Infection of the bovine mammary gland by avian H5N1 subclade 2.3.4.4b influenza viruses

Abstract

The emergence of the panzootic clade of highly pathogenic avian influenza H5N1 (2.3.4.4b) in 2020 marked a major expansion in the host range of influenza A viruses (IAVs), raising concerns about further cross-species transmission events and zoonotic spillover. Introduction of 2.3.4.4b viruses into U.S. dairy herds has resulted in widespread circulation, accompanied by reduced milk yield, mastitis, and high viral loads in milk. Notably, virus circulation in dairy cattle represents a novel route for mammalian adaptation and transmission that has already led to more than 40 human cases in the U.S. since 2024. Here, we investigated whether avian clade 2.3.4.4b viruses could infect mammary tissue from Aberdeen Angus, Holstein Friesian, and Limousin cattle, three breeds commonly farmed in Europe, the Americas, and Oceania. Using mammary gland explants, we inoculated tissues with attenuated reassortant viruses expressing the haemagglutinin and neuraminidase glycoproteins of three 2.3.4.4b viruses that predated the emergence of H5N1 in US cattle: A/chicken/England/053052/2021 (AIV07), A/chicken/Scotland/054477/2021 (AIV09), and A/chicken/England/085598/2022 (AIV48). Infected epithelial cells were identified using immunohistochemistry in explants from both the teat and gland cistern for all three breeds following infection with AIV09 and AIV48, indicating that mammary tissue from each of the three tested cattle breeds cattle is permissive to H5N1 infection. Lectin staining showed expression of both 2,3-linked and 2,6-linked sialic acids in the mammary tissue of all donors showing that all three breeds have the potential to support infection with both avian-adapted and mammalian adapted IAVs. Together, these findings demonstrate that mammary glands from both beef and dairy cattle breeds are permissive to infection with avian-adapted and mammalian-adapted H5N1 viruses and highlight the potential for this tissue to act as a mixing vessel for IAV reassortment, underscoring the need to include cattle in ongoing H5N1 surveillance and risk-assessment frameworks. Impact StatementThe emergence of highly pathogenic avian influenza H5N1 in dairy cattle has expanded the recognised host range of influenza A viruses. Further, the ability of the virus to infect the mammary gland and transmit via milk revealed a novel interface for transmission to humans and animals. Although sustained circulation in US dairy herds has been reported, the susceptibility of mammary tissue from other breeds (including beef cattle) commonly used in different countries has been largely unexplored. Here, we show that avian-origin H5N1 viruses can infect tissues derived from the mammary gland of three common cattle breeds (Aberdeen Angus, Holstein Friesian, and Limousin). Virus was detected in epithelial cells from both dairy and beef breeds, indicating that H5N1 can infect multiple breeds. Receptor profiling showed abundant 2,3-linked and 2,6-linked sialic acids, consistent with a tissue environment that may support infection with both avian-adapted and mammalian-adapted viruses. These findings demonstrate that multiple cattle breeds are permissive to H5N1 infection and strengthens the evidence base for including cattle in H5N1 surveillance and risk-assessment frameworks.