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Abstract An unprecedented outbreak of a highly pathogenic avian influenza virus, H5 clade 2.3.4.4b, was reported in United States dairy cattle during the spring of 2024. It has now spread to hundreds of herds across multiple states. In humans, antibodies to the hemagglutinin (HA) protein confer the strongest protection against infection. Human herd immunity limits viral spread but also drives the emergence of antigenic variants that escape dominant antibody responses. We used store-bought milk to profile the collective H5N1 antibody response of dairy cattle herds . We detected HA binding antibodies in specific samples from states with recent/ongoing outbreaks. These antibodies present in milk neutralized replicating virus expressing dairy cattle HA and neuraminidase (NA). Despite originating from independent vendors, dairies/plants, geographic regions, and time, antibodies present in these samples are remarkably similar in activity and HA binding specificity. The dominant antibody resp...

Abstract Highly pathogenic avian influenza A(H5N1) virus has caused a multistate outbreak among US dairy cattle , spreading across 16 states and infecting hundreds of herds since its onset. We rapidly developed and optimized PCR-based detection assays and sequencing protocols to support H5N1 molecular surveillance . Using 214 retail milk samples from 20 states for methods development, we found that H5N1 virus concentrations by digital PCR strongly correlated with quantitative PCR cycle threshold values ; digital PCR exhibited greater sensitivity. Metagenomic sequencing after hybrid selection was best for higher concentration samples, whereas amplicon sequencing performed best for lower concentrations. By establishing these methods, we were able to support the creation of a statewide surveillance program to perform monthly testing of bulk milk samples from all dairy cattle farms in Massachusetts , USA, which remain negative to date. The methods, workflow, and recommendations described p...

ABSTRACT Unexpected outbreaks caused by the H5N1 highly pathogenic avian influenza virus (HPAIV) in dairy cows in the United States (US) have raised significant veterinary and public health concerns . When and how the H5N1 HPAIV was introduced into dairy cows and the broader epidemiology of influenza A virus (IAV) infections in cattle in the US remain unclear. Herein, we performed a retrospective study to screen more than 1,700 cattle serum samples collected from different bovine breeds in the US from January 2023 to May 2024 using an enzyme-linked immunosorbent assay (ELISA) targeting the nucleoprotein (NP) to detect IAV infections, and the positive samples were further tested by hemagglutination inhibition (HI) assay . Results showed that 586 of 1,724 samples (33.99%) from 15 US states were seropositive by the NP ELISA assay , including 78 samples collected in 2024 and 508 samples collected in 2023 . Moreover, the HI assay revealed that 45 of these ELISA-positive samples were positiv...

Abstract Testing approved antivirals against A(H5N1) influenza viruses circulating in peridomestic species, including dairy cows , is critical to public health and pre-pandemic planning . It cannot be tested in humans due to A(H5N1) disease severity. Here, in mice, we demonstrate that US FDA-approved baloxavir treatment mediates improved disease outcomes ( survival and viral dissemination ) over oseltamivir after lethal intranasal and ocular challenge with A(H5N1)-contaminated cow milk. Source: Nature Microbiology,  https://www.nature.com/articles/s41564-025-01961-5 ____

Abstract H5N1 influenza has been circulating in birds from Eurasia and Africa for more than 146 years , but human infection has been sporadic. H5N1 (clade 2.3.4.4b) has recently infected hundreds of species of wild and domestic birds and mammals in North America . Furthermore, as of February 26, 2025, H5N1 has infected 70 humans in the United States , and one infection proved lethal . Furthermore, in attempts to control H5N1 in the United States, 10s of millions of egg-laying chickens have died or been culled . These efforts have led to very high egg prices in the United States. We have developed an analytical bioinformatics and genomics workflow to understand better how H5N1 is circulating in North America and adapting to new host species . Our workflow consists of: 1) Phylogenetic analyses of large viral sequence datasets to identify subclades of viral lineages causing the current outbreaks in humans and farm animals and closely related viral background lineages. 2) Next, we transfer...

