.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013863","type":"journal-article","created":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T18:32:04Z","timestamp":1769193124000},"page":"e1013863","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Intra-patient neuraminidase mutations in avian H5N1 influenza virus reduce sialidase activity to complement weaker hemagglutinin binding and facilitate human infection"],"prefix":"10.1371","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8305-6952","authenticated-orcid":true,"given":"Yohei","family":"Watanabe","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madiha 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