Influenza A(H5N1) Viruses Isolated From Dairy Cattle Demonstrate High Virulence in Laboratory Models, but Retain Avian Virus-like Properties

Influenza A(H5N1) Viruses Isolated From Dairy Cattle Demonstrate High Virulence in Laboratory Models, but Retain Avian Virus-like Properties | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Influenza A(H5N1) Viruses Isolated From Dairy Cattle Demonstrate High Virulence in Laboratory Models, but Retain Avian Virus-like Properties Richard Webby, Thomas Fabrizio, Ahmed Kandeil, Walter Harrington, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5806806/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract In March 2024, clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses were first detected in U.S. dairy cattle. Similar viruses have since caused 66 zoonotic human infections. To assess changes to zoonotic potential, we characterized A(H5N1) clade 2.3.4.4b viruses isolated from cows’ milk and birds. Bovine-derived viruses were lethal in mice and ferrets and transmitted to direct but not airborne contact ferrets. All viruses replicated in human bronchial epithelial cells despite preferentially binding avian virus-like receptors. The bovine-derived viruses remained susceptible to FDA-approved antivirals and were neutralized by sera from ferrets vaccinated with WHO CVVs or humans vaccinated with clade 2.3.4.4c vaccine. While 2.3.4.4b viruses induce severe disease in mammalian models, they retain many avian virus-like characteristics. Combined, we conclude that the risk of contemporary bovine-derived viruses to humans not in contact with affected animals is low. However, heightened vigilance remains essential to promptly detect and respond to any changes. Biological sciences/Microbiology/Virology/Influenza virus Biological sciences/Microbiology/Pathogens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Since its initial identification in 1996, the A/goose/Guangdong/1/1996 (gs/Gd) lineage of highly pathogenic avian influenza (HPAI) A(H5N1) virus has undergone dramatic genetic diversification with the emergence of multiple genetic clades, some of which spread globally. Viruses of this lineage pose a significant public health threat and have a case-fatality rate of around 52% following zoonotic infection. Their impact on avian species has been particularly dramatic, with roughly 110 million poultry culled in the United States (U.S.) alone since 2022 1,2 . Between 2020–2021, viruses with hemagglutinin (HA) clade 2.3.4.4b [A(H5N1) 2.3.4.4b] dominated in many countries 3 , 4 . Following widespread circulation in Africa, Asia, and Europe, 2.3.4.4b viruses entered North America in late 2021, representing only the second introduction of gs/Gd-lineage viruses into the continent. Soon after introduction into the Americas, the viruses quickly diversified and acquired internal gene segments from endemic low pathogenicity avian influenza viruses through reassortment 5 , 6 . Further, there was an increase in the number of identified hosts, particularly non-poultry avians and terrestrial and aquatic mammals 7 – 11 . The diversification of hosts coupled with a steady isolation of this lineage since 2021 is a concerning pattern with the potential to alter viral evolution and host range. On March 25, 2024, an A(H5N1) virus-positive milk sample was collected from a dairy cow in the U.S. with subsequent confirmation in cats and wild birds associated with this index farm 12 , 13 . The virus was identified as HPAI A(H5N1) 2.3.4.4b, genotype B3.13. To date, the virus has been reported on 923 dairy farms spanning 16 states, and the presence of non-infectious viral RNA has been detected in pasteurized commercial milk products sourced from at least 15 states 14 ,15 . Sixty-six human cases have been reported, in California, Colorado, Iowa, Michigan, Oregon, Texas, Washington, and Wisconsin as a result of occupational exposure to infected cattle or poultry 16 – 18 . Two infections with unknown sources have been detected in California and Missouri, while one exposure to backyard poultry occurred in Louisiana 19 . These zoonotic infections have primarily manifested with conjunctivitis, accompanied by limited respiratory symptoms. Though at least two cases have led to severe disease in patients with potential co-morbidities. As the outbreak among dairy cows continues to spread across the U.S., there is concern the viruses may accumulate mammalian adaptive markers, increasing their risk to humans. In this study, we compared the phenotypic properties of 17 avian and bovine influenza A(H5N1) clade 2.3.4.4b viruses (Table 1 ), with an overarching goal to address the potential risks they pose to human health. We investigated various viral characteristics, including replication kinetics in primary human cells, receptor binding preferences, pathogenesis, transmission dynamics in mice and ferrets, and levels of population immunity. Our findings indicate that these viruses retain avian-like features despite sustained transmission through dairy cattle. RESULTS Replication of A(H5N1) 2.3.4.4b viruses in vitro Replication capacity of emerging influenza viruses in mammalian cells can be an early indicator of viral fitness in non-avian hosts. Accordingly, we assessed viral replication kinetics of A(H5N1) 2.3.4.4b viruses detected in cows (genotype B3.13) and avian hosts (genotypes Minor60 and B3.7) in two different mammalian cell models, the madin-darby canine-kidney cell line (MDCK) and primary differentiated normal human bronchial epithelial cells (NHBEs). All viruses replicated efficiently in both cell types. A(H5N1) 2.3.4.4b viruses grew to high titers in MDCK cells, peaking by 36 hours post-infection (hpi) with median titers of 8.1 to 9.4 log 10 TCID 50 /mL, significantly higher ( P ≤ 0.01) than those of the seasonal CA/09 (H1N1)pdm09 (Fig. 1 a). In differentiated NHBE cell cultures, all bovine viruses also replicated well reaching peak titers by ≈ 72 hpi (8.0 to 8.9 log 10 TCID 50 /mL). However, no significant differences were observed in replication kinetics between avian-, bovine- or the human-origin influenza viruses in these cells (Fig. 1 b). Receptor binding specificity of A(H5N1) 2.3.4.4b viruses To determine whether a shift in receptor preference from the avian virus-like α2,3 linked sialic acid receptor to the human virus-like α2,6 linkage followed transmission in dairy cattle, we evaluated the binding of the bovine viruses to both receptor ligands in solid phase binding assays 20 . All bovine A(H5N1) viruses tested bound exclusively to the avian virus-like α2,3 linked sialic acid receptors and not to human virus-like receptors (Fig. 2 a-e). As expected, the human CA/09 (H1N1)pdm09 virus preferentially bound to α2,6 sialic acids (Fig. 2 g) and the avian goose/KS/930F (H5N1) was restricted to α2,3 sialic acids (Fig. 2 f). Consistent with the observed binding patterns, the bovine and avian A(H5N1) viruses maintained the avian virus preferred glutamine at HA position 226 (H3 numbering), a position recently shown to be important for receptor preference of the bovine A(H5N1) viruses 21 . Acquiring or losing N-glycosylation in the globular head of the HA protein can directly influence or alter the affinity of influenza virus towards specific receptors. One of our tested viruses (TX/97794) had an amino acid substitution suggesting potential additional N-glycosylation (caused by HA-T156A), but it retained patterns similar to the other bovine viruses lacking the glycosylation consensus site (Supplementary Table 1). Pathogenicity of A(H5N1) 2.3.4.4b viruses in mice The high viral loads detected in raw milk from infected cows present potential risk of human exposure. To investigate virus pathogenicity directly from milk, we modeled exposure in mice via intranasal inoculation. BALB/c mice inoculated with either bovine/OH/439 (H5N1) or bovine/TX/98638 (H5N1) viruses were susceptible to infection (Fig. 3 a, b). Signs of morbidity were observed starting 2–3 days post-inoculation (dpi), including ruffled fur, loss of body weight (Supplementary Fig. 1), and neurologic symptoms associated with movement including ataxia, paralysis, and tremors. All mice succumbed to disease by 5 to 8 dpi. To further investigate virus pathogenicity, the 50% mouse lethal dose (MLD 50 ) was determined for both viruses. Bovine/OH/439 (H5N1) and bovine/TX/98638 (H5N1) viruses were exceptionally lethal to mice with MLD 50 values of 1.25 and 1.5 log 10 TCID 50 , respectively (Fig. 3 a, b). Both A(H5N1) viruses spread systemically in mice, with viral detection in nasal turbinates, lungs, brains, spinal cord, livers, and brown adipose tissue at 3 and 4 dpi (Fig. 3 d-i). Infected mice exhibited widespread and disseminated microscopic lesions consistent with severe viral infection. The upper respiratory tract displayed extensive infection of nasal respiratory epithelia characterized by antigen staining and loss of cilia (Fig. 3 d) but surprisingly limited inflammatory cell infiltrates or nasal exudates (Fig. 3 e). The lower respiratory tract exhibited extensive necrotizing bronchiolitis and diffuse alveolar damage characterized by necrotic cell debris, septal thickening and necrosis, alveolar collapse, alveolar edema and fibrin. Antigen-positive cells included both type I and type I pneumocytes (Fig. 3 e). Virus dissemination to neural or neural-adjacent tissues was evident by abundant viral antigen in the olfactory epithelia (Supplementary Fig. 2a), randomly distributed throughout the brain (Fig. 3 f), proximal spinal cord (Fig. 3 g), ganglia, and liver (Fig. 3 h). Examination of whole sections of the mouse heads revealed viral antigen staining in other epithelial areas including the maxillary sinus and Eustachian tubes of the inner ear (Supplementary Fig. 2b-d). Antigen staining was also observed in brown adipose tissues (Fig. 3 i), eyes, pituitary gland, incisor teeth, and bone marrow (Supplementary Fig. 2e-i). These data suggest that bovine viruses are highly virulent in mice due to extrapulmonary dispersion and efficient replication soon after inoculation. Pathogenicity and transmission of A(H5N1) 2.3.4.4b viruses in ferrets To understand if the A(H5N1) 2.3.4.4b viruses are transmissible in mammals, donor ferrets were inoculated with 10 4 TCID 50 /mL of bovine/OH/439. At 1 dpi, each donor ferret was housed with a naïve direct contact (DC) ferret and one ferret was placed in an adjacent cage separated by a perforated barrier as an airborne contact (AC). Donor ferrets displayed elevated temperatures (> 40°C) at 2–3 dpi, which coincided with the onset of weight loss; humane endpoints were met by 8 dpi (Fig. 4 a,b). Donor ferrets shed virus in nasal washes with peak titers ranging from 6.3–6.7 log 10 TCID 50 /mL (Fig. 4 c). Two of three naive DC ferrets also became infected, met human endpoints, and shed virus in nasal washes to peak titers of 3.5–4.3 log 10 TCID 50 /mL between 5–7 days post-contact (dpc) (Fig. 4 c). The third DC ferret did not show clinical signs, and the virus was not detected in nasal washes. Virus was not detected in any of the AC ferrets, and they displayed no signs of disease. Tissues were taken from donor and DC ferrets that had reached human endpoints (6 to 10 dpi) for virus isolation. Infectious virus was detected in all tissues collected, including the nasal turbinate, trachea, lungs, brain, liver, and duodenum portion of the small intestine, from two of the three infected donors and two infected DCs. One donor ferret did not have any viable virus detected in the liver or trachea, but virus was recovered from the other tissues (Fig. 4 d). Thus, bovine/OH/439 (H5N1) clade 2.3.4.4b virus spread systemically in multiple tissues of donor ferrets and transmitted to most naive ferrets by contact route but failed to transmit via the airborne route. Replication and transmission of A(H5N1) 2.3.4.4b viruses in chickens While avian HPAI A(H5N1) 2.3.4.4b viruses cause 100% mortality in chickens 22 , the pathogenicity of bovine-origin A(H5N1) viruses in experimentally infected poultry is unknown; therefore, we investigated the replication and transmissibility of bovine/OH/439 (H5N1) and goose/KS/930F (H5N1) in chickens to determine if mammalian passage had impacted the viruses’ ability to infect and transmit in poultry. All donor virus-inoculated chickens died or reached humane endpoints at 2 dpi. In the avian virus group, five contact chickens died or were euthanized at 2 dpc and the remaining four chickens at 3 dpc. In the bovine virus group, two contact chickens died or were euthanized at 2 dpc, four chickens likewise at 3 dpc and two chickens survived until 6 dpc (Fig. 5 ). Therefore, the bovine A(H5N1) 2.3.4.4b virus was highly lethal to chickens and was comparable in pathogenicity to the avian-origin virus. Antiviral susceptibility of A(H5N1) 2.3.4.4b viruses To assess the potential efficacy of available therapeutic options for the control of bovine A(H5N1) 2.3.4.4b infections in humans, we determined the frequencies of genetic markers associated with reduced or highly reduced inhibition (RI/HRI) by FDA-approved influenza neuraminidase (NA) inhibitors (NAIs, oseltamivir, zanamivir, peramivir) or the cap dependent endonuclease inhibitor (CENI, baloxavir). Sequence-based analysis of NA and PA proteins available in public databases revealed low frequencies of bovine A(H5N1) influenza viruses with NAI (0.07%, 1/1489) and CENI (0.21%, 3/1406) RI/HRI-associated substitutions (Supplementary Table 2). A single bovine A(H5N1) virus had NA-T438I, which mediates zanamivir/peramivir RI 23 . One bovine A(H5N1) virus had PA-A37T that was shown to cause RI by baloxavir in human A(H3N2) viruses 24 . Two viruses had PA-A36T, which has been recently shown to confer baloxavir RI in avian influenza A viruses 25 . Phenotypic testing confirmed that all bovine A(H5N1) viruses tested were susceptible to NAIs and CENI baloxavir at sub-nanomolar concentrations (Table 2 ). The results were consistent between bovine viruses from Texas and Ohio. Oseltamivir IC 50s of bovine A(H5N1) viruses were slightly lower than contemporary A(H5N1) clade 2.3.4.4.b viruses circulating in 2022–2023 in birds 23 . Overall, highly pathogenic influenza A(H5N1) viruses isolated from dairy cattle retain susceptibility to FDA-approved antiviral drugs. Antigenic relationship of A(H5N1) viruses to WHO candidate vaccine viruses Analysis of the antigenic relationships between the bovine A(H5N1) clade 2.3.4.4b viruses and WHO-recommended candidate vaccine viruses (CVVs) was accomplished using post-infection ferret antiserum generated against three clade 2.3.4.4b CVVs [Astrakhan/3212 (H5N8), ck/Ghana/39, and wigeon/SC/345], a well-characterized representative avian clade 2.3.4.4b virus 5 [eagle/FL/W22], and the clade 2.3.4.4c CVV [gyrfalcon/41088 (H5N8)] (Fig. 6 a, Supplementary Table 3). All viruses tested, including TX/38161, TX/42041, TX43134, and TX/97794 with the additional glycosylation site in HA, reacted to within two-fold of homologous titers to at least one of the three CVVs. These data suggest that vaccines made using available clade 2.3.4.4b CVVs may protect against bovine A(H5N1) viruses in the event of a pandemic. H5 HA subtype neutralizing antibodies in human sera To further evaluate the utility of existing stockpiled vaccines, we tested a set of human sera obtained from a Phase 1 clade 2.3.2.1c H5 vaccine trial with donors aged 18 to 50 years. 26 We examined the presence of cross-reactive HA antibodies against bovine A(H5N1) 2.3.4.4b viruses. Previous studies have shown that HI antibody titers of > 40 are protective against disease. 27, 28 Antibodies induced by vaccination of individuals with an adjuvanted vaccine containing 15 µg of the HA derived from gyrfalcon/41088 (H5N8) 2.3.4.4c antigen reacted with GMTs ranging from 45.9 to 88.8 to the bovine A(H5N1) viruses (Fig. 6 b). Twenty of 20 individuals achieved a seroprotective HI titer of 40 against ​bovine/OH/342, 13 of 20 against bovine/OH/368, 18 of 20 against bovine/TX/40106, and 15 of 20 against bovine/TX/97794 (Supplementary Table 4). Thus, vaccination of humans with an adjuvanted vaccine using an A(H5N8) 2.3.4.4c antigen induces sufficient HA antibody responses that should protect against HA clade-mismatched virus. N1 NA subtype neutralizing antibodies in human sera Using a panel of 24 human sera selected based on existing A(H1N1)pdm09 virus HA antibody titers (HI ≥ 40), we investigated the extent of cross-reactivity of NA antibodies generated against seasonal influenza virus/vaccine exposure against A(H5N1) 2.3.4.4b NA (Fig. 6 c). As expected, all individuals demonstrated neutralization of the CA/09 (H1N1pdm09) NA with a mean NI titer of 29.0 regardless of adjuvant included with the vaccine. While mean NI titers were lower for both bovine/OH/439 and eagle/FL/W22 viruses, 17.6 and 14.6, respectively, 14 of 24 sera neutralized the bovine N1 from these viruses to roughly 1.5 times above assay background (NI titer of 9), while 13 of 24 sera neutralized the avian N1 (Fig. 6 c). The N1 NA proteins of the A(H5N1) 2.3.4.4b and A(H1N1) pdm09 viruses have 89.6% amino acid identity, with considerable conservation at some antigenic sites 5 . Although the levels of protective NA antibodies have not been established, these data suggest that some level of cross-protection against infection with bovine A(H5N1) viruses may be achieved based on NA antibodies generated against seasonal A(H1N1)pdm09. DISCUSSION The introduction of HPAI A(H5N1) 2.3.4.4b viruses into U.S. dairy cattle, followed by their spread and zoonotic infections, is unprecedented 29, 30 . The primary objective of our study was to understand if, and to what extent, these viruses have acquired properties suggestive of increased zoonotic risk following replication in dairy cattle. Critically, while the bovine A(H5N1) 2.3.4.4b viruses tested were pathogenic and highly virulent in ferrets and mice, we observed some contact transmission but no airborne transmission. This transmission profile may be partially attributed to the strict preference for avian-like α2,3 linked sialic acid ligands observed in the receptor binding assay. The lack of binding to α2,6 linked sialic acids that we observed contrasts with a study of an early bovine virus, A/dairy cattle/New Mexico/A24920343-93/2024 (H5N1), that demonstrated some degree of human-receptor binding 31 . These differences may in part be due to assay variations, which measure a limited number of glycans, as studies utilizing large glycan array panels continue to show a clear preference for avian-like ligands 32 – 34 . Further evidence of avian host preference of the bovine A(H5N1) viruses was demonstrated by the poultry inoculation studies shown here, lack of cow-to-cow transmission in experimentally infected calves 35 , and the robust presence of avian-like receptor ligands in the bovine mammary gland 36 which are readily bound by contemporary H5 proteins 37 . The degree of airborne transmission of the bovine A(H5N1) viruses to exposed ferrets has varied between viruses and studies. While we were unable to detect airborne transmission, studies of the human isolate A/Texas/37/2024 have demonstrated an inability to transmit, albeit inefficiently, via this route 31 , 32 , 38 . Nevertheless, there has been no evidence for human-to-human transmission in human infection. The acquisition of the mammalian adaptation marker PB2-E627K in some human isolates, along with a recent severe human infection with no poultry or bovine epidemiological link 39 does, however, raise concern and highlights the importance of continued laboratory testing of these and future isolates. While current data suggests A(H5N1) 2.3.4.4b viruses do not spread efficiently between humans, they do meet two other criteria that are broadly accepted as requirements for the generation of pandemic viruses: productive replication in humans and antigenic disparity from circulating viruses leading to a lack of population immunity to the novel virus antigens 40 . It is therefore important to understand the effectiveness of pharmacological interventions against 2.3.4.4b viruses in terms of both prophylaxis and treatment. The WHO Global Influenza and Response System (GISRS) routinely consider CVVs for zoonotic influenza, including A(H5N1). In some countries, A(H5N1) vaccines have been stockpiled. A recent study identified some degree of heterologous antibody recognition of Astrakhan/3212 from sera from clade 1 or 2.1 vaccinated individuals 41 indicating these earlier stockpiled vaccines may have some efficacy against currently circulating 2.3.4.4b viruses. However, a closely matched CVV is likely to provide better protection. Our data indicate that ferret post-infection antisera raised against three 2.3.4.4b CVVs, including Astrakhan/3212, effectively neutralize a majority of bovine A(H5N1) viruses we tested. Further, sera from individuals receiving gyrfalcon/41088 (H5N8) clade 2.3.4.4c adjuvanted vaccine also broadly inhibited 2.3.4.4b bovine viruses, with minor exceptions, including seven participants who had HI titers < 40 to bovine/OH/368 which has a novel mutation in antigenic site B. However, most participants had HI titers ≥ 40, which has long been the standard correlate for protection against seasonal influenza viruses 28 . Humoral protective immunity is dominated by antibody responses to the HA surface glycoprotein, but evidence suggests that some protective immunity may be generated against the lesser abundant NA glycoprotein. While NA-based immunity may still be permissive to viral infection, it may lessen disease severity, decrease viral loads in tissues, and reduce viral shedding 42 . Importantly, seasonal A(H1N1)pdm09 viruses have an N1 NA as do circulating A(H5N1) viruses, although the genes encoding the two proteins are genetically distinct. Correspondingly, we observed neutralization of avian and bovine 2.3.4.4b virus NAs with serum from healthy adults, similar to our previous studies of avian 2.3.4.4b viruses 5 . While this suggests that N1-based antibody immunity may be effective against circulating A(H5N1) viruses, further in vivo testing is urgently required to fully address this hypothesis. Collectively, current seasonal vaccination strategies, stockpiled pre-pandemic vaccines, and WHO CVVs appear to provide cross-reactivity against currently circulating bovine HPAI A(H5N1) 2.3.4.4b viruses and may be useful if vaccination efforts are deemed necessary for high-risk groups such as those with direct exposure to affected agricultural species. Additionally, our phenotypic testing indicated that circulating bovine viruses remained susceptible to currently available antiviral therapies at sub-nanomolar concentrations, and genotypic testing revealed a low frequency of substitutions associated with RI/HRI. Many of the initial human 2.3.4.4b human infections were treated with the NAI oseltamivir, but current in vitro data show higher oseltamivir IC 50 values for A(H5N1) 2.3.4.4b viruses as compared to viruses circulating before 2021 25,43 . It is not understood if this translates to decreased drug efficacy in humans, but it may increase the importance of alternative drug classes. This includes CENI baloxavir marboxil, which targets PA protein and has the potential to treat severe human infections if A(H5N1) 2.3.4.4b acquires genetic signatures of RI by oseltamivir 31 , 32 , 35 , 38 , 44 . Based on the data generated through this assessment, the risk to human health posed by the bovine A(H5N1) viruses in their current form is low, especially for those not exposed to dairy cows, their raw milk, or culling infected poultry flocks. The viruses show inefficient transmission within laboratory models and more importantly, no human-to-human transmission has been reported. We have also demonstrated that currently available antiviral drugs remain effective and there are vaccines available that offer cross protection in the event they are needed. Despite this work, the 2.3.4.4b A(H5N1) viruses continue circulating in wild and domestic animals at a large-scale, and human infections continue to be reported. This includes a severe infection in British Columbia, Canada, in an obese teenager with a history of mild asthma with no link to poultry or other affected species 39 as well as a recent fatal case in Louisiana in an