Bovine-derived influenza A virus (H5N1) shows efficient replication in well-differentiated human nasal epithelial cells without requiring genetic adaptation

Highly pathogenic avian influenza (H5N1) viruses of clade 2.3.4.4b have caused significant losses among bird populations worldwide and have repeatedly crossed the species barrier, infecting mammals, including humans. However, efficient human-to-human transmission has not yet been observed. Here, we demonstrate that an H5N1 virus isolated from bovine milk in Texas in 2024 (H5N1 Tex/24 ) replicates in differentiated human nasal epithelial cells as efficiently as a 2009 pandemic H1N1 virus strain (H1N1 HH4/09 ) at both 37 °C and 33 °C. The adaptive mutations PB2-M631L and PA-K497R do not affect H5N1 Tex/24 replication at 37 °C but promote replication at 33 °C. Conversely, H5N1 BE/22 , a virus from the same clade isolated from a pelican in 2022 that lacks these mutations, replicates in human nasal epithelial cells at 37 °C as efficiently as H5N1 Tex/24 but exhibits limited replication at 33 °C. Introducing the two mutations PB2-M631L and PA-K497R did not overcome this limitation. Furthermore, nasal epithelial cells express receptors for both human and avian influenza viruses. Accordingly, no mutations were detected in HA which are known to switch receptor preference. Finally, we demonstrate that H5N1 Tex/24 remains sensitive to the antiviral effects of interferon-λ (IFN-λ), however, infected nasal epithelial cells secrete only small amounts of this cytokine. Overall, our results suggest that H5N1 Tex/24 possesses intrinsic traits enabling efficient replication in the human upper airways.