In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA ¹ . As of mid-2025 the epidemic is ongoing, resulting in spillbacks into poultry, wild birds and other mammals including humans ² . Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells and tissues, as well as cells of other mammalian species including humans and pigs. We find evidence of several mammalian adaptations gained early in the evolution of these viruses in cattle including PB2 M631L, which is found in all cattle sequences, and PA K497R, which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication. We show that this mutation adapts the polymerase to better interact with bovine ANP32 proteins, particularly ANP32A, and thereby enhances virus replication in bovine mammary systems and primary human airway cultures. Importantly, we show that ongoing evolution during 2024 and 2025 in the PB2 gene, including E627K and a convergently arising D740N substitution, further increase polymerase activity and virus replication in a range of mammalian cells. Thus, the continued circulation of H5N1 in dairy cattle not only allows virus adaption improving replicative ability in cattle, but also increases the risk of zoonotic spillover.

Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ ATPase pump, promptly elicits an extended antiviral state that dramatically blocks influenza A virus production. Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.

Since 2020, the United Kingdom (UK) has suffered repeated epizootics of clade 2.3.4.4b H5 high pathogenicity avian influenza viruses (HPAIVs) in wild birds and poultry, resulting in substantial economic losses due to enforced statutory control. The rapid evolution of H5 HPAIVs continues to raise concern with heightened zoonotic and pandemic risks. The immunodominant haemagglutinin glycoprotein (HA) is crucial for influenza virus receptor binding and pH-induced fusion of viral and cellular membranes. Mutations in HA are frequent due to polymerase error, immune pressure and host adaptation, resulting in antigenic modulation and/or an expansion of host tropism, respectively, ultimately hindering control strategies. We evaluated a comprehensive panel of H5 viruses representing prevalent genotypes from UK outbreaks spanning 2020 to 2022 for HA functionality. HA genes from each genotype were assessed through receptor binding, pH of fusion, thermostability and HA inhibition assays to evaluate factors contributing to zoonotic potential, stability, and antigenicity. The viruses only bound to avian receptors and exhibited fusion at a pH of 5.8, above the pH range (pH 5.0 to 5.5) associated with efficient human-to-human transmission. Therefore, these H5 viruses have low immediate zoonotic threat. Contemporary H5 viruses were more thermostable and showed antigenic drift compared to the earlier 2017-2018 clade 2.3.4.4b H5N8 viruses, and N236D in HA was identified as a significant antigenic epitope. The findings of this study underscore the evolving nature of the HA of these viruses and highlight the importance of ongoing surveillance and characterisation efforts to identify factors that might contribute to zoonotic risk.

In late 2023 an H5N1 lineage of high pathogenicity avian influenza virus (HPAIV) began circulating in American dairy cattle ¹ . Concerningly, high titres of virus were detected in cows’ milk, raising the concern that milk could be a route of human infection. Cows’ milk is typically pasteurised to render it safe for human consumption, but the effectiveness of pasteurisation on influenza viruses in milk was uncertain. To assess this, we evaluated heat inactivation in milk for a panel of different influenza viruses. This included human and avian influenza A viruses (IAVs), an influenza D virus that naturally infects cattle, and recombinant IAVs carrying contemporary avian or bovine H5N1 glycoproteins. At pasteurisation temperatures, viral infectivity was rapidly lost and became undetectable before the times recommended for pasteurisation. We then showed that an H5N1 HPAIV in milk was effectively inactivated by a comparable treatment, even though its genetic material remained detectable. We conclude that industry standard pasteurisation conditions should effectively inactivate H5N1 HPAIV in cows’ milk, but that unpasteurised milk could carry infectious influenza viruses.

Sustained mammal-to-mammal transmission of high pathogenicity H5N1avian influenza viruses is reshaping the host range of these pathogens. One of the longest-running mammalian transmission chains involves the B3.13 genotype circulating in U.S. dairy cattle which was detected early in 2024. Genomic analysis revealed selection and rapid fixation of haemagglutinin mutations D104G and V147M. We demonstate, via glycomic profiling, that bovine tissues, including the mammary gland, are enriched in N- and O-linked glycans capped with N-glycolylneuraminic acid (NeuGc), a sialic acid absent in humans and birds, which instead express only N-acetylneuraminic acid (NeuAc). Early cattle H5 viruses poorly recognized NeuGc, but D104G and V147M enabled efficient engagement of both NeuAc- and NeuGc-containing receptors. These mutations enhanced replication in bovine mammary tissue without major attenuation of replication in human lung and primary nasal epithelial cells. NeuGc-driven receptor adaptation therefore promotes viral fitness in cattle while potentially limiting immediate zoonotic risk. Deep mutational scanning further identifies alternative haemagglutinin substitutions that confer NeuGc usage and represent surveillance markers for emerging cattle H5 lineages.

The incursion of high pathogenicity avian influenza A virus (IAV) into US dairy cows is unprecedented in the era of molecular diagnosis and pathogen sequencing. This raises questions over the likelihood of further outbreaks and whether dairy cattle could be a “mixing vessel” for novel strains of IAV. Using a panel of BSL2-safe reassortant viruses representing clade 2.3.4.4b H5 epizootic lineages circulating since 2020, we found that a cow B3.13 isolate displayed enhanced replication in cow mammary gland cells, along with increased viral polymerase activity and stronger interferon antagonism in cow cells compared to an earlier EA-2020-C genotype virus. However, multiple avian and mammalian IAV strains, including other clade 2.3.4.4b high pathogenicity genotypes, were replication competent in bovine cells, particularly those of the mammary gland, suggesting that there is a diverse circulating IAV pool with the potential to infect cows. Moreover, we show that cow mammary cells co-express α-2,3 and α-2,6 – linked sialic acids, and are susceptible to co-infection with human and avian IAVs. We conclude that the US cow influenza outbreak does not simply reflect a unique adaptation of the B3.13 genotype virus; rather, the bovine udder represents a permissive niche for IAV and a plausible site for reassortment, underscoring its potential role in generating novel influenza viruses with pandemic risk.