Abstract The current outbreak of highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype clade 2.3.4.4b in dairy cattle in the United States has affected nearly 900 dairy farms and resulted in at least 39 human infections, putting health authorities and the scientific community on high alert. Here we characterize the virus growth properties and host-pathogen interactions of an isolate obtained from a sick dairy cow in Texas in vitro and in vivo and compare it to an older HPAI isolate. Despite so far being associated with mild disease in human patients, the cattle H5N1 virus showed superior growth capability and rapid replication kinetics in a panel of human lung cell lines in vitro . In vivo, cattle H5N1 exhibited more intense pathogenicity in mice , with rapid lung pathology and high virus titers in the brain , accompanied by high mortality after challenge via different inoculation routes. Additionally, the cattle H5N1 demonstrated efficient antagonism of overexpressed RI...

ABSTRACT Background In 2021, the H5N1 clade 2.3.4.4b Avian Influenza Viruses (AIVs) emerged on the American continent. At the same time, a further global spread took place. Infections have been reported in avian species as well as in over 50 mammalian species in 26 countries , and often result in severe disease with notable neurological pathology . Outbreaks in dairy cattle in the United States in 2024 illustrate viral transmission at a non-traditional interface and cross-species transmission. This development raises significant global concern regarding the virus's potential for wider spread. Given that H5N1 infections in birds reached record-high levels in Germany by late 2022, it is important to investigate whether Influenza A Virus (IAV) infections were also occurring in mammals sharing habitats with wild birds. Methods and Results Selected wild and domestic mammal populations were monitored over a two-year period (from January 2022 to December 2023), which coincided with a majo...

Abstract We measured stability of infectious influenza A(H5N1) virus in irradiated raw milk and wastewater and on surfaces . We found a relatively slow decay in milk , indicating that contaminated milk and fomites pose transmission risks . Although the risk is low, our results call for caution in milk handling and disposal from infected cattle. Source: US Centers for Disease Control and Prevention,  https://wwwnc.cdc.gov/eid/article/31/4/24-1615_article ____

Abstract Highly pathogenic avian influenza viruses (HPAIV) pose a serious public health concern . In March 2024, a first-time outbreak of HPAIV H5N1 in dairy cattle herds was reported in the United States (US). Since then, the virus has continued to spread in cattle herds and spilt over into humans . We recently showed that the first human isolate reported in the US in Texas (HPhTX) from a dairy worker in an affected cattle farm has enhanced replication kinetics and pathogenicity in mice compared to a closely related bovine isolate (HPbTX). However, the molecular determinants of differential pathogenicity have not yet been identified. Herein, we show that HPhTX has enhanced polymerase activity , compared with HPbTX, in human cells and that the polymerase basic 2 (PB2) protein is the main factor responsible for this difference. Through single and combined site-directed mutagenesis and swapping the three amino acids different between HPhTX and HPbTX, we found that PB2 mutation E627K is t...

Summary -- What is already known about this topic? -  Highly pathogenic avian influenza A(H5N1) outbreaks have emerged in U.S. cattle and poultry . Wastewater surveillance detects influenza A(H5) subtype but does not currently distinguish between human and animal sources. -- What is added by this report? -  During September 15, 2021–July 11, 2024 , retrospective analysis of wastewater surveillance data revealed 21 avian influenza A(H5) subtype detections across 12 Oregon communities . No association was found between detections in a community’s wastewater and history of a poultry outbreak or presence of dairy processing facilities or dairy farms within the sewershed. Avian influenza A(H5) was detected most frequently in two communities with important wild bird habitats . -- What are the implications for public health practice? -  Wastewater surveillance was an early indicator of avian influenza emergence in Oregon. Nonhuman and noncattle animal inputs , including wild bir...

ABSTRACT H5N1 subtype influenza A viruses represent a long-standing pandemic concern . Owing to their global occurrence in poultry, humans are routinely exposed to these viruses, and hundreds of human cases have been documented worldwide since 2003. The relevant viral lineages are not static, however, and have recently undergone a massive expansion of host range and geographic distribution . Within this expansion, the introduction of H5N1 viruses into dairy cattle in the United States has spawned a novel animal-human interface . In response, public health agencies have sought to evaluate the risk of an H5N1 pandemic stemming from the bovine outbreak. These assessments draw on evidence from the field and the laboratory to score a series of recognized risk factors . As such, their utility hinges on fundamental understanding of the processes that drive pandemic emergence and the availability of relevant data. Advancing this understanding and gathering data prior to and during an outbreak ...

