Attachment and replication of clade 2.3.4.4b influenza A (H5N1) viruses in human respiratory epithelium: an in-vitro study

Background:

Highly pathogenic avian influenza H5N1 viruses of the A/Goose/Guangdong/1/1996 lineage pose a global threat to wildlife, domestic animals, and humans. Cross-species transmission events to mammals, including humans, in the past 4 years highlight this threat. For influenza A viruses, crucial determinants of cross-species and intraspecies transmission to and among mammals include attachment to and replication in respiratory airway epithelial cells. Although these determinants have been studied for H5N1 viruses in the past, limited studies for clade 2.3.4.4b viruses exist. Therefore, the aim of this study was to determine the ability of recent clade 2.3.4.4b H5N1 viruses to attach to human respiratory tissues, to replicate in human airway epithelial cells and the associated immune response. 

Methods:

In this in-vitro study, we investigated three H5N1 clade 2.3.4.4b viruses (H5N1^Gull2022, H5N1^Polecat2022, and H5N1^Bovine2024) in comparison with previously studied 2.1.3.2 H5N1 (H5N1²⁰⁰⁵) and a seasonal H3N2 virus. First, we compared virus attachment patterns by virus histochemistry. Second, we investigated the infection and replication efficiency, and innate immune responses in infected human respiratory epithelium in vitro. Third, we measured polymerase complex activity using a minigenome assay. 

Findings:

Clade 2.3.4.4b viruses and H5N1²⁰⁰⁵ virus differed by five amino acids located near the receptor binding site of the haemagglutinin. All clade 2.3.4.4b viruses attached more efficiently to cells of the human upper and lower respiratory tract compared with H5N1²⁰⁰⁵ virus. All clade 2.3.4.4b viruses replicated in human nasal and tracheobronchial respiratory epithelium cultures. In the tracheobronchial respiratory epithelium cultures, H5N1^Gull2022 virus replicated more efficiently than H5N1²⁰⁰⁵ virus (p=0·0050) and reached titres similar to H3N2²⁰⁰³ virus. Polymerase complex activity of H5N1^Gull2022 virus was not significantly different from that of H5N1²⁰⁰⁵ and was significantly lower compared with H3N2²⁰⁰³ virus (p≤0·0001). Infection with H5N1^Gull2022 virus induced a broader antiviral immune response than H5N1²⁰⁰⁵ virus. 

Interpretation:

Clade 2.3.4.4b H5N1 viruses have phenotypic characteristics that are different from a clade 2.1.3.2 H5N1²⁰⁰⁵ virus. The ability of clade 2.3.4.4b viruses to attach to and replicate in respiratory epithelium likely contributes to an increased risk for both human infection and virus adaptation to humans.