The highly Pathogenic Avian Influenza H5N1 (HPAI H5N1) was first detected in chickens in Scottland in 1959 and has since circulated globally1. In 1996, the HPAI H5N1 virus was reported in domestic waterfowl in Southern China, known as A/Goose/Guangdong/1/1996 or Gs/Gd lineage, resulting in the first known human infections, responsible of 18 human cases, including 6 deaths in 1997 in Hong Kong2. For almost 30 years, the virus spread via migratory birds in waves to the terrestrial avian hosts and mammalian species throughout the world leading to significant diversities in evolutionary virus lineages and clades2.

Since 2013, ancestral HPAI H5Nx viruses belonging to clade 2.3.4.4 have been circulating in Southeast Asia, combined with three neuraminidase subtypes (N1, N6, and N8). In late 2013, clade 2.3.4.4b H5N8 viruses were initially detected in China and subsequently in South Korea in early 20143. In early 2020, a novel variant of the clade 2.3.4.4b H5N1 virus emerged in Europe and rapidly disseminated, primarily via wild birds, across Europe, Asia, and Africa4,5,6. By late 2020, this novel H5N1 clade 2.3.4.4b variant became the predominant HPAI H5N1 virus worldwide, causing a panzootic in wild birds and considerable outbreaks in domestic birds, and occasional spillover into mammals7,8,9,10. In December 2021, a transatlantic incursion of HPAI H5N1 of the prevalent clade 2.3.4.4b occurred via wild birds from Europe to Canada and then to the United States (US) in early 202211,12. After the introduction of several different genotypes of the virus into North America, specific Eurasian avian lineages A1 and A3 began reassorting with low pathogenic avian influenza (LPAI) viruses in migratory birds in the Americas, leading to the emergence of two new 2.3.4.4b HPAI H5N1 genotypes, B3.13 in late 2023/early 2024 and D1.1 in September 2024, respectively10,11,13,14,15. Briefly, both B3.13 and D1.1 genotypes are 4 + 4 reassortant strains, where B3.13 retains PA, HA, NA, and M genes from the Eurasian lineage A1 (ea1) lineage, whereas the D1.1 retains the PB1, HA, M and NS genes from the Eurasian lineage A3 (ea3) lineage (Fig. 1a)13,14. The remaining segments for each genotype were closely related to LPAI viruses that were circulating in the migratory birds in the Americas12,14,15 (Fig. 1a)13,14. Despite first appearing in late 2024, the D1.1 is currently the prevalent 2.3.4.4b HPAI H5N1 genotype in migratory wild birds in the Americas14. Notably, in late 2024, the avian-derived D1.1 genotype of the 2.3.4.4b HPAI H5N1 virus was associated with severe illnesses in two cases, one in British Columbia (Canada) and the other in Louisiana (US), with the Louisiana case resulting in fatality16,17. Further concerns about the potential impact on human health is due to the recent introduction of the D1.1 genotype into dairy cattle in January 2025, and spillover cases in humans and other mammals.

Fig. 1: The origin for 2.3.4.4b HPAI H5N1 genotypes B3.13 and D1.1 and associated zoonotic events.
figure 1

a Genomic composition of the 2.3.4.4b HPAI H5N1 genotypes B3.13 and D1.1 showing the retained 4 segments from the ancestor Eurasian A1 (Cyan) and A3 (Red) genotypes, respectively. The other segments were acquired due to reassortments with LPAI H5N1 strains from migratory birds in the Americas (Orange and purple, respectively). b Schematic representation showing 2.3.4.4b HPAI H5N1 genotypes B3.13 and D1.1 dissemination from wild birds into avian hosts, dairy cattle, and other mammals, including humans. From March 2024 to June 2025, the U.S. has reported 70 human infections with HPAI H5N1 including 1 fatal case (Case fatality rate = 1.43%)23. Of these, 41 were linked to contact with sick dairy cows (B3.13 genotype), 24 were connected to exposure to poultry infected with HPAI H5N1 virus (mainly D1.1 genotype) and the source of exposure remains unknown or linked with dealing with other animals in five cases23. A large majority of B3.13 H5N1 from cattle show the PB2 M631L mutation at the cattle-to-cattle transmission interface. However, few B3.13 and D1.1 H5N1 showed other/additional mammalian adaptation markers, such as E627K and D701N, at the cattle-human interface. The figure has been created/assembled with BioRender.com.