elderly male with exposure to sick and dying poultry 45 . Limited data are available for these cases and virus characteristics. It is imperative that we maintain increased surveillance within wild birds, domestic animals, and humans for potential genetic and phenotypic changes that could have a profound impact on human health. METHODS Cells Madin–Darby Canine Kidney cells (MDCK, ATCC, #CCL-34) were maintained in complete growth medium [MEM (CellGro), 5% FBS (HyClone), 1 mM L-glutamine, 1× penicillin/streptomycin/amphotericin B (Gibco)] at 37°C, 5% CO 2 . Differentiated primary normal human bronchial epithelial (NHBE) cells (Mattek, AIR-100) were cultured at an air-liquid interface in manufacturer provided media at 37°C, 5% CO2. Virus isolation A total of 15 influenza A(H5N1) 2.3.4.4b genotype B3.13 viruses were isolated from raw milk samples collected on two geographically distinct farms, one in Ohio and one in Texas (Table 1 ) 46 . Primary samples were inoculated into the allantoic cavities of 10-day-old embryonated chicken eggs (eggs), incubated at 35°C for ≤ 48 h, harvested, and stored at − 80°C. Seasonal human CA/09 (H1N1)pdm09 was propagated in MDCK cells at 37°C for 48 h. CVVs [Astrakhan/3212 (H5N8) and wigeon/SC/345 (H5N1), a recombinant 6 + 2 ck/Ghana/21 (H5N1) AVL-763 (all kindly shared by CDC)], and an early North American reassortment 6 + 2 clade 2.3.4.4b virus eagle/FL/W22 (H5N1) was generated by reverse genetics with A/Puerto Rico/8/1934 (H1N1) internal gene segments and all were propagated in eggs at 37°C for 48 h. 50% tissue culture infectious dose (TCID 50 ) or 50% egg infectious dose (EID 50 ) were calculated by Reed and Muench method 47 using limiting dilution in MDCK cells or eggs. Replication kinetics Multi-round replication curves were performed in MDCK cells (5 × 10 5 cells/well, 12-well plates), primary NHBE cell (≈ 1.2 x 10 6 cells/insert) at MOI of 0.005 of indicated viruses. Monolayer supernatants or a 200 µL apical layer wash for airway cultures were collected at indicated timepoints and titrated by TCID 50 assay in MDCK cells. Receptor binding assay Fetuin coated immunoassay plates were incubated with 32 HA units of A(H5N1) 2.3.4.4b viruses in blocking buffer (PBS, 1% BSA) then the serially diluted biotinylated sialylglycopolymers [Neu5Acα3`Lac-Gly-PAA, 3`SLN-C3-PAA, Neu5Acα6`Lac-C2-PAA, and 6`SLN-C3-PAA (Sigma-Aldrich ] in the reaction buffer [PBS, 0.02% Tween-80, 0.02 % BSA, 5 µM oseltamivir carboxylate (MedChem Express)] were added and incubated for 2 h at 4°C. After washing, HRP-conjugated streptavidin (Invitrogen; 1:2000) was added for 1 h at 4°C. Plates were washed and TMB (3,3′, 5,5′ tetramethylbenzidine dihydrochloride, Sigma-Aldrich) substrate was added for 10 min at room temperature (RT). Reactions were stopped with 1 N H 2 SO 4 and absorbances were measured at 450 nm using a Synergy H1 microplate reader (BioTek Instruments). Pathogenicity in mice Six to eight-week-old female BALB/c mice (Jackson Laboratory, Bar Harbor, ME, USA) were lightly anesthetized with isoflurane and intranasally (IN) inoculated with 10-fold serial dilutions containing 10 1 to 10 6 TCID 50 /mouse of bovine/OH/439 (H5N1) and bovine/TX/98638 (H5N1) viruses (n = 3/virus dose). Mice were weighed and monitored daily for clinical signs for 14 dpi. The 50% mouse lethal dose (MLD 50 ) was calculated for both tested A(H5N1) viruses 48 . To determine viral replication and pathological features in BALB/c mice, a group of mice (n = 9/virus) was lightly anesthetized with isoflurane and inoculated intranasally with 10 4 TCID 50 in 20 µL of PBS. At 3 and 4 dpi, three mice from each tested A(H5N1) virus were euthanized, and tissues (nasal turbinates, lungs, intestines, livers, and brains) were collected, and virus titration was determined by TCID 50 assay in MDCK cells. Histopathology and immunohistochemistry BALB/c mice (n = 3/group) were sacrificed at 3 and 4 dpi and tissues (nasal turbinates, lungs, brain, liver, and small intestine) were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4–6 µm thickness and stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) staining for viral antigen detection was performed using the Ventana Discovery Ultra Autostainer (Roche Ventana, Tucson, Arizona). Sections were initially heated for 4 min at 72°C and placed in EZ prep solution (Roche Ventana) for deparaffinization. Antigen retrieval was performed for 56 min at 95°C in Cell Conditioning Solution 1 (Roche Ventana). A rabbit primary monoclonal antibody (GeneTex, Irvine, CA) raised against NP protein of A/Kansas/14/2017(H3N2) virus was applied at 1:8000, and the OmniMap anti-rabbit HRP (Roche Life Science) and ChromoMap DAB detection kits (Roche Ventana) were used to label virus-positive cells. Lung sections were then counterstained with hematoxylin and examined by a pathologist blinded to the experimental group assignments. Pathogenicity and transmission in ferrets Eight-week-old influenza-seronegative male ferrets (Triple F Farms) were IN inoculated with 10 4 TCID 50 units of bovine/OH/439 (H5N1) virus in 500 µL of PBS. One direct contact ferret was placed in the same cage as each inoculated animal 24 hpi (n = 3/group) and one ferret each in an adjacent cage with a perforated barrier allowing airflow and excluding contact. Animals were monitored daily for clinical illness (temperature, weight loss, relative inactivity indices, ataxia, respiratory symptoms, neuropathologic signs) 49 . Nasal washes were collected by intramuscular (IM) ketamine injection (25 mg/kg) and IN instillation of 1 mL PBS to induce sneezing starting 1 dpi and alternating days thereafter for a total of 7 collections. Any ferret reaching the humane endpoint or found dead had tissues collected for virus isolation and pathology. Tissues (nasal turbinate, trachea, lung, brain, liver, and small intestine) were homogenized and subjected to titration by TCID 50 in MDCK cells. Experimental infection of chickens White leghorn chickens (AVS Bio, Norwich, CT) were inoculated with 10 6 EID 50 /0.3 mL via intra-tracheal route (IT) with goose/KS/930F (H5N1) or bovine/OH/439 (H5N1) viruses (n = 6/group). After 16 hpi, two donor chickens from each experimental group were placed in contact with three naive direct contact chickens in replicates of three cages, except for one replicate of the avian virus, for which only two contact chickens were available. Chickens were monitored twice daily for disease symptoms and euthanized according to IACUC approved humane endpoint protocols if disease signs were observed. Antiviral susceptibility Sequence data for NA (n = 1489) and PA (n = 1406) proteins of bovine influenza viruses available publicly through the Global Initiative on Sharing All Influenza Data (GISAID) database ( https://gisaid.org , accessed November 8, 2024) were screened for genetic markers associated with RI/HRI by NAIs and CENI baloxavir according to the World Health Organization (WHO) Global Influenza Programme 2024 guidelines 50 , 51 . Nucleotide sequences were aligned using progressive (FFT-NS-2) and iterative (FFT-NS-i) algorithms of Multiple Alignment Fast Fourier Transform 52 . Amino acid sequences were analyzed with BioEdit (v.7.7.1) software. Phenotypic susceptibility to NAIs (oseltamivir carboxylate [oseltamivir] and zanamivir [MedChem Express]) was assessed with a fluorescence-based assay with 2′-(4-methylumberlliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate (Sigma-Aldrich) 53 . NA activity of each virus was standardized to relative fluorescent unit equivalents of 10 µM 4-methylumbelliferone (4-MU) 54 . After 30 min of incubation with NAI (5 pM − 50 µM) at 37ºC, fluorescent NA-cleaved MUNANA substrate was measured with a Synergy 2 multimode microplate reader (BioTek Instruments) at Ex/Em 360/460 nm. Half-maximal inhibitory concentrations (IC 50s ) were estimated from dose–response curves by using the sigmoidal, four-parameter logistic non-linear regression equation (GraphPad Prism v.10.1.2). Phenotypic susceptibility to CENI baloxavir marboxil active metabolite baloxavir acid (baloxavir [MedChem Express]) was determined by Influenza Replication Inhibition NA-based Assay (IRINA) in MDCK cells 55 . Virus inoculum was standardized to 1.9 nM/well of 4-MU and incubated with baloxavir (6 pM − 111 nM) on cell monolayer (96-well microplates, 8 h at 37°C) without TPCK-treated trypsin to achieve a single cycle of virus replication. NA activity of the infected cells was measured, and baloxavir half-maximal effective concentrations (EC 50s ) were calculated as described for NAI assay. Serologic testing and virus antigenicity Antigenic cross-reactivity and/or seroconversion of post-challenge animal (mouse, chicken, ferret) sera was assessed using the cattle-derived viral antigens with post-infection ferret antisera (Supplementary Table 3) in an HI assay. Briefly, sera samples were treated with receptor-destroying enzyme II (Denka Seiken Co.) and were serially diluted and incubated with 4 HA units of respective virus for 45 min at RT before addition of 0.5% chicken erythrocytes (Rockland Immunochemicals). HI titers were recorded after 30 min incubation at RT as the reciprocal of the highest serum dilution with complete inhibition of hemagglutination. Enzyme-linked lectin assay The presence of NA-specific antibodies in human was determined in enzyme-linked lectin assays (ELLAs) as previously described 56 . The influenza A(H6N1) viruses used in ELLA assay were generated by reverse genetics, all having the same HA gene from A/Teal/Hong Kong/w312/1997 (H6N1) virus, six internal segments from A/Puerto Rico/8/1934 (H1N1) virus and the NA gene of bovine/OH/439 (H5N1) virus, eagle/FL/W22 (H5N1), or CA/09 (H1N1)pdm09 viruses. The flat-bottom, 96-well plates (Thermo Scientific) were coated with fetuin (Sigma-Aldrich) at 25 g/mL in 0.1 M PBS at 4ºC for 48 h. Heat-inactivated human sera (56ºC for 1 h) that were commercially obtained from BioIVT were titrated by serially diluted in Dulbecco's phosphate-buffered saline (DPBS), 1% BSA, 0.5% Tween-20 and added to plates. An optimized amount of rg–derived H6N1 virus was added, and the plates were incubated for 18 h at 37ºC. After three washing, horseradish peroxidase–conjugated peanut agglutinin (Sigma-Aldrich) was added, and the plates were incubated for 2 h at RT. After washing, 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) substrate was added. Reactions were stopped after 10 min by adding 1 N H 2 SO 4 and the plates were read at 490 nM using a Synergy H1 microplate reader. The NI titers were defined as the reciprocal of the last dilution that resulted in at least 50% inhibition using nonlinear regression analysis of GraphPad Prism software (version 10.1.2). Statistical analysis Data were analyzed using two-way Anova with multiple comparisons in GraphPad Prism v.10.1.2. Replicates, group comparisons, and P values are listed in each figure legend. Declarations Data Availability Data generated in this study are provided in the main manuscript, figures, supplemental figures, and in source data files. Corresponding Author Richard J. Webby Department of Host–Microbe Interactions, St. Jude Children’s Research Hospital, 262 Danny Thomas Place MS330, Memphis, Tennessee 38105-3678, USA Phone: +1 (901) 595-3014 E-mail: [email protected] Acknowledgements & Funding This project was funded in whole or in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract 75N93021C00016 and by St. Jude ALSAC. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We gratefully acknowledge the efforts of the GISAID Data Science Initiative and associated public databases that contributed to this study, and the US CDC for reagents including Astrakhan/3212 (H5N8) and wigeon/SC/345 (H5N1), and recombinant 6+2 ck/Ghana/21 (H5N1) AVL-763. References Global Influenza Program, W. H. O. (https://www.who.int/publications/m/item/cumulative-number-of-confirmed-human-cases-for-avian-influenza-a(h5n1)-reported-to-who-2003-2024, 2024). CDC. H5N1 Bird Flu Detections across the United States in Backyard and Commercial Poultry , (2024). Caliendo, V. et al. Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021. Sci Rep 12 , 11729 (2022). https://doi.org:10.1038/s41598-022-13447-z Pohlmann, A. et al. Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021. mBio 13 , e0060922 (2022). https://doi.org:10.1128/mbio.00609-22 Kandeil, A. et al. Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America. Nat Commun 14 , 3082 (2023). https://doi.org:10.1038/s41467-023-38415-7 Youk, S. et al. H5N1 highly pathogenic avian influenza clade 2.3.4.4b in wild and domestic birds: Introductions into the United States and reassortments, December 2021-April 2022. Virology 587 , 109860 (2023). https://doi.org:10.1016/j.virol.2023.109860 Plaza, P. I., Gamarra-Toledo, V., Eugui, J. R. & Lambertucci, S. A. Recent Changes in Patterns of Mammal Infection with Highly Pathogenic Avian Influenza A(H5N1) Virus Worldwide. Emerg Infect Dis 30 , 444-452 (2024). https://doi.org:10.3201/eid3003.231098 National Wildlife Health Center, U. Highly pathogenic avian influenza H5N1 virus (HPAIv) has been present in North America since its first detection in wild birds in November 2021 (Newfoundland and Labrador, Canada), and has subsequently spread across the Americas., (2023). Puryear, W. et al. Highly Pathogenic Avian Influenza A(H5N1) Virus Outbreak in New England Seals, United States. Emerg Infect Dis 29 , 786-791 (2023). https://doi.org:10.3201/eid2904.221538 Harvey, J. A., Mullinax, J. M., Runge, M. C. & Prosser, D. J. The changing dynamics of highly pathogenic avian influenza H5N1: Next steps for management & science in North America. Biol Conserv 282 (2023). https://doi.org:ARTN 110041 10.1016/j.biocon.2023.110041 Murawski, A. et al. Highly pathogenic avian influenza A(H5N1) virus in a common bottlenose dolphin (Tursiops truncatus) in Florida. Commun Biol 7 (2024). https://doi.org:ARTN 476 10.1038/s42003-024-06173-x Burrough, E. R. et al. Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024. Emerg Infect Dis 30 , 1335-1343 (2024). https://doi.org:10.3201/eid3007.240508 Hu, X. et al. Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle. Emerg Microbes Infect 13 , 2380421 (2024). https://doi.org:10.1080/22221751.2024.2380421 Spackman, E. et al. Characterization of highly pathogenic avian influenza virus in retail dairy products in the US. J Virol (2024). https://doi.org:10.1128/jvi.00881-24 FDA. Updates on Highly Pathogenic Avian Influenza (HPAI) , (2024). Uyeki, T. M. et al. Highly Pathogenic Avian Influenza A(H5N1) Virus Infection in a Dairy Farm Worker. New Engl J Med (2024). https://doi.org:10.1056/NEJMc2405371 CDC. CDC Confirms Second Human H5 Bird Flu Case in Michigan; Third Case Tied to Dairy Outbreak , (2024). CDC. CDC Reports Fourth Human Case of H5 Bird Flu Tied to Dairy Cow Outbreak , (2024). CDC. H5 Bird Flu: Current Situation , (2024). Rogers, G. N. & Paulson, J. C. Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin. Virology 127 , 361-373 (1983). https://doi.org:10.1016/0042-6822(83)90150-2 Lin, T. H. et al. A single mutation in bovine influenza H5N1 hemagglutinin switches specificity to human receptors. Science 386 , 1128-1134 (2024). https://doi.org:10.1126/science.adt0180 Swayne, D. E. & Suarez, D. L. Highly pathogenic avian influenza. Rev Sci Tech 19 , 463-482 (2000). https://doi.org:10.20506/rst.19.2.1230 Andreev, K. et al. Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Circulating Globally in 2022-2023. J Infect Dis 229 , 1830-1835 (2023). https://doi.org:10.1093/infdis/jiad418 Omoto, S. et al. Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. Sci Rep 8 , 9633 (2018). https://doi.org:10.1038/s41598-018-27890-4 Andreev, K. et al. Genotypic and phenotypic susceptibility of emerging avian influenza A viruses to neuraminidase and cap-dependent endonuclease inhibitors. Antiviral Res 229 , 105959 (2024). https://doi.org:10.1016/j.antiviral.2024.105959 Neuzil, K. M. et al. Safety and Immunogenicity of Influenza A/H5N8 Virus Vaccine in Healthy Adults: Durability and Cross-reactivity of Antibody Responses. Clin Infect Dis (2023). https://doi.org:10.1093/cid/ciac982 Hobson, D., Curry, R. L., Beare, A. S. & Ward-Gardner, A. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. J Hyg (Lond) 70 , 767-777 (1972). https://doi.org:10.1017/s0022172400022610 Coudeville, L. et al. Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model. BMC Med Res Methodol 10 , 18 (2010). https://doi.org:10.1186/1471-2288-10-18 Kozlov, M. & Mallapaty, S. Bird flu outbreak in US cows: why scientists are concerned. Nature 628 , 484-485 (2024). https://doi.org:10.1038/d41586-024-01036-1 Nguyen, T.-Q. et al. Emergence and interstate spread of highly pathogenic avian influenza A(H5N1) in dairy cattle. bioRxiv , 2024.2005.2001.591751 (2024). https://doi.org:10.1101/2024.05.01.591751 Gu, C. et al. A human isolate of bovine H5N1 is transmissible and lethal in animal models. Nature (2024). https://doi.org:10.1038/s41586-024-08254-7 Pulit-Penaloza, J. A. et al. Transmission of a human isolate of clade 2.3.4.4b A(H5N1) virus in ferrets. Nature (2024). https://doi.org:10.1038/s41586-024-08246-7 Santos, J. J. S. et al. Bovine H5N1 influenza virus binds poorly to human-type sialic acid receptors. bioRxiv , 2024.2008.2001.606177 (2024). https://doi.org:10.1101/2024.08.01.606177 Chopra, P. et al. Receptor Binding Specificity of a Bovine A(H5N1) Influenza Virus. bioRxiv , 2024.2007.2030.605893 (2024). https://doi.org:10.1101/2024.07.30.605893 Halwe, N. J. et al. H5N1 clade 2.3.4.4b dynamics in experimentally infected calves and cows. Nature (2024). https://doi.org:10.1038/s41586-024-08063-y Nelli, R. K. et al. Sialic Acid Receptor Specificity in Mammary Gland of Dairy Cattle Infected with Highly Pathogenic Avian Influenza A(H5N1) Virus. Emerg Infect Dis 30 , 1361-1373 (2024). https://doi.org:10.3201/eid3007.240689 Rios Carrasco, M., Grone, A., van den Brand, J. M. A. & de Vries, R. P. The mammary glands of cows abundantly display receptors for circulating avian H5 viruses. J Virol 98 , e0105224 (2024). https://doi.org:10.1128/jvi.01052-24 Eisfeld, A. J. et al. Pathogenicity and transmissibility of bovine H5N1 influenza virus. Nature 633 , 426-432 (2024). https://doi.org:10.1038/s41586-024-07766-6 Jassem, A. N. et al. Critical Illness in an Adolescent with Influenza A(H5N1) Virus Infection. N Engl J Med (2024). https://doi.org:10.1056/NEJMc2415890 McCullers, J. A. Preparing for the next influenza pandemic. Pediatr Infect Dis J 27 , S57-59 (2008). https://doi.org:10.1097/INF.0b013e3181684d41 Khurana, S. et al. Licensed H5N1 vaccines generate cross-neutralizing antibodies against highly pathogenic H5N1 clade 2.3.4.4b influenza virus. Nat Med 30 , 2771-2776 (2024). https://doi.org:10.1038/s41591-024-03189-y Zhang, X. & Ross, T. M. Anti-neuraminidase immunity in the combat against influenza. Expert Rev Vaccines 23 , 474-484 (2024). https://doi.org:10.1080/14760584.2024.2343689 Nguyen, H. T. et al. Antiviral susceptibility of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses isolated from birds and mammals in the United States, 2022. Antiviral Res 217 , 105679 (2023). https://doi.org:10.1016/j.antiviral.2023.105679 Baker, A. L. et al. Dairy cows inoculated with highly pathogenic avian influenza virus H5N1. Nature (2024). https://doi.org:10.1038/s41586-024-08166-6 LDH. LDH reports first U.S. H5N1-related human death , (2025). Scott Krauss, D. W., Robert G. Webster. in Influenza Virus Vol. 865 Methods in Molecular Biology (ed Gabriele Neumann Yoshihiro Kawaoka) Ch. 2, 234 (Humana Totowa, 2012). Reed, L. J., Muench, H. A Simple Method of Estimating Fifty Per Cent Endpoints. American Journal of Epidemiology 27 , 493-497 (1938). https://doi.org:10.1093/oxfordjournals.aje.a118408 REED, L. J. & MUENCH, H. A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS12. American Journal of Epidemiology 27 , 493-497 (1938). https://doi.org:10.1093/oxfordjournals.aje.a118408 Maher, J. A. & DeStefano, J. The ferret: An animal model to study influenza virus. Lab Animal 33 , 50-+ (2004). https://doi.org:DOI 10.1038/laban1004-50 WHO. Summary of polymerase acidic (PA) protein amino acid substitutions analysed for their effects on baloxavir susceptibility , (2024). WHO. Summary of neuraminidase (NA) amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors (NAIs) among avian influenza viruses of Group 1 and Group 2 NAs , <https://www.who.int/publications/m/item/summary-of-neuraminidase-(na)-amino-acid-substitutions-associated-with-reduced-inhibition-by-neuraminidase-inhibitors-(nais)-among-avian-influenza-viruses-of-group-1-and-group-2-nas> (2024). Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30 , 3059-3066 (2002). https://doi.org:10.1093/nar/gkf436 Leang, S. K. & Hurt, A. C. Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals. Jove-J Vis Exp (2017). https://doi.org:ARTN e55570 10.3791/55570 Marathe, B. M., Lévêque, V., Klumpp, K., Webster, R. G. & Govorkova, E. A. Determination of Neuraminidase Kinetic Constants Using Whole Influenza Virus Preparations and Correction for Spectroscopic Interference by a Fluorogenic Substrate. Plos One 8 (2013). https://doi.org:ARTN e71401 10.1371/journal.pone.0071401 Patel, M. C. et al. An optimized cell-based assay to assess influenza virus replication by measuring neuraminidase activity and its applications for virological surveillance. Antivir Res 208 (2022). https://doi.org:ARTN 105457 10.1016/j.antiviral.2022.105457 Couzens, L. et al. An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. J Virol Methods 210 , 7-14 (2014). https://doi.org:10.1016/j.jviromet.2014.09.003 Tables Table 1 Influenza viruses, including bovine A(H5N1) clade 2.3.4.4b viruses used in this study Influenza A virus Subtype HA clade Abbreviation Genotypes a Reference CVV A/Astrakhan/3212/2020 H5N8 2.3.4.4b Astrakhan/3212 NA b A/chicken/Ghana/AVL-76321VIR7050-39/2021 H5N1 2.3.4.4b ck/Ghana/39 NA A/American wigeon/South Carolina/22-000345-001/2021 H5N1 2.3.4.4b wigeon/SC/345 A1 A/bald eagle/Florida/W22-134-OP/2022 H5N1 2.3.4.4b eagle/FL/W22 B1.1 A/gyrfalcon/Washington/41088-6/2014 H5N8 2.3.4.4c gyrfalcon/41088 NA Bovine and avian influenza viruses A/bovine/Ohio/B24OSU-302/2024 H5N1 2.3.4.4b bovine/OH/302 B3.13 A/bovine/Ohio/B24OSU-342/2024 H5N1 2.3.4.4b bovine/OH/342 B3.13 A/bovine/Ohio/B24OSU-358/2024 H5N1 2.3.4.4b bovine/OH/358 B3.13 A/bovine/Ohio/B24OSU-368/2024 H5N1 2.3.4.4b bovine/OH/368 B3.13 A/bovine/Ohio/B24OSU-432/2024 H5N1 2.3.4.4b bovine/OH/432 B3.13 A/bovine/Ohio/B24OSU-439/2024 H5N1 2.3.4.4b bovine/OH/439 B3.13 A/bovine/Ohio/B24OSU-497/2024 H5N1 2.3.4.4b bovine/OH/497 B3.13 A/bovine/Ohio/B24OSU-541/2024 H5N1 2.3.4.4b bovine/OH/541 B3.13 A/bovine/Texas/3301/2024 H5N1 2.3.4.4b bovine/TX/38301 B3.13 A/bovine/Texas/38161/2024 H5N1 2.3.4.4b bovine/TX/38161 B3.13 A/bovine/Texas/40106/2024 H5N1 2.3.4.4b bovine/TX/40106 B3.13 A/bovine/Texas/42041/2024 H5N1 2.3.4.4b bovine/TX/42041 B3.13 A/bovine/Texas/43134/2024 H5N1 2.3.4.4b bovine/TX/43134 B3.13 A/bovine/Texas/97794/2024 H5N1 2.3.4.4b bovine/TX/97794 B3.13 A/Ross’s goose/Kansas/W23-930F/2023 H5N1 2.3.4.4b goose/KS/930F Minor60 A/snow goose/Louisiana/W23-957/2023 H5N1 2.3.4.4b goose/LA/957 B3.7 Human influenza virus A/California/04/2009 H1N1pdm09 NA CA/09 NA a Genotype of A(H5N1) virus was conducted using GenoFLU 6 . b NA, not assigned. Table 2 Phenotypic NAI and CENI susceptibility of bovine HPAI A(H5N1) 2.3.4.4b viruses Influenza A virus CENI baloxavir EC 50 ± SD [nM] (fold change) a NAI, IC 50 ± SD [nM] (fold change) a Oseltamivir Zanamivir Peramivir bovine/TX/38161 0.52 ± 0.19 (2) 3.08 ± 0.08 (2) 0.29 ± 0.03 (1) 0.08 ± 0.00 (1) bovine/TX/97794 0.19 ± 0.07 (1) 0.70 ± 0.07 (1) 0.14 ± 0.01 (1) 0.07 ± 0.00 (1) bovine/TX/43134 0.34 ± 0.08 (1) 0.71 ± 0.07 (1) 0.14 ± 0.01 (1) 0.07 ± 0.01 (1) bovine/TX/42041 0.65 ± 0.03 (2) 0.91 ± 0.05 (1) 0.15 ± 0.00 (1) 0.07 ± 0.01 (1) bovine/OH/302 0.41 ± 0.16 (1) 0.80 ± 0.13 (1) 0.16 ± 0.02 (1) 0.08 ± 0.01 (1) bovine/OH/358 0.52 ± 0.14 (2) 0.84 ± 0.10 (1) 0.20 ± 0.04 (1) 0.09 ± 0.00 (1) bovine/OH/439 0.45 ± 0.05 (2) 0.78 ± 0.05 (1) 0.15 ± 0.00 (1) 0.15 ± 0.06 (1) bovine/OH/541 0.17 ± 0.01 (1) 0.98 ± 0.12 (1) 0.19 ± 0.01 (1) 0.09 ± 0.00 (1) Median (n = 8) 0.43 ± 0.07 (1) 0.82 ± 0.08 (1) 0.15 ± 0.01 (1) 0.08 ± 0.00 (1) Mean values from two independent experiments are provided ± the standard deviation (SD). a Relative to the baseline susceptibility of North American HPAI A(H5N1) clade 2.3.4.4b viruses 25 . 