Abstract The spillover of highly pathogenic avian influenza H5N1 into dairy cattle has raised concerns over the safety of fluid milk . While no human foodborne infection has been reported , this strain has infected dozens of people and milk from infected cows is known to be infectious by ingestion in multiple other species. Investigation into the public health threat of this outbreak is critical . This study uses quantitative risk assessment (QRA) models to represent the United States raw and pasteurized fluid milk supply chains to estimate the risk of human infection from consumption of fluid cow's milk. These models were parameterized with literature emerging from this outbreak, then employed to estimate the H5N1 infection risk and evaluate multiple potential interventions aimed at reducing this risk. The median (5th, 95th percentiles) probabilities of infection per 240-mL serving of pasteurized, farmstore-purchased raw, or retail-purchased raw milk were 5.68E-15 (1.77E-16, 2.98E...

ABSTRACT Since 2020, H5N1 highly pathogenic avian influenza (HPAI) viruses of clade 2.3.4.4b have been rapidly spreading in wild birds but have also caused a large number of mammalian infections and more than 70 known human cases . Importantly, this H5N1 clade has also crossed the species barrier into dairy cattle in the US in late 2023/early 2024. The neuraminidase (NA) protein of the N1 subtype can feature truncations in its stalk domain, which have been identified as putative virulence factors in poultry but seem to have a negative impact on transmission in mammals. Since its emergence, the vast majority of HPAI H5N1 A/goose/Guangdong/1/1996-lineage isolates have featured this truncated version of the NA stalk domain. Here, we report that this changed with the 2020 expansion of clade 2.3.4.4b H5N1 and that the majority of isolates—including the strains circulating in dairy cattle—feature a long NA stalk domain . Source: mBio,  https://journals.asm.org/doi/10.1128/mbio.03989-24 _...

Abstract In late 2021, Eurasian-lineage highly pathogenic avian influenza (HPAI) A(H5N1) viruses from HA clade 2.3.4.4b were first detected in the United States . These viruses have caused severe morbidity and mortality in poultry and have been detected in numerous wild and domestic animals , including cows and humans . Notably, infected cows transmitted the virus to cats , causing extreme pathogenicity and death. While human-to-human spread of the virus has not been recorded, efficient transmission of the bovine-origin virus has also led to extreme pathogenicity and death in ferret models . Recently, markers in PB2 (E627K) and HA (E186D, Q222H), indicating mammalian adaptation mutations , were detected in an H5N1-infected patient manifesting critical illness in Canada . These, combined with instances of interspecies spread of the virus , have raised global public health concerns. This could highlight the potential for the virus to successfully adapt to mammals, posing a serious risk o...

Abstract Highly Pathogenic Avian Influenza H5N1 (HPAI H5N1) was first detected in chickens in Scottland in 1959 and has since circulated globally, causing regular outbreaks among different animal species, as well as incidental infections in humans . In this scoping review, the epidemiology and impact of HPAI H5N1 among migratory birds, poultry, and cattle in the United States were analyzed, with a particular focus on outbreaks since January 2022. Following PRISMA guidelines, a total of 27 articles were identified for this review. Publicly available data and reports from the USDA and CDC were also evaluated and summarized. The identified articles primarily included epidemiological studies of detections in wild birds, mammals, and case reports on H5N1 and transmission dynamics among cattle , with a notable absence of poultry-focused reports . Wild birds, especially migratory species, have played an important role in virus dissemination. Studies among mammals , including seals, bears, and...