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In the US, reports have demonstrated the wide distribution and transmissibility, for the first time, of 2.3.4.4b HPAI genotypes B3.13 (March 2024) and D1.1 (January 2025) to cattle, expanding the wide host range of this HPAI H5N1 clade14,18 (Fig. 1b). Although the 2.3.4.4b HPAI H5N1 virus has been detected in various mammalian species, including marine animals, the emergence of B3.13 and D1.1 genotypes of the 2.3.4.4b HPAI H5N1 virus and their spread to dairy cattle is particularly concerning. This highlights the ability of the B3.13 and D1.1 genotypes to adapt to and proliferate in new host species such as ruminants, which were not previously considered susceptible to active infections with influenza A viruses (IAVs)12. These genotypes exhibit a notable capacity to infect the respiratory tract of calves and the mammary glands of dairy cows, leading to significant mastitis and a rapid and severe decline in milk production with large amounts of infectious virus in the produced milk12,19. The ongoing outbreaks in cattle and birds also raise safety concerns for food and could feature the virus’s potential to adapt and transmit from dairy cattle to humans and non-human mammals such as domestic cats, alpacas, goats12, rodents20,21 (Fig. 1b), and possibly pigs22. Since the emergence of 2.3.4.4b HPAI H5N1 outbreaks in March 2024 until June 2025, the virus was detected in approximately 13,225 wild birds, 175 million poultry, and 1075 dairy herds in 17 states in the US23. Importantly, the B3.13 transmission to cattle was associated with several virulence-determinant amino acid mutations in PB2 (e.g., 631 L)13,24, HA (e.g., Q134K, Q154R, Q234K, S336N and P337L), MP (e.g.,R77K and S207G) and NS1 (E229K and D125N/G) that may increase the virulence in mammals13. In addition, specific B3.13 and D1.1 strains acquired known mammalian-type PB2-627K and -701N residues at the bovine-human or birds-bovine-human interfaces that could improve virus fitness, replication, pathogenicity, and potentially transmissibility in mammals14,15,25,26. Unlike point mutations, the segmented nature of IAVs may facilitate the genetic shift or reassortment with other IAVs in wild birds or mammals, creating viral progeny with major genetic changes, altered viral replication and/or pathogenicity2,27. Moreover, the likelihood and the impact of genetic reassortments of B3.13 or D1.1 genotypes with each other, or with other LPAI viruses in wild birds, swine, or seasonal viruses in unvaccinated poultry or dairy farmers on virus adaptation to humans and pandemic potential are also unknown17. In fact, IAV represents an important zoonotic risk to humans, since throughout the last hundred years, avian-origin IAVs played an important role in the last four human influenza pandemics2,28. The complexity of virus-virus and virus-host interactions and limited research make it challenging to predict the fitness outcomes of such possible reassortments. This underscores the potential evolution scenarios of these B3.13 or D1.1 genotypes post-infection to variants of concern with greater infection and transmission potential, highlighting the need for continued surveillance to monitor their prevalence and potential public health consequences.

In the US, to mitigate the risk of HPAI H5N1 transmission across state lines, all dairy cattle intended for interstate movement must be tested at a National Animal Health Laboratory Network (NAHLN) facility that is properly accredited. Only animals with negative test results are permitted to be transported between states. In cases where cattle test positive, herd owners are required to provide detailed epidemiological data, including movement histories, to facilitate effective disease tracking and containment29. Additionally, dairy calves intended for interstate movement must meet specific requirements set by the Animal and Plant Health Inspection Service (APHIS) to ensure compliance with biosecurity regulations and prevent further spread of the virus29,30. With these measures in place, a notable decrease in the interstate spread of HPAI H5N1 among dairy cattle was observed, along with fewer reported outbreaks compared to earlier periods in cattle and no new reported cases in human since March 202523.

Unlike poultry farms, dairy cattle are usually housed and fed in outdoor feedlot pens or grazing areas maximizing the opportunity to contact directly with migratory birds. Fortunately, these genotypes remain geographically confined to North America. However, the potential for their spread to other regions, particularly Latin America, through direct contact involving migratory birds or infected cattle herds underscores the need for global vigilance and calls for enhanced serological and etiological surveillance efforts31. Although HPAI H5N1 genotypes already circulating in Europe have been shown to infect cattle in both experimental and field settings19, the recent cross-species transmission events observed in the U.S. may reflect specific epidemiological conditions or gaps in biosecurity and surveillance systems of cattle farms. Another challenge that makes the situation worse is the fears that surveillance research might affect the dairy business, limiting the collaboration with dairy farms in collecting comprehensive epidemiological data that are necessary for the design of the proper control measures against subsequent HPAI H5N1 outbreaks in dairy farms32.