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"","lastName":"Jones","suffix":""},{"id":410966051,"identity":"f6b40778-2efd-4580-bcdf-45408d86240d","order_by":5,"name":"Trushar Jeevan","email":"","orcid":"","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Trushar","middleName":"","lastName":"Jeevan","suffix":""},{"id":410966052,"identity":"6f4afc3e-6799-4a9c-9441-4ebe3923a387","order_by":6,"name":"Konstantin Andreev","email":"","orcid":"https://orcid.org/0000-0002-1757-4532","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Konstantin","middleName":"","lastName":"Andreev","suffix":""},{"id":410966053,"identity":"8a660bd8-49d4-4d24-9965-ccb66c2f4073","order_by":7,"name":"Jon seiler","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jon","middleName":"","lastName":"seiler","suffix":""},{"id":410966054,"identity":"7ca63288-6b46-4bb8-bb91-5a3e4c24ec8d","order_by":8,"name":"Jonathan Fogo","email":"","orcid":"","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Fogo","suffix":""},{"id":410966055,"identity":"61ea1f4f-397d-4677-9a24-19fe9f414833","order_by":9,"name":"Morgan Davis","email":"","orcid":"","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Morgan","middleName":"","lastName":"Davis","suffix":""},{"id":410966056,"identity":"4d797053-af1c-4075-83c2-1cae0fd730b1","order_by":10,"name":"Jeri-Carol Crumpton","email":"","orcid":"","institution":"Department of Infectious Diseases, St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jeri-Carol","middleName":"","lastName":"Crumpton","suffix":""},{"id":410966057,"identity":"995af60e-7368-417f-9b03-7c7877be7332","order_by":11,"name":"John Franks","email":"","orcid":"","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Franks","suffix":""},{"id":410966058,"identity":"c629fe36-4c6f-4d17-9785-7f0661cc09dc","order_by":12,"name":"Jennifer DeBeauchamp","email":"","orcid":"","institution":"Department of Infectious Diseases, St. Jude Children’s Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"DeBeauchamp","suffix":""},{"id":410966059,"identity":"759d3cc8-3e79-4ddc-9dfd-002fd5ef6329","order_by":13,"name":"Peter Vogel","email":"","orcid":"https://orcid.org/0000-0002-7535-0545","institution":"St Judes Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Vogel","suffix":""},{"id":410966060,"identity":"332f985b-e79c-4e20-8489-2d331feb8967","order_by":14,"name":"C. Scanlon Daniels","email":"","orcid":"","institution":"Circle H Headquarters LLC","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"Scanlon","lastName":"Daniels","suffix":""},{"id":410966061,"identity":"82e7e5c4-6dce-4135-b99c-4a28a0e02609","order_by":15,"name":"Rebecca Poulson","email":"","orcid":"","institution":"College of Veterinary Medicine, The University of Georgia","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Poulson","suffix":""},{"id":410966062,"identity":"9f8fbff0-da7f-4845-833e-eaac09f4603c","order_by":16,"name":"Andrew Bowman","email":"","orcid":"https://orcid.org/0000-0002-0738-8453","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Bowman","suffix":""},{"id":410966063,"identity":"f42d8f7a-9e8c-419f-b135-6c568b7b535d","order_by":17,"name":"Elena Govorkova","email":"","orcid":"https://orcid.org/0000-0001-9067-5682","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Govorkova","suffix":""}],"badges":[],"createdAt":"2025-01-11 02:00:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5806806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5806806/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-61757-3","type":"published","date":"2025-07-23T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75484002,"identity":"4ed55844-0af7-41db-a4f9-61c1f382136f","added_by":"auto","created_at":"2025-02-05 06:05:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":155892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReplication growth kinetics of bovine HPAI A(H5N1) 2.3.4.4b viruses in vitro\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, MDCK or \u003cstrong\u003eb\u003c/strong\u003e, NHBE cells were inoculated at MOI of 0.005 with three representative bovine A(H5N1) 2.3.4.4b, concurrently circulating avian A(H5N1), and human A(H1N1)pdm09 viruses and incubated at 37ºC. The supernatants from MDCK virus-infected cells and apical surface washes from NHBE inserts were collected at the indicated time points and virus titers were determined by TCID50 assay. The data are shown as the mean ± SD from quadruplicate wells.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/33f628de1cc0e0d5239fe0d7.png"},{"id":75484947,"identity":"534ae270-3201-4a21-951b-be0f31bea52c","added_by":"auto","created_at":"2025-02-05 06:13:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":191701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReceptor binding specificity of bovine HPAI A(H5N1) 2.3.4.4b viruses. a-e\u003c/strong\u003e, The solid-phase binding assay with five representative bovine A(H5N1) 2.3.4.4b viruses, \u003cstrong\u003ef\u003c/strong\u003e, concurrently circulating avian A(H5N1) virus, \u003cstrong\u003eg\u003c/strong\u003e, and human A(H1N1)pdm09 viruses to biotinylated sialylglycopolymers (Neu5Acα3`Lac-Gly-PAA, and 3`SLN-C3-PAA) that are the avian influenza virus preferred receptors and biotinylated sialylglycopolymers (Neu5Acα6`Lac-C2-PAA, and 6`SLN-C3-PAA) that are the human influenza virus preferred receptors. The data are shown as the mean ± SD from duplicate wells and represent one of two independent experiments.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/fd9fceb775d4b214450d26c6.png"},{"id":75483998,"identity":"4b2ef24b-2fe7-4707-b429-700cdb9caa33","added_by":"auto","created_at":"2025-02-05 06:05:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1159845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathogenicity of bovine HPAI A(H5N1) 2.3.4.4b viruses in mice. a\u003c/strong\u003e,\u003cstrong\u003e b\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eBALB/c mice (6-8 weeks old) were lightly anesthetized and inoculated IN with 10-fold-serial dilutions of bovine/OH/439 (H5N1) and bovine/TX/98638 (H5N1) viruses to determine MLD\u003csub\u003e50\u003c/sub\u003e for each virus (n = 3/virus dose).\u0026nbsp;\u003cstrong\u003ec\u003c/strong\u003e, virus titer in tissues from infected mice was determined by TCID\u003csub\u003e50\u003c/sub\u003e in MDCK cells. Immunohistochemistry of fixed tissues from A(H5N1) virus-infected mice showed virus antigen in nasal epithelium (\u003cstrong\u003ed, \u003c/strong\u003escale bar, 50 µm), lung (\u003cstrong\u003ee, \u003c/strong\u003escale bar, 200 µm), brain (\u003cstrong\u003ef, \u003c/strong\u003escale bar, 2 mm), spinal cord (\u003cstrong\u003eg,\u003c/strong\u003e scale bar, 1 mm\u003cstrong\u003e \u003c/strong\u003e), liver (\u003cstrong\u003eh, \u003c/strong\u003escale bar, 100 µm), and brown adipose tissue (BAT) (\u003cstrong\u003ei,\u003c/strong\u003e scale bar, 100 µm\u003cstrong\u003e \u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/9e54fadb9db392688ebc4a31.png"},{"id":75484950,"identity":"3e6fbf40-25a3-445e-b744-b77cac68f289","added_by":"auto","created_at":"2025-02-05 06:13:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathogenicity and transmission of bovine HPAI A(H5N1) 2.3.4.4b viruses in ferrets. \u003c/strong\u003eFerrets (n = 3) were lightly anesthetized and IN inoculated with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e units of bovine/OH/439 (H5N1) virus in 500 μl of PBS. \u003cstrong\u003ea\u003c/strong\u003e, Body weights and \u003cstrong\u003eb\u003c/strong\u003e, survival were monitored daily. \u003cstrong\u003ec\u003c/strong\u003e, Virus titers in nasal washes and \u003cstrong\u003ed\u003c/strong\u003e, various tissues were determined by TCID\u003csub\u003e50\u003c/sub\u003e in MDCK cells. At 24 hpi, donor ferrets were placed in direct contact (DC) or aerosol contact (AC) with naïve uninfected ferrets (n = 3). Shapes and colors differentiate between inoculated, DC and AC ferrets. The data are shown as the mean ± SD. The dotted line indicates limit of detection for the assay (1.0 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL). Data for non-infected animals was excluded from panels a and c for clarity.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/a42383aecded8e952bfabb8c.png"},{"id":75484008,"identity":"dc2b62f3-42b1-463b-bbcf-fb53c0bd92ab","added_by":"auto","created_at":"2025-02-05 06:05:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental infection of chickens with bovine HPAI A(H5N1) 2.3.4.4b viruses. \u003c/strong\u003eChickens (6 weeks old) were inoculated intra-tracheally with 10\u003csup\u003e6\u003c/sup\u003e EID\u003csub\u003e50\u003c/sub\u003e/0.3 mL of\u0026nbsp;goose/KS/930F (H5N1) or bovine/OH/439 (H5N1) viruses (n = 6/group). After 16 hpi, two donor chickens from each experimental group were placed in contact with three naive direct contact chickens. The chickens were monitored twice daily for disease symptoms and survival.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/fd1c53b7ee410800b0c79942.png"},{"id":75484951,"identity":"e7acdd6d-1019-4e6e-935e-0bafd760cc7e","added_by":"auto","created_at":"2025-02-05 06:13:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":145745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntigenic and serologic characterization of bovine HPAI A(H5N1) 2.3.4.4b viruses. a\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eAntigenic properties of bovine HPAI A(H5N1) 2.3.4.4b viruses (n = 14) were determined in HI assay with post-infection ferret antisera generated against the WHO-recommended CVVs of clade 2.3.4.4b [Astrakhan/3212 (H5N8), wigeon/SC/345 (H5N1), ck/Ghana/21 (H5N1)], and 2.3.4.4c [gyrfalcon/41088 (H5N8] and representative avian eagle/FL/W22 (H5N1) virus. The data are shown as the mean ± SD. The dotted line indicates limit of detection for the assay (HI titer = 10). \u003cstrong\u003eb\u003c/strong\u003e, The presence of cross-reactive HA antibodies against bovine A(H5N1) 2.3.4.4b viruses was examined in a set of human sera obtained from a Phase 1 clade 2.3.2.1c H5 vaccine trial. Individuals (aged 18 to 50 years) were vaccinated with 15 µg of the HA derived from gyrfalcon/41088 (H5N8) 2.3.4.4c antigen in combination with AS03 (n = 10) or MF59 (n = 10) adjuvant. The data are shown as the mean ± SD. \u003cstrong\u003ec\u003c/strong\u003e, NA neutralizing (NI) antibody levels in human serum samples (n = 24) against N1 NA protein derived from CA/09 (H1N1)pdm09, bovine/OH/439 (H5N1), and eagle/FL/W22 (H5N1) viruses (as measured by ELLA assay). The data are shown as geometric mean titer (line) of the individual IC\u003csub\u003e50\u003c/sub\u003e NI titers (dots). **** represents significance at p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/73862a2158efc9bc2c50b8fc.png"},{"id":87469988,"identity":"0d5f3a6a-cf6c-4a59-8b87-e6b2622921bc","added_by":"auto","created_at":"2025-07-24 08:28:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3259613,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/5c8cd4b6-ad7c-43bf-b7d2-28f508cbb342.pdf"},{"id":75484000,"identity":"13e4f46b-c28c-4bfa-b469-c4a9e85e3333","added_by":"auto","created_at":"2025-02-05 06:05:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":423158,"visible":true,"origin":"","legend":"SUPPLEMENTARY INFORMATION","description":"","filename":"BovineH5N1Paperv8SupplementaryFinal1.10.25.docx","url":"https://assets-eu.researchsquare.com/files/rs-5806806/v1/42da03e8f6eaa719039d4c13.