Abstract The emergence of highly pathogenic avian influenza A(H5N1) virus in dairy cattle herds across the United States in 2024 caused several human infections . Understanding the risk for spillover infections into humans is crucial for protecting public health. We investigated whether immunity from influenza A(H1N1)pdm09 (pH1N1) virus would provide protection from death and severe clinical disease among ferrets intranasally infected with H5N1 virus from dairy cows from the 2024 outbreak. We observed differential tissue tropism among pH1N1-immune ferrets. pH1N1-immune ferrets also had little H5N1 viral dissemination to organs outside the respiratory tract and much less H5N1 virus in nasal secretions and the respiratory tract than naive ferrets. In addition, ferrets with pH1N1 immunity produced antibodies that cross-reacted with H5N1 neuraminidase protein. Taken together, our results suggest that humans with immunity to human seasonal influenza viruses may experience milder disease fro...

Summary -- What is already known about this topic? -  Outdoor cats on U.S. dairy farms have been infected with highly pathogenic avian influenza (HPAI) A(H5N1) virus; infection has not been reported in indoor cats. -- What is added by this report? -  HPAI A(H5N1) virus was detected in two indoor domestic cats with respiratory and neurologic illness that lived in homes of dairy workers but had no known direct exposure to HPAI A(H5N1)–affected farms. Both dairy workers declined testing ; other household members received negative test results for influenza A. -- What are the implications for public health practice? -  Veterinarians in states with confirmed HPAI A(H5N1) in livestock should consider obtaining household occupational information , testing for influenza A viruses , and wearing personal protective equipment when evaluating companion cats with respiratory or neurologic illness . Suspected cases should be reported to public and animal health officials. Abstract High...

{Automatic Translation} Since 2020, the world has seen an increase in the number of outbreaks of avian influenza caused by the highly pathogenic avian influenza A (H5N1) virus and an expansion of the geography of circulation of this virus. In 2024, more than 1.2 thousand outbreaks of influenza A (H5N1) among wild birds and about 2 thousand outbreaks among poultry were registered in the world. The infection affected 68 countries in Asia, Europe, Africa, North and South America, and also reached Antarctica. In addition, in 2024, a large number of cases of detection of the influenza A (H5N1) virus among wild and domestic mammals were recorded, including with subsequent infection of people. These include outbreaks among cattle on US dairy farms, which by the end of the year affected more than 900 dairy farms in 16 states. The avian influenza A (H5N1) virus, isolated from cows, also caused illness in 40 people - farm workers. At the same time, the influenza A(H5N1) viruses detected in cows ...

Summary -- What is already known about this topic? -  Highly pathogenic avian influenza (HPAI) A(H5) virus infections have been detected in humans exposed to infected dairy cattle. -- What is added by this report? -  Public health officials conducted a serosurvey among 150 bovine veterinary practitioners. Three practitioners had evidence of recent infection with HPAI A(H5 ) virus, including two without exposures to animals with known or suspected HPAI A(H5) virus infections and one who did not practice in a U.S. state with known HPAI A(H5) virus–infected cattle. -- What are the implications for public health practice? -  These findings suggest the possible benefit of systematic surveillance for rapid identification of HPAI A(H5) virus in dairy cattle, milk, and humans who are exposed to cattle to ensure appropriate hazard assessments. Abstract The current outbreak of highly pathogenic avian influenza (HPAI) A(H5) clade 2.3.4.4.b viruses, genotype B3.13, among dairy cattle...

Abstract In this article, we present an assessment of the risk of the potential introduction and spread of highly pathogenic avian influenza (HPAI) in cows in Bulgaria . In the spring of 2024, we witnessed an unprecedented spread of the virus in dairy herds in the USA . This crossing of interspecies barriers by the virus creates a real danger of pandemic manifestations in humans . The continued spread of H5N1 clade 2.3.4.4b in dairy populations and other mammalian species and efficient animal-to-animal transmission increases the risk of infection and subsequent spread of the virus in human populations. According to registers, as of 1 November 2022, a total of 559,544 cattle were bred in Bulgaria . The total number of dairy cows decreased by 5.2% year-on-year to 197,996. Farms breeding dairy cows as of 1 November 2022 were 12,439, which is 22.1% less than the previous year. The production of cow’s milk in 2022 amounted to 748,278 thousand liters. Traditionally, the largest share in the ...