The current interventions for human infections with HPAI H5N1 in the US primarily involve antiviral treatments using FDA-approved anti-influenza medications, surveillance of high-risk individuals for potential exposure and infection, and the implementation of enhanced biosecurity measures to limit virus transmission and spread to healthy individuals. In lactating dairy cattle, measures focus on improving biosecurity, restricting animal movements, conducting H5N1 testing before interstate transport, and isolating infected herds. In poultry farms, controlling AIVs typically involves culling or depopulating infected and exposed flocks, alongside robust biosecurity practices and ongoing surveillance33. To date, no vaccination programs have been approved in the US for dairy cattle or high-risk dairy farm workers to control IAV infections, even in the presence of widespread circulation of the B3.13 and D1.1 genotypes of the 2.3.4.4b HPAI H5N1 viruses. However, the US Department of Agriculture (USDA) has recently granted a conditional license for an inactivated H5N2 vaccine for use in chickens. While neuraminidase N1 is crucial for providing additional specific antibody protection34,35, the N2 designation in this inactivated H5N2 vaccine is used as a DIVA (Differentiating Infected from Vaccinated Animals) strategy to recognize vaccinated poultry from naturally infected birds. Although poultry vaccination plays a critical role in reducing the risk of virus transmission to mammals, dairy cattle herds with substantial asymptomatic infections and economic losses in milk production36, along with the high-risk to farm workers, remains a priority for a conditional vaccination license. The USDA and the Center for Disease Control and Prevention (CDC) are actively supporting the development and evaluation of vaccines for dairy cattle and humans, respectively. The recommended prepandemic candidate vaccine viral (CVV) strains by The World Health Organization (WHO) against HPAI H5N1 clade 2.3.4.4b strains currently circulating in birds and mammals in North America include the A/Astrakhan/3212/2020 (H5N8)-like strain and the A/American wigeon/South Carolina/22-000345-001/2021 (H5N1)-like strain37. Stockpiles of these CVVs are currently available to potentially vaccinate people who may be at an increased exposure risk to the virus through direct contact with infected animals. So far, only Finland has actively implemented a vaccination program for the high-risk groups against HPAI H5N138. Beside the WHO-recommended CVVs, more than 20 inactivated H5 influenza vaccines were licensed by regulatory bodies worldwide and several live-attenuated and mRNA-based vaccines are currently under research and/or advanced to clinical trials39,40. In parallel, four FDA-approved anti-influenza drugs are currently available and acting by either inhibiting viral neuraminidase (NA) activity (oseltamivir, zanamivir, and peramivir) or inhibiting the cap-dependent endonuclease activity of the viral PA polymerase subunit (baloxavir)2. Except for one isolate from California that showed reduced susceptibility to baloxavir, all human B3.13 and D1.1 isolates to date do not have concerning substitutions that were previously reported to be associated with drug-resistance to FDA-approved drugs14,41,42. Experimentally, baloxavir treatment of mice following lethal intranasal and ocular challenge with H5N1-contaminated cow milk mediates improved disease outcomes over oseltamivir43. These findings may strengthen and enhance the global capacity to respond to a potential avian influenza pandemic.

Taken together, the large epizootic of the two B3.13 and D1.1 genotypes of the 2.3.4.4b HPAI H5N1 marks a challenging new chapter in the ongoing battle against zoonotic avian influenza viruses since 1996. Certainly, the effective implementation of the “One Health” approach and transparency by providing detailed epidemiological data during systemic surveillance studies and the proper adoption of effective preventive measures like vaccination could reduce the risk of the ongoing deadly and costly outbreaks in dairy cattle, while also minimizing zoonotic transmission of these 2.3.4.4b HPAI H5N1 viruses to humans. In addition, wild bird-to-poultry (D1.1), wild bird-to-cow (B3.13) and the new avian-to-cow (D1.1) spillovers challenge the food supply system, especially in the cow industry that is no yet familiarized to HPAI H5N1 infections, that would benefit of strategies to control HPAI H5N1 infections, and their continuous spillover to humans. This situation might be a reminder to reinforce support for the ongoing research and surveillance programs and to enhance collaboration with international organizations to avoid a recurrence of another pandemic crisis.