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eInfluenza A(H5N1) Viruses Isolated From Dairy Cattle Demonstrate High Virulence in Laboratory Models, but Retain Avian Virus-like Properties\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSince its initial identification in 1996, the A/goose/Guangdong/1/1996 (gs/Gd) lineage of highly pathogenic avian influenza (HPAI) A(H5N1) virus has undergone dramatic genetic diversification with the emergence of multiple genetic clades, some of which spread globally. Viruses of this lineage pose a significant public health threat and have a case-fatality rate of around 52% following zoonotic infection. Their impact on avian species has been particularly dramatic, with roughly 110\u0026nbsp;million poultry culled in the United States (U.S.) alone since 2022\u003csup\u003e1,2\u003c/sup\u003e. Between 2020\u0026ndash;2021, viruses with hemagglutinin (HA) clade 2.3.4.4b [A(H5N1) 2.3.4.4b] dominated in many countries\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Following widespread circulation in Africa, Asia, and Europe, 2.3.4.4b viruses entered North America in late 2021, representing only the second introduction of gs/Gd-lineage viruses into the continent. Soon after introduction into the Americas, the viruses quickly diversified and acquired internal gene segments from endemic low pathogenicity avian influenza viruses through reassortment\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Further, there was an increase in the number of identified hosts, particularly non-poultry avians and terrestrial and aquatic mammals\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The diversification of hosts coupled with a steady isolation of this lineage since 2021 is a concerning pattern with the potential to alter viral evolution and host range.\u003c/p\u003e \u003cp\u003eOn March 25, 2024, an A(H5N1) virus-positive milk sample was collected from a dairy cow in the U.S. with subsequent confirmation in cats and wild birds associated with this index farm\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The virus was identified as HPAI A(H5N1) 2.3.4.4b, genotype B3.13. To date, the virus has been reported on 923 dairy farms spanning 16 states, and the presence of non-infectious viral RNA has been detected in pasteurized commercial milk products sourced from at least 15 states\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e14\u003c/span\u003e,15\u003c/sup\u003e. Sixty-six human cases have been reported, in California, Colorado, Iowa, Michigan, Oregon, Texas, Washington, and Wisconsin as a result of occupational exposure to infected cattle or poultry\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR17\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Two infections with unknown sources have been detected in California and Missouri, while one exposure to backyard poultry occurred in Louisiana\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These zoonotic infections have primarily manifested with conjunctivitis, accompanied by limited respiratory symptoms. Though at least two cases have led to severe disease in patients with potential co-morbidities. As the outbreak among dairy cows continues to spread across the U.S., there is concern the viruses may accumulate mammalian adaptive markers, increasing their risk to humans.\u003c/p\u003e \u003cp\u003eIn this study, we compared the phenotypic properties of 17 avian and bovine influenza A(H5N1) clade 2.3.4.4b viruses (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with an overarching goal to address the potential risks they pose to human health. We investigated various viral characteristics, including replication kinetics in primary human cells, receptor binding preferences, pathogenesis, transmission dynamics in mice and ferrets, and levels of population immunity. Our findings indicate that these viruses retain avian-like features despite sustained transmission through dairy cattle.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReplication of A(H5N1) 2.3.4.4b viruses in vitro\u003c/h2\u003e \u003cp\u003eReplication capacity of emerging influenza viruses in mammalian cells can be an early indicator of viral fitness in non-avian hosts. Accordingly, we assessed viral replication kinetics of A(H5N1) 2.3.4.4b viruses detected in cows (genotype B3.13) and avian hosts (genotypes Minor60 and B3.7) in two different mammalian cell models, the madin-darby canine-kidney cell line (MDCK) and primary differentiated normal human bronchial epithelial cells (NHBEs). All viruses replicated efficiently in both cell types. A(H5N1) 2.3.4.4b viruses grew to high titers in MDCK cells, peaking by 36 hours post-infection (hpi) with median titers of 8.1 to 9.4 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL, significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01) than those of the seasonal CA/09 (H1N1)pdm09 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In differentiated NHBE cell cultures, all bovine viruses also replicated well reaching peak titers by \u0026asymp;\u0026thinsp;72 hpi (8.0 to 8.9 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL). However, no significant differences were observed in replication kinetics between avian-, bovine- or the human-origin influenza viruses in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReceptor binding specificity of A(H5N1) 2.3.4.4b viruses\u003c/h3\u003e\n\u003cp\u003eTo determine whether a shift in receptor preference from the avian virus-like α2,3 linked sialic acid receptor to the human virus-like α2,6 linkage followed transmission in dairy cattle, we evaluated the binding of the bovine viruses to both receptor ligands in solid phase binding assays\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. All bovine A(H5N1) viruses tested bound exclusively to the avian virus-like α2,3 linked sialic acid receptors and not to human virus-like receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-e). As expected, the human CA/09 (H1N1)pdm09 virus preferentially bound to α2,6 sialic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg) and the avian goose/KS/930F (H5N1) was restricted to α2,3 sialic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Consistent with the observed binding patterns, the bovine and avian A(H5N1) viruses maintained the avian virus preferred glutamine at HA position 226 (H3 numbering), a position recently shown to be important for receptor preference of the bovine A(H5N1) viruses\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Acquiring or losing N-glycosylation in the globular head of the HA protein can directly influence or alter the affinity of influenza virus towards specific receptors. One of our tested viruses (TX/97794) had an amino acid substitution suggesting potential additional N-glycosylation (caused by HA-T156A), but it retained patterns similar to the other bovine viruses lacking the glycosylation consensus site (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePathogenicity of A(H5N1) 2.3.4.4b viruses in mice\u003c/h3\u003e\n\u003cp\u003eThe high viral loads detected in raw milk from infected cows present potential risk of human exposure. To investigate virus pathogenicity directly from milk, we modeled exposure in mice via intranasal inoculation. BALB/c mice inoculated with either bovine/OH/439 (H5N1) or bovine/TX/98638 (H5N1) viruses were susceptible to infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Signs of morbidity were observed starting 2\u0026ndash;3 days post-inoculation (dpi), including ruffled fur, loss of body weight (Supplementary Fig.\u0026nbsp;1), and neurologic symptoms associated with movement including ataxia, paralysis, and tremors. All mice succumbed to disease by 5 to 8 dpi. To further investigate virus pathogenicity, the 50% mouse lethal dose (MLD\u003csub\u003e50\u003c/sub\u003e) was determined for both viruses. Bovine/OH/439 (H5N1) and bovine/TX/98638 (H5N1) viruses were exceptionally lethal to mice with MLD\u003csub\u003e50\u003c/sub\u003e values of 1.25 and 1.5 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Both A(H5N1) viruses spread systemically in mice, with viral detection in nasal turbinates, lungs, brains, spinal cord, livers, and brown adipose tissue at 3 and 4 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-i).\u003c/p\u003e \u003cp\u003eInfected mice exhibited widespread and disseminated microscopic lesions consistent with severe viral infection. The upper respiratory tract displayed extensive infection of nasal respiratory epithelia characterized by antigen staining and loss of cilia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) but surprisingly limited inflammatory cell infiltrates or nasal exudates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). The lower respiratory tract exhibited extensive necrotizing bronchiolitis and diffuse alveolar damage characterized by necrotic cell debris, septal thickening and necrosis, alveolar collapse, alveolar edema and fibrin. Antigen-positive cells included both type I and type I pneumocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Virus dissemination to neural or neural-adjacent tissues was evident by abundant viral antigen in the olfactory epithelia (Supplementary Fig.\u0026nbsp;2a), randomly distributed throughout the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), proximal spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), ganglia, and liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Examination of whole sections of the mouse heads revealed viral antigen staining in other epithelial areas including the maxillary sinus and Eustachian tubes of the inner ear (Supplementary Fig.\u0026nbsp;2b-d). Antigen staining was also observed in brown adipose tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), eyes, pituitary gland, incisor teeth, and bone marrow (Supplementary Fig.\u0026nbsp;2e-i). These data suggest that bovine viruses are highly virulent in mice due to extrapulmonary dispersion and efficient replication soon after inoculation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePathogenicity and transmission of A(H5N1) 2.3.4.4b viruses in ferrets\u003c/h3\u003e\n\u003cp\u003eTo understand if the A(H5N1) 2.3.4.4b viruses are transmissible in mammals, donor ferrets were inoculated with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL of bovine/OH/439. At 1 dpi, each donor ferret was housed with a na\u0026iuml;ve direct contact (DC) ferret and one ferret was placed in an adjacent cage separated by a perforated barrier as an airborne contact (AC). Donor ferrets displayed elevated temperatures (\u0026gt;\u0026thinsp;40\u0026deg;C) at 2\u0026ndash;3 dpi, which coincided with the onset of weight loss; humane endpoints were met by 8 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Donor ferrets shed virus in nasal washes with peak titers ranging from 6.3\u0026ndash;6.7 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Two of three naive DC ferrets also became infected, met human endpoints, and shed virus in nasal washes to peak titers of 3.5\u0026ndash;4.3 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL between 5\u0026ndash;7 days post-contact (dpc) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The third DC ferret did not show clinical signs, and the virus was not detected in nasal washes. Virus was not detected in any of the AC ferrets, and they displayed no signs of disease.\u003c/p\u003e \u003cp\u003eTissues were taken from donor and DC ferrets that had reached human endpoints (6 to 10 dpi) for virus isolation. Infectious virus was detected in all tissues collected, including the nasal turbinate, trachea, lungs, brain, liver, and duodenum portion of the small intestine, from two of the three infected donors and two infected DCs. One donor ferret did not have any viable virus detected in the liver or trachea, but virus was recovered from the other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Thus, bovine/OH/439 (H5N1) clade 2.3.4.4b virus spread systemically in multiple tissues of donor ferrets and transmitted to most naive ferrets by contact route but failed to transmit via the airborne route.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eReplication and transmission of A(H5N1) 2.3.4.4b viruses in chickens\u003c/h3\u003e\n\u003cp\u003eWhile avian HPAI A(H5N1) 2.3.4.4b viruses cause 100% mortality in chickens\u003csup\u003e22\u003c/sup\u003e, the pathogenicity of bovine-origin A(H5N1) viruses in experimentally infected poultry is unknown; therefore, we investigated the replication and transmissibility of bovine/OH/439 (H5N1) and goose/KS/930F (H5N1) in chickens to determine if mammalian passage had impacted the viruses\u0026rsquo; ability to infect and transmit in poultry. All donor virus-inoculated chickens died or reached humane endpoints at 2 dpi. In the avian virus group, five contact chickens died or were euthanized at 2 dpc and the remaining four chickens at 3 dpc. In the bovine virus group, two contact chickens died or were euthanized at 2 dpc, four chickens likewise at 3 dpc and two chickens survived until 6 dpc (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore, the bovine A(H5N1) 2.3.4.4b virus was highly lethal to chickens and was comparable in pathogenicity to the avian-origin virus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntiviral susceptibility of A(H5N1) 2.3.4.4b viruses\u003c/h2\u003e \u003cp\u003eTo assess the potential efficacy of available therapeutic options for the control of bovine A(H5N1) 2.3.4.4b infections in humans, we determined the frequencies of genetic markers associated with reduced or highly reduced inhibition (RI/HRI) by FDA-approved influenza neuraminidase (NA) inhibitors (NAIs, oseltamivir, zanamivir, peramivir) or the cap dependent endonuclease inhibitor (CENI, baloxavir). Sequence-based analysis of NA and PA proteins available in public databases revealed low frequencies of bovine A(H5N1) influenza viruses with NAI (0.07%, 1/1489) and CENI (0.21%, 3/1406) RI/HRI-associated substitutions (Supplementary Table\u0026nbsp;2). A single bovine A(H5N1) virus had NA-T438I, which mediates zanamivir/peramivir RI\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. One bovine A(H5N1) virus had PA-A37T that was shown to cause RI by baloxavir in human A(H3N2) viruses\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Two viruses had PA-A36T, which has been recently shown to confer baloxavir RI in avian influenza A viruses\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Phenotypic testing confirmed that all bovine A(H5N1) viruses tested were susceptible to NAIs and CENI baloxavir at sub-nanomolar concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results were consistent between bovine viruses from Texas and Ohio. Oseltamivir IC\u003csub\u003e50s\u003c/sub\u003e of bovine A(H5N1) viruses were slightly lower than contemporary A(H5N1) clade 2.3.4.4.b viruses circulating in 2022\u0026ndash;2023 in birds\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Overall, highly pathogenic influenza A(H5N1) viruses isolated from dairy cattle retain susceptibility to FDA-approved antiviral drugs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntigenic relationship of A(H5N1) viruses to WHO candidate vaccine viruses\u003c/h3\u003e\n\u003cp\u003eAnalysis of the antigenic relationships between the bovine A(H5N1) clade 2.3.4.4b viruses and WHO-recommended candidate vaccine viruses (CVVs) was accomplished using post-infection ferret antiserum generated against three clade 2.3.4.4b CVVs [Astrakhan/3212 (H5N8), ck/Ghana/39, and wigeon/SC/345], a well-characterized representative avian clade 2.3.4.4b virus\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e [eagle/FL/W22], and the clade 2.3.4.4c CVV [gyrfalcon/41088 (H5N8)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, Supplementary Table\u0026nbsp;3). All viruses tested, including TX/38161, TX/42041, TX43134, and TX/97794 with the additional glycosylation site in HA, reacted to within two-fold of homologous titers to at least one of the three CVVs. These data suggest that vaccines made using available clade 2.3.4.4b CVVs may protect against bovine A(H5N1) viruses in the event of a pandemic.\u003c/p\u003e\n\u003ch3\u003eH5 HA subtype neutralizing antibodies in human sera\u003c/h3\u003e\n\u003cp\u003eTo further evaluate the utility of existing stockpiled vaccines, we tested a set of human sera obtained from a Phase 1 clade 2.3.2.1c H5 vaccine trial with donors aged 18 to 50 years.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e We examined the presence of cross-reactive HA antibodies against bovine A(H5N1) 2.3.4.4b viruses. Previous studies have shown that HI antibody titers of \u0026gt;\u0026thinsp;40 are protective against disease.\u003csup\u003e27,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Antibodies induced by vaccination of individuals with an adjuvanted vaccine containing 15 \u0026micro;g of the HA derived from gyrfalcon/41088 (H5N8) 2.3.4.4c antigen reacted with GMTs ranging from 45.9 to 88.8 to the bovine A(H5N1) viruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Twenty of 20 individuals achieved a seroprotective HI titer of 40 against ​bovine/OH/342, 13 of 20 against bovine/OH/368, 18 of 20 against bovine/TX/40106, and 15 of 20 against bovine/TX/97794 (Supplementary Table\u0026nbsp;4). Thus, vaccination of humans with an adjuvanted vaccine using an A(H5N8) 2.3.4.4c antigen induces sufficient HA antibody responses that should protect against HA clade-mismatched virus.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eN1 NA subtype neutralizing antibodies in human sera\u003c/h2\u003e \u003cp\u003eUsing a panel of 24 human sera selected based on existing A(H1N1)pdm09 virus HA antibody titers (HI\u0026thinsp;\u0026ge;\u0026thinsp;40), we investigated the extent of cross-reactivity of NA antibodies generated against seasonal influenza virus/vaccine exposure against A(H5N1) 2.3.4.4b NA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). As expected, all individuals demonstrated neutralization of the CA/09 (H1N1pdm09) NA with a mean NI titer of 29.0 regardless of adjuvant included with the vaccine. While mean NI titers were lower for both bovine/OH/439 and eagle/FL/W22 viruses, 17.6 and 14.6, respectively, 14 of 24 sera neutralized the bovine N1 from these viruses to roughly 1.5 times above assay background (NI titer of 9), while 13 of 24 sera neutralized the avian N1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The N1 NA proteins of the A(H5N1) 2.3.4.4b and A(H1N1) pdm09 viruses have 89.6% amino acid identity, with considerable conservation at some antigenic sites\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although the levels of protective NA antibodies have not been established, these data suggest that some level of cross-protection against infection with bovine A(H5N1) viruses may be achieved based on NA antibodies generated against seasonal A(H1N1)pdm09.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe introduction of HPAI A(H5N1) 2.3.4.4b viruses into U.S. dairy cattle, followed by their spread and zoonotic infections, is unprecedented\u003csup\u003e29,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The primary objective of our study was to understand if, and to what extent, these viruses have acquired properties suggestive of increased zoonotic risk following replication in dairy cattle.\u003c/p\u003e \u003cp\u003eCritically, while the bovine A(H5N1) 2.3.4.4b viruses tested were pathogenic and highly virulent in ferrets and mice, we observed some contact transmission but no airborne transmission. This transmission profile may be partially attributed to the strict preference for avian-like α2,3 linked sialic acid ligands observed in the receptor binding assay. The lack of binding to α2,6 linked sialic acids that we observed contrasts with a study of an early bovine virus, A/dairy cattle/New Mexico/A24920343-93/2024 (H5N1), that demonstrated some degree of human-receptor binding\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These differences may in part be due to assay variations, which measure a limited number of glycans, as studies utilizing large glycan array panels continue to show a clear preference for avian-like ligands\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Further evidence of avian host preference of the bovine A(H5N1) viruses was demonstrated by the poultry inoculation studies shown here, lack of cow-to-cow transmission in experimentally infected calves\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and the robust presence of avian-like receptor ligands in the bovine mammary gland\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e which are readily bound by contemporary H5 proteins\u003csup\u003e37\u003c/sup\u003e. The degree of airborne transmission of the bovine A(H5N1) viruses to exposed ferrets has varied between viruses and studies. While we were unable to detect airborne transmission, studies of the human isolate A/Texas/37/2024 have demonstrated an inability to transmit, albeit inefficiently, via this route\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Nevertheless, there has been no evidence for human-to-human transmission in human infection. The acquisition of the mammalian adaptation marker PB2-E627K in some human isolates, along with a recent severe human infection with no poultry or bovine epidemiological link\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e does, however, raise concern and highlights the importance of continued laboratory testing of these and future isolates.\u003c/p\u003e \u003cp\u003eWhile current data suggests A(H5N1) 2.3.4.4b viruses do not spread efficiently between humans, they do meet two other criteria that are broadly accepted as requirements for the generation of pandemic viruses: productive replication in humans and antigenic disparity from circulating viruses leading to a lack of population immunity to the novel virus antigens\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. It is therefore important to understand the effectiveness of pharmacological interventions against 2.3.4.4b viruses in terms of both prophylaxis and treatment. The WHO Global Influenza and Response System (GISRS) routinely consider CVVs for zoonotic influenza, including A(H5N1). In some countries, A(H5N1) vaccines have been stockpiled. A recent study identified some degree of heterologous antibody recognition of Astrakhan/3212 from sera from clade 1 or 2.1 vaccinated individuals\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e indicating these earlier stockpiled vaccines may have some efficacy against currently circulating 2.3.4.4b viruses. However, a closely matched CVV is likely to provide better protection. Our data indicate that ferret post-infection antisera raised against three 2.3.4.4b CVVs, including Astrakhan/3212, effectively neutralize a majority of bovine A(H5N1) viruses we tested. Further, sera from individuals receiving gyrfalcon/41088 (H5N8) clade 2.3.4.4c adjuvanted vaccine also broadly inhibited 2.3.4.4b bovine viruses, with minor exceptions, including seven participants who had HI titers\u0026thinsp;\u0026lt;\u0026thinsp;40 to bovine/OH/368 which has a novel mutation in antigenic site B. However, most participants had HI titers\u0026thinsp;\u0026ge;\u0026thinsp;40, which has long been the standard correlate for protection against seasonal influenza viruses\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Humoral protective immunity is dominated by antibody responses to the HA surface glycoprotein, but evidence suggests that some protective immunity may be generated against the lesser abundant NA glycoprotein. While NA-based immunity may still be permissive to viral infection, it may lessen disease severity, decrease viral loads in tissues, and reduce viral shedding\u003csup\u003e42\u003c/sup\u003e. Importantly, seasonal A(H1N1)pdm09 viruses have an N1 NA as do circulating A(H5N1) viruses, although the genes encoding the two proteins are genetically distinct. Correspondingly, we observed neutralization of avian and bovine 2.3.4.4b virus NAs with serum from healthy adults, similar to our previous studies of avian 2.3.4.4b viruses\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. While this suggests that N1-based antibody immunity may be effective against circulating A(H5N1) viruses, further in vivo testing is urgently required to fully address this hypothesis. Collectively, current seasonal vaccination strategies, stockpiled pre-pandemic vaccines, and WHO CVVs appear to provide cross-reactivity against currently circulating bovine HPAI A(H5N1) 2.3.4.4b viruses and may be useful if vaccination efforts are deemed necessary for high-risk groups such as those with direct exposure to affected agricultural species. Additionally, our phenotypic testing indicated that circulating bovine viruses remained susceptible to currently available antiviral therapies at sub-nanomolar concentrations, and genotypic testing revealed a low frequency of substitutions associated with RI/HRI. Many of the initial human 2.3.4.4b human infections were treated with the NAI oseltamivir, but current in vitro data show higher oseltamivir IC\u003csub\u003e50\u003c/sub\u003e values for A(H5N1) 2.3.4.4b viruses as compared to viruses circulating before 2021\u003csup\u003e25,43\u003c/sup\u003e. It is not understood if this translates to decreased drug efficacy in humans, but it may increase the importance of alternative drug classes. This includes CENI baloxavir marboxil, which targets PA protein and has the potential to treat severe human infections if A(H5N1) 2.3.4.4b acquires genetic signatures of RI by oseltamivir\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on the data generated through this assessment, the risk to human health posed by the bovine A(H5N1) viruses in their current form is low, especially for those not exposed to dairy cows, their raw milk, or culling infected poultry flocks. The viruses show inefficient transmission within laboratory models and more importantly, no human-to-human transmission has been reported. We have also demonstrated that currently available antiviral drugs remain effective and there are vaccines available that offer cross protection in the event they are needed. Despite this work, the 2.3.4.4b A(H5N1) viruses continue circulating in wild and domestic animals at a large-scale, and human infections continue to be reported. This includes a severe infection in British Columbia, Canada, in an obese teenager with a history of mild asthma with no link to poultry or other affected species\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e as well as a recent fatal case in Louisiana in an elderly male with exposure to sick and dying poultry\u003csup\u003e45\u003c/sup\u003e. Limited data are available for these cases and virus characteristics. It is imperative that we maintain increased surveillance within wild birds, domestic animals, and humans for potential genetic and phenotypic changes that could have a profound impact on human health.\u003c/p\u003e \u003c/div\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eCells\u003c/h2\u003e \u003cp\u003eMadin\u0026ndash;Darby Canine Kidney cells (MDCK, ATCC, #CCL-34) were maintained in complete growth medium [MEM (CellGro), 5% FBS (HyClone), 1 mM L-glutamine, 1\u0026times; penicillin/streptomycin/amphotericin B (Gibco)] at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Differentiated primary normal human bronchial epithelial (NHBE) cells (Mattek, AIR-100) were cultured at an air-liquid interface in manufacturer provided media at 37\u0026deg;C, 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eVirus isolation\u003c/h2\u003e \u003cp\u003eA total of 15 influenza A(H5N1) 2.3.4.4b genotype B3.13 viruses were isolated from raw milk samples collected on two geographically distinct farms, one in Ohio and one in Texas (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Primary samples were inoculated into the allantoic cavities of 10-day-old embryonated chicken eggs (eggs), incubated at 35\u0026deg;C for \u0026le;\u0026thinsp;48 h, harvested, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Seasonal human CA/09 (H1N1)pdm09 was propagated in MDCK cells at 37\u0026deg;C for 48 h. CVVs [Astrakhan/3212 (H5N8) and wigeon/SC/345 (H5N1), a recombinant 6\u0026thinsp;+\u0026thinsp;2 ck/Ghana/21 (H5N1) AVL-763 (all kindly shared by CDC)], and an early North American reassortment 6\u0026thinsp;+\u0026thinsp;2 clade 2.3.4.4b virus eagle/FL/W22 (H5N1) was generated by reverse genetics with A/Puerto Rico/8/1934 (H1N1) internal gene segments and all were propagated in eggs at 37\u0026deg;C for 48 h. 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) or 50% egg infectious dose (EID\u003csub\u003e50\u003c/sub\u003e) were calculated by Reed and Muench method\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e using limiting dilution in MDCK cells or eggs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eReplication kinetics\u003c/h2\u003e \u003cp\u003eMulti-round replication curves were performed in MDCK cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well, 12-well plates), primary NHBE cell (\u0026asymp;\u0026thinsp;1.2 x 10\u003csup\u003e6\u003c/sup\u003e cells/insert) at MOI of 0.005 of indicated viruses. Monolayer supernatants or a 200 \u0026micro;L apical layer wash for airway cultures were collected at indicated timepoints and titrated by TCID\u003csub\u003e50\u003c/sub\u003e assay in MDCK cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eReceptor binding assay\u003c/h2\u003e \u003cp\u003eFetuin coated immunoassay plates were incubated with 32 HA units of A(H5N1) 2.3.4.4b viruses in blocking buffer (PBS, 1% BSA) then the serially diluted biotinylated sialylglycopolymers [Neu5Acα3`Lac-Gly-PAA, 3`SLN-C3-PAA, Neu5Acα6`Lac-C2-PAA, and 6`SLN-C3-PAA (Sigma-Aldrich ] in the reaction buffer [PBS, 0.02% Tween-80, 0.02 % BSA, 5 \u0026micro;M oseltamivir carboxylate (MedChem Express)] were added and incubated for 2 h at 4\u0026deg;C. After washing, HRP-conjugated streptavidin (Invitrogen; 1:2000) was added for 1 h at 4\u0026deg;C. Plates were washed and TMB (3,3\u0026prime;, 5,5\u0026prime; tetramethylbenzidine dihydrochloride, Sigma-Aldrich) substrate was added for 10 min at room temperature (RT). Reactions were stopped with 1 N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and absorbances were measured at 450 nm using a Synergy H1 microplate reader (BioTek Instruments).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePathogenicity in mice\u003c/h2\u003e \u003cp\u003eSix to eight-week-old female BALB/c mice (Jackson Laboratory, Bar Harbor, ME, USA) were lightly anesthetized with isoflurane and intranasally (IN) inoculated with 10-fold serial dilutions containing 10\u003csup\u003e1\u003c/sup\u003e to 10\u003csup\u003e6\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mouse of bovine/OH/439 (H5N1) and bovine/TX/98638 (H5N1) viruses (n\u0026thinsp;=\u0026thinsp;3/virus dose). Mice were weighed and monitored daily for clinical signs for 14 dpi. The 50% mouse lethal dose (MLD\u003csub\u003e50\u003c/sub\u003e) was calculated for both tested A(H5N1) viruses\u003csup\u003e48\u003c/sup\u003e. To determine viral replication and pathological features in BALB/c mice, a group of mice (n\u0026thinsp;=\u0026thinsp;9/virus) was lightly anesthetized with isoflurane and inoculated intranasally with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e in 20 \u0026micro;L of PBS. At 3 and 4 dpi, three mice from each tested A(H5N1) virus were euthanized, and tissues (nasal turbinates, lungs, intestines, livers, and brains) were collected, and virus titration was determined by TCID\u003csub\u003e50\u003c/sub\u003e assay in MDCK cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eBALB/c mice (n\u0026thinsp;=\u0026thinsp;3/group) were sacrificed at 3 and 4 dpi and tissues (nasal turbinates, lungs, brain, liver, and small intestine) were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4\u0026ndash;6 \u0026micro;m thickness and stained with hematoxylin and eosin (H\u0026amp;E). Immunohistochemical (IHC) staining for viral antigen detection was performed using the Ventana Discovery Ultra Autostainer (Roche Ventana, Tucson, Arizona). Sections were initially heated for 4 min at 72\u0026deg;C and placed in EZ prep solution (Roche Ventana) for deparaffinization. Antigen retrieval was performed for 56 min at 95\u0026deg;C in Cell Conditioning Solution 1 (Roche Ventana). A rabbit primary monoclonal antibody (GeneTex, Irvine, CA) raised against NP protein of A/Kansas/14/2017(H3N2) virus was applied at 1:8000, and the OmniMap anti-rabbit HRP (Roche Life Science) and ChromoMap DAB detection kits (Roche Ventana) were used to label virus-positive cells. Lung sections were then counterstained with hematoxylin and examined by a pathologist blinded to the experimental group assignments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePathogenicity and transmission in ferrets\u003c/h2\u003e \u003cp\u003eEight-week-old influenza-seronegative male ferrets (Triple F Farms) were IN inoculated with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e units of bovine/OH/439 (H5N1) virus in 500 \u0026micro;L of PBS. One direct contact ferret was placed in the same cage as each inoculated animal 24 hpi (n\u0026thinsp;=\u0026thinsp;3/group) and one ferret each in an adjacent cage with a perforated barrier allowing airflow and excluding contact. Animals were monitored daily for clinical illness (temperature, weight loss, relative inactivity indices, ataxia, respiratory symptoms, neuropathologic signs)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Nasal washes were collected by intramuscular (IM) ketamine injection (25 mg/kg) and IN instillation of 1 mL PBS to induce sneezing starting 1 dpi and alternating days thereafter for a total of 7 collections. Any ferret reaching the humane endpoint or found dead had tissues collected for virus isolation and pathology. Tissues (nasal turbinate, trachea, lung, brain, liver, and small intestine) were homogenized and subjected to titration by TCID\u003csub\u003e50\u003c/sub\u003e in MDCK cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eExperimental infection of chickens\u003c/h2\u003e \u003cp\u003eWhite leghorn chickens (AVS Bio, Norwich, CT) were inoculated with 10\u003csup\u003e6\u003c/sup\u003e EID\u003csub\u003e50\u003c/sub\u003e/0.3 mL via intra-tracheal route (IT) with goose/KS/930F (H5N1) or bovine/OH/439 (H5N1) viruses (n\u0026thinsp;=\u0026thinsp;6/group). After 16 hpi, two donor chickens from each experimental group were placed in contact with three naive direct contact chickens in replicates of three cages, except for one replicate of the avian virus, for which only two contact chickens were available. Chickens were monitored twice daily for disease symptoms and euthanized according to IACUC approved humane endpoint protocols if disease signs were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAntiviral susceptibility\u003c/h2\u003e \u003cp\u003eSequence data for NA (n\u0026thinsp;=\u0026thinsp;1489) and PA (n\u0026thinsp;=\u0026thinsp;1406) proteins of bovine influenza viruses available publicly through the Global Initiative on Sharing All Influenza Data (GISAID) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gisaid.org\u003c/span\u003e\u003cspan address=\"https://gisaid.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed November 8, 2024) were screened for genetic markers associated with RI/HRI by NAIs and CENI baloxavir according to the World Health Organization (WHO) Global Influenza Programme 2024 guidelines\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Nucleotide sequences were aligned using progressive (FFT-NS-2) and iterative (FFT-NS-i) algorithms of Multiple Alignment Fast Fourier Transform\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Amino acid sequences were analyzed with BioEdit (v.7.7.1) software. Phenotypic susceptibility to NAIs (oseltamivir carboxylate [oseltamivir] and zanamivir [MedChem Express]) was assessed with a fluorescence-based assay with 2\u0026prime;-(4-methylumberlliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate (Sigma-Aldrich)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. NA activity of each virus was standardized to relative fluorescent unit equivalents of 10 \u0026micro;M 4-methylumbelliferone (4-MU)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. After 30 min of incubation with NAI (5 pM \u0026minus;\u0026thinsp;50 \u0026micro;M) at 37\u0026ordm;C, fluorescent NA-cleaved MUNANA substrate was measured with a Synergy 2 multimode microplate reader (BioTek Instruments) at Ex/Em 360/460 nm. Half-maximal inhibitory concentrations (IC\u003csub\u003e50s\u003c/sub\u003e) were estimated from dose\u0026ndash;response curves by using the sigmoidal, four-parameter logistic non-linear regression equation (GraphPad Prism v.10.1.2). Phenotypic susceptibility to CENI baloxavir marboxil active metabