The hemagglutinin proteins of clades 1 and 2.3.4.4b H5N1 highly pathogenic avian influenza viruses exhibit comparable attachment patterns to avian and mammalian tissues.

The global spread of the A/goose/Guangdong/1/96-lineage H5N1 highly pathogenic avian influenza (HPAI) viruses has been accompanied by an expanded host range and the establishment of sustained viral transmission among dairy cattle. To evaluate if the evolving H5N1 viruses have changed tissue tropism over time, we compared the binding patterns of recombinant hemagglutinin (HA) proteins derived from clade 1 (A/Vietnam/1203/04, H5VN) and circulating clade 2.3.4.4b viruses detected from wild bird (A/Eurasian Teal/Hong Kong/AFCD-HKU-23-14009-01020/2023, H5HK) and dairy cattle (A/bovine/Ohio/B24OSU-439/2024, H5OH). The HA protein of A(H1N1)pdm09 virus was included for comparison. Using bio-layer interferometry, H1 protein preferentially bound to the α2,6-linked sialoside 6'SLNLN, while H5 proteins preferentially bound to the α2,3-linked sialoside 3'SLN. H5OH showed higher binding affinity to 3'SLN than H5HK and H5VN. The attachment patterns of H1 and H5 proteins to the respiratory tissues of different species and dairy cattle mammary glands were evaluated. Compared to the H1 protein, H5 proteins showed stronger binding to the lung epithelial cells of cat, cattle, chicken, ferret, human, and pig, and the clade 2.3.4.4b H5 proteins exhibited increased binding to pig and cattle bronchial epithelial cells. All H5 proteins were attached to the alveolar and cistern epithelial cells in mammary glands, where α2,3-linked and α2,6-linked sialyl glycans were detected by Maackia amurensis lectin II and Sambucus nigra lectin, respectively. Taken together, the HA proteins of clade 1 and 2.3.4.4b H5N1 viruses generally share comparable attachment patterns to avian and mammalian tissues, despite evolving into antigenically distinct clades over the past 3 decades. IMPORTANCE: The outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus among US dairy cattle since 2024 have raised concerns of the potential changes in HA receptor binding specificity and tissue tropism. Using insect-cell-expressed recombinant HA proteins derived from clade 1 and circulating clade 2.3.4.4b H5N1 viruses, we showed that the dairy cattle H5 protein retained binding specificity for the avian-like α2,3-linked sialoside 3'SLN over the human-like α2,6-linked sialoside 6'SLNLN, with higher binding affinity to 3'SLN than the other H5 proteins. Clade 1 and 2.3.4.4b H5 proteins showed comparable attachment patterns to the mammary tissues of lactating dairy cattle, which showed high expression of α2,3-linked and α2,6-linked sialyl glycans. All H5 proteins also showed comparable attachment patterns to the lungs of cat, cattle, chicken, ferret, human, and pig. Our results suggest that the recent H5N1 outbreaks in dairy cattle may be related to ecological factors rather than changes in HA receptor binding specificity.