Highly pathogenic avian H5N1 influenza A virus replication in ex vivo cultures of bovine mammary gland and teat tissues

Since early 2024, highly pathogenic avian influenza H5N1 viruses have been causing outbreaks in dairy cattle in the United States. Here, we compared the replicative capacity of A/dairy cattle/Texas/24-008749-001/2024 (H5N1; Cow-H5N1) isolated from a dairy cow, A/chicken/Ghana/AVL-76321VIR7050-39/2021 (H5N1; Chicken-H5N1) isolated from a chicken, and a human H1N1 2009 pandemic virus in ex vivo explant cultures of mammary gland and teat from lactating cows. We also examined the expression of influenza virus receptors in these organs. We observed that human influenza virus receptors are widely distributed throughout the epithelium of alveoli, ducts, and gland cisterns within the mammary gland, and in the teat cistern epithelium of dairy cattle, whereas avian influenza virus receptors are distributed on the alveolar, ductal, and teat cistern epithelium. We also found that Cow-H5N1 virus replicates more efficiently than Chicken-H5N1 or human H1N1pdm viruses in the gland cistern epithelium of dairy cattle. Notably, bovine H5N1 viruses replicated efficiently in the epithelium of the bovine teat cistern. These findings suggest that H5N1 viruses invade the mammary gland through the teat canal, which is easily accessed by viruses.