Primary bovine embryonic fibroblasts demonstrate variable fitness following infection with highly pathogenic avian influenza H5N1 strains and are susceptible to a recently circulating human 2009 pandemic lineage H1N1 strain.

The recent emergence of highly pathogenic avian influenza (HPAI) H5N1 (clade 2.3.4.4b, genotype B3.13) in dairy cattle presents substantial challenges to the agricultural sector and public health. Mechanistic studies of infection and transmission in cattle have proven difficult due to animal handling restrictions and the limited availability of established cell culture models. Primary bovine embryonic fibroblasts (BeEFs) were isolated and investigated here as a model to study influenza A virus (IAV) infection dynamics. We compared sialylation profiles, infectious virus production, viral replication, and plaque morphology in BeEFs following infection with the bovine HPAI H5N1 and an earlier 2.3.4.4b genotype (B1.1) isolated in 2022. The data presented here demonstrate increased expression of α-2,3 sialic acids compared to α-2,6 sialic acids in BeEFs, similar to sialylation profiles previously reported in bovine mammary tissue. These data also display increased viral fitness of the bovine origin HPAI H5N1 strains across bovine and avian cell lines, consistent with previous characterization in bovine mammary tissue. Furthermore, BeEFs were fully susceptible to a 2022 H1N1pdm09-like IAV strain while maintaining resistance to the 2009 H1N1pdm09 IAV as previously characterized in mammary cells. This study highlights the ongoing zoonotic adaptation of HPAI H5N1 in mammals and the potential for coinfection with select human H1N1 2009 pandemic lineage strains, enabling the potential development of reassortant strains. These data support the ability of BeEFs to serve as a complementary <i>in vitro</i> system for studying IAV infections in bovine hosts. Zoonotic spillover to humans with avian influenza A subtypes, such as H5N1, can have extraordinarily high mortality rates. Recently, highly pathogenic avian influenza (HPAI) H5N1 has spread to dairy cattle and caused widespread disease in over a thousand herds across the United States. This widespread infection not only poses notable economic challenges to agricultural industries but also represents a notable concern to human public health. While studies of infection dynamics of HPAI H5N1 in cattle remain crucial, animal handling restrictions and a lack of well-characterized cell culture models make this work challenging. The significance of our research lies in identifying an <i>in vitro</i> system-primary bovine embryonic fibroblasts (BeEFs)-as a physiologically relevant <i>in vitro</i> system for studying these infection dynamics, helping to mitigate the limitations imposed by stringent animal handling requirements.