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Abstract Highly pathogenic avian influenza (HPAI) viruses with H5 hemagglutinin (HA) genes (clade 2.3.4.4b) are causing an ongoing panzootic in wild birds . Circulation of these viruses is associated with spillover infections in multiple species of mammals , including a large, unprecedented outbreak in American dairy cattle . Before widespread on-farm testing, there was an unmet need for genomic surveillance. Infected cattle can shed high amounts of HPAI H5N1 viruses in milk , allowing detection in pasteurized retail dairy samples. Over a 2-month sampling period in one Midwestern city , we obtained dairy products processed in 20 different states. Here we demonstrate that a tiled-amplicon sequencing approach produced over 90% genome coverage at greater than 20x depth from 5 of 13 viral RNA positive samples, with higher viral copies corresponding to better sequencing success. The sequences clustered phylogenetically within the rest of the cattle outbreak sequences reported. A combination...

Abstract Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosph...

Abstract The 2024 outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in U.S. dairy cattle presented an unprecedented scenario where the virus infected bovine mammary glands and was detected in milk , raising serious concerns for public health and the dairy industry. Unlike previously described subclinical influenza A virus (IAV) infections in cattle, H5N1 infection induced severe clinical symptoms , including respiratory distress, mastitis, and abnormal milk production . To understand the host immune responses and changes, particularly in the mammary gland, we performed single-cell RNA sequencing analysis on bovine milk somatic cells (bMSCs) in vitro exposed to an H5N1 isolate from an infected dairy farm. We identified ten distinct cell clusters and observed a shift toward type-2 immune responses , characterized by T cells expressing IL13 and GATA3 , and three different subtypes of epithelial cells based on the expression of genes associated with milk production. Our stud...

Abstract We confirmed influenza A virus (IAV) by PCR in serum from 18 cows on 3 affected dairy farms in California, USA . Our findings indicate the presence of viremia and might help explain IAV transmission dynamics and shedding patterns in cows. An understanding of those dynamics could enable development of IAV mitigation strategies. Source: US Centers for Disease Control and Prevention,  https://wwwnc.cdc.gov/eid/article/31/7/25-0134_article ____

Abstract In late 2023, H5N1 high pathogenicity avian influenza (HPAIV) started circulating in dairy cattle in the USA . High viral titres were detected in milk from infected cows , raising concerns about onwards human infections . Although pasteurisation was shown to effectively inactivate influenza viruses in milk, unpasteurised milk still poses a risk of infection, both from occupational exposure in dairies and from the consumption of raw milk. We therefore assessed how long influenza viruses could remain infectious for in milk without heat inactivation. We examined the stability of a panel of influenza viruses in milk , including a contemporary H5N1 HPAIV and a variety of other influenza A and D viruses. We incubated viruses in cows' milk under laboratory conditions : at room temperature to simulate exposure in dairies and at 4°C to simulate exposure to refrigerated raw milk. Following an isolated report of H5N1 viral RNA being detected in milk from a sheep in the UK , we also c...

Abstract Since the first emergence of highly pathogenic avian influenza (HPAI) H5N1 viruses in dairy cattle , the virus has continued to spread, reaching at least 17 states and at least 950 dairy herds in the United States. Subsequently, spillovers of the virus from dairy cattle to humans have been reported. Pigs are an important reservoir in influenza ecology because they serve as a mixing vessel in which novel reassortant viruses with pandemic potential can be generated. Here, we show that oro-respiratory infection of pigs resulted in productive replication of a bovine-derived HPAI H5N1 B3.13 virus . Infectious virus was mainly identified in the lower respiratory tract of principal infected pigs, and sero-conversion was observed in most of the principal pigs at later time points, suggesting limited replication of the bovine-derived HPAI H5N1 B3.13 virus in pigs. In one animal, we detected the emergence of a mutation in hemagglutinin (HA) previously associated with increased affinity ...

ABSTRACT The recent emergence of the H5N1 influenza virus in dairy cattle has raised significant public health concerns . Using a previously established pseudovirus-based neutralization assay , we evaluated the impact of emerging hemagglutinin (HA) mutations on the efficacy of current candidate vaccine viruses (CVVs). Neutralization analysis revealed that the cow-derived H5N1 virus showed up to a 2.2-fold reduction in sensitivity compared to the CVV homologous neutralization titers. Among the 1,453 HA sequences analyzed from cow-derived H5N1 viruses, we identified four major mutations (E2K, D104G, V147M, and S336N) that emerged after the initial isolation, with 134 isolates (9.22%) harboring all four mutations . These multi-mutation variants exhibited up to a 3.3-fold reduction compared with the CVV homologous neutralization titers. Single-mutation analysis demonstrated that the D104G mutation , present in 47.8% of sequences, markedly contributed to antibody escape . Our findings highl...

Abstract Highly pathogenic avian influenza (HPAI) A(H5N1) virus emerged in lactating dairy cattle in March 2024, causing mastitis-related disease and infections in other farm animals and workers . Recent work identified α2,6 and α2,3-linked sialic acids (SA), which serve as influenza virus receptors, in the lactating bovine mammary gland ; however, their distribution across stages of mammary growth and development remains unknown. We compared the distribution of tissue sialylation in mammary glands of prepubertal dairy calves , pregnant dairy heifers , and lactating cows . Mammary glands at all physiological stages expressed both α2,6 SA, the preferred receptor linkage for human influenza viruses, and α2,3 SA, the preferred receptor linkage for avian influenza viruses. Importantly, mammary glands of pregnant dairy heifers exhibited the highest overall expression of α2,3 SA, observed in both tissue and alveolar lumens. Our results suggest that pregnant dairy heifers, like lactating dair...

Abstract In February 2024, H5N1 highly pathogenic avian influenza viruses (HPAIVs) of clade 2.3.4.4b were first reported in dairy cows in the USA. Subsequent multiple outbreaks on dairy farms and sporadic human infections have raised substantial public health concerns . In the same year, four H5N1 HPAIVs of clade 2.3.4.4b were isolated from ducks and geese in live poultry markets (LPMs) spanning seven provinces in China . Evolutionary analysis demonstrated that these viruses had undergone two genetic reassortments with H5 influenza viruses from wild birds in different countries. Except for 565/H5N1, the other three viruses exhibited over 99% genetic homology with avian-origin H5N1 HPAIVs from South Korea and Japan . Notably, 571/H5N1 demonstrated high replication efficiency in bovine-derived cells , particularly in bovine mammary epithelial (MAC-T) cells, and caused 16.7% (1/6) mortality in mice at a dose of 10⁵ EID₅₀/50 μL, indicating its zoonotic potential . Given the potential cross...

ABSTRACT Since 2020, the clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 viruses have caused unprecedented outbreaks in wild birds and domestic poultry globally , resulting in significant ecological damage and economic losses due to the disease and enforced stamp-out control. In addition to the avian hosts, the H5N1 viruses have expanded their host range to infect many mammalian species , potentially increasing the zoonotic risk . Here, we review the current knowns and unknowns of clade 2.3.4.4b HPAI H5N1 viruses, and we highlight common challenges in prevention. By integrating our knowledge of viral evolution and ecology, we aim to identify discrepancies and knowledge gaps for a more comprehensive understanding of the virus. Ultimately, this review will serve as a theoretical foundation for researchers involved in related avian influenza virus studies, aiding in improved control and prevention of H5N1 viruses. Source: Journal of Virology,  https://journals.asm.org/doi...

Abstract Background .  Influenza A virus (IAV) is a major cause of morbidity and mortality globally , causing seasonal influenza in humans and infecting birds and some mammals. In 2024, IAV H5N1 highly pathogenic avian influenza (HPAI) in the United States moved into cattle. While the outbreak is currently of low risk to the general public, there is an urgent need to monitor the disease and prevent spread.  Methods .  We conducted a nationwide study evaluating the relationship between H5 hemagglutinin gene RNA concentrations in wastewater and reported outbreaks of IAV H5N1 in animals and humans . We utilized an H5-specific droplet digital RT-PCR test to quantify H5 RNA in wastewater in 40 states across the United States, and 1) examined the temporal association between outbreaks and wastewater detections and 2) utilized linear mixed models (LMM) to determine the relationship between measurements in wastewater and outbreak-related factors in the local area.  Results ....

Abstract Influenza A(H5N1) viruses have been detected in US dairy cow herds since 2024 . We assessed the pathogenesis, transmission, and airborne release of A/Michigan/90/2024, an H5N1 isolate from a dairy farm worker in Michigan , in the ferret model. Results show this virus caused airborne transmission with moderate pathogenicity , including limited extrapulmonary spread , without lethality. Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses have displayed unprecedented global spread among wild birds leading to numerous spillover infections in mammalian species. Of note, outbreaks in dairy cattle and gallinaceous birds have resulted in human infections in the United States during 2024–2025 (1). Increased frequency of H5N1 viruses crossing species barriers has caused concern that the avian influenza viruses are adapting to mammals . A critical component of influenza pandemic preparedness is early identification of emerging novel influenza viruses that cause disease and t...

Abstract Highly pathogenic avian influenza (HPAI) clade 2.3.4.4b H5N1 is circulating widely in lactating cows in the United States . Due to the critical need for intervention strategies for this outbreak, we evaluated antibody and cellular immune responses of a clade 2.3.4.4b H5 mRNA-LNP vaccine in calves . We found that the H5 mRNA-LNP vaccine induced a robust antibody and CD8+ T cellular-mediated immune response and conferred protection against clade 2.3.4.4b H5N1 infection. Source: BioRxIV,  https://www.biorxiv.org/content/10.1101/2025.05.01.651548v1 ____

ABSTRACT The outbreak of H5N1 in dairy cattle in United States revealed challenges in identification and management of a novel disease . The virus showed an exceptional ability to spread between farms and among cows within a farm. The impact of the virus on dairy cattle varied from nonclinical to severe clinical signs and death. Many dairy producers did not report clinical signs in their cows or test for the virus. Cats and peridomestic birds on many affected dairies died from viral exposure . Dairy workers showed signs of conjunctivitis , which was confirmed to be due to H5N1. With the disease affecting multiple species and showing efficient cow-to-cow transmission , the situation only worsened. There was a negative impact on the relationships among dairy producers, dairy workers, poultry producers, and veterinarians, in which professional and personal relationships were severed and some experienced loss of employment. The regulatory response varied by geographic location, and in some...

Editor’s summary High-pathogenicity avian influenza subtype H5N1 is now present throughout the US , and possibly beyond. More cattle infections elevate the risk of the virus evolving the capacity to transmit between humans , potentially with high fatality rates . Nguyen et al. show that from a single transmission event from a wild bird to dairy cattle in December 2023, there has been cattle-to-poultry, cattle-to-peridomestic bird , and cattle-to-other mammal transmission. The movement of asymptomatic dairy cattle has facilitated the rapid dissemination of H5N1 from Texas across the US . Evolution within cattle, assessed using deep-sequencing data, has detected low-frequency sequence variants that had previously been associated with mammalian adaptation and transmission efficiency. —Caroline Ash Structured Abstract INTRODUCTION Highly pathogenic avian influenza (HPAI) viruses have critical consequences for animal health and the agricultural economy—and may have pandemic potential. HPAI ...

Abstract The spread of the highly pathogenic avian influenza (HPAI) H5N1 virus among dairy cattle illustrates the adaptability of influenza A viruses (IAV) to infect non-traditional species . While IAV-specific sialic acid (SA) receptors have been identified in the mammary glands of dairy cattle , their presence in pigs, sheep, goats, and alpacas has not been studied until now. The zoonotic transmission of HPAI H5N1 to dairy and poultry farm workers during outbreaks raises public health concerns . This study employed lectin histochemistry to examine the mammary glands of livestock and humans . We found that these tissues were rich in SA α2,6-Gal receptors, followed by SA α2,3-Gal receptors , essential for IAV binding . Notably, the A(H5N1) clade 2.3.4.4b virus could bind to mammary tissue from both cattle and pigs . These findings highlight the potential for HPAI H5N1 to infect and spread within the mammary glands of production animals and humans. Source: BioRxIV,  https://www.bior...

Key points   -- At the present time , based on available information, FAO-WHO-WOAH assess the global public health risk of influenza A(H5) viruses to be low , while the risk of infection for occupationally exposed persons is low to moderate depending on the risk mitigation measures in place and the local avian influenza epidemiological situation.  -- Transmission between animals continues to occur and, to date, a growing yet still limited number of human infections are being reported. Although additional human infections associated with exposure to infected animals or contaminated environments are expected to occur, the overall public health impact of such infections at a global level, at the present time, is considered minor .  -- The assessment could change if and when additional epidemiological or virological information becomes available.  -- This risk assessment from FAO, WHO and WOAH updates the assessment of the risk of zoonotic transmission (for example, ani...

{Excerpt} Highly pathogenic H5N1 avian influenza (HPAI) viruses started circulating in lactating dairy cattle in the USA at the end of 2023 (ref. 1) and these viruses are now rapidly spreading between cows2. Eisfeld et al.3 found that a clade 2.3.4.4b H5N1 virus from this cattle outbreak can bind to α2,6-linked sialyl-glycopolymers on microtitre plates . Here we show that the haemagglutinin from a clade 2.3.4.4b H5N1 virus binds poorly to glycans that terminate with α2-6 sialic acids. This is an important finding, as α2,6 sialic acid is abundant in the upper respiratory tract of humans , and acquisition of α2,6 sialic acid receptor specificity is believed to be required for efficient transmission of influenza virus in humans and is considered a risk factor for the emergence of a new pandemic virus4. (...) Source: Nature,  https://www.nature.com/articles/s41586-025-08821-6 ____