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 α2,3‑linked and α2,6‑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.
Competing Interest Statement
The authors have declared no competing interest.
Funder Information Declared
Medical Research Council, https://ror.org/03x94j517, MR/Y03368X/1, MR/Y03368X/1, MC_UU_0034/2, MC_UU_0034/3, MC_UU_0034/1
Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/V004697/1
Source:
Link: https://www.biorxiv.org/content/10.64898/2026.04.16.718897v1
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