Receptor basis of unusual tissue tropism of avian influenza H5N1 clade 2.3.4.4b virus in cattle

The unusual tropism of H5N1 clade 2.3.4.4b for cattle mammary glands, causing necrotizing mastitis without major respiratory involvement, raises critical questions about its underlying mechanisms. We conducted glycomics and linkage-specific lectin histochemistry to characterize sialic acid (SA) receptor diversity and anatomical distribution, and virus binding assays and high-resolution electron microscopy (EM) to visualize virus-receptor interactions. Cattle mammary gland exhibited an abundance of N- and O-linked SA glycans, showing a stronger binding affinity to clade 2.3.4.4b H5 than clade 2.2 H5. In contrast, the cattle trachea contained only O-linked but not N-linked SAs and showed no detectable H5 binding, indicating limited compatible influenza A virus (IAV) receptor availability in the tracheal epithelium. EM of virus-bound tissues further validated the receptor basis of H5N1 infection in cattle. As H5N1 continues infecting unusual hosts, as evidenced by the first case in sheep, our study offers a methodological framework for assessing IAV susceptibility.