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

## TL;DR

This study shows that mutations in the neuraminidase (NA) protein of H5N1 avian influenza viruses work with hemagglutinin (HA) mutations to help the virus adapt to human hosts.

## Contribution

The study reveals that NA mutations, in combination with HA mutations, play a cooperative role in early human adaptation of H5N1 influenza viruses.

## Key findings

- NA mutations modestly reduce sialidase activity to support replication in human-like α2,6 sialylglycan environments.
- Naturally occurring HA/NA mutation pairs cooperatively enhance α2,6-associated replication in human airway-like conditions.
- NA mutations co-occur with HA mutations that confer weak α2,6 binding, suggesting coordinated adaptation.

## 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.

Avian influenza viruses rarely infect humans because they bind poorly to receptors in the human airway. However, clade 2.2 H5N1 has caused an unusually high number of human infections, providing a unique opportunity to investigate how these viruses begin adapting to human hosts. While most research has focused on hemagglutinin (HA), which mediates host receptor binding, less is understood about the role of neuraminidase (NA), the other viral surface glycoprotein. In this study, we analyzed NA mutations detected in viruses isolated from clade 2.2-infected patients and compared them with mutations that emerged during virus replication in human airway cell cultures. We found that these NA mutations modestly adjusted enzyme activity in ways that supported replication under receptor conditions resembling those in the human airway. Importantly, many of the NA mutations identified in patients occurred alongside HA mutations previously shown to confer only weak increases in binding to human-type receptors. Using reverse-genetics-derived viruses, we found that these naturally co-occurring HA and NA mutations exhibited modest cooperative effects under human airway-like receptor conditions. These findings suggest that coordinated, incremental changes in both genes may contribute to the earliest stages of viral adaptation to humans. Together, this work highlights NA as an important—but previously underappreciated—component of early human adaptation in avian influenza viruses. Monitoring these coordinated genetic changes may strengthen genomic surveillance and pandemic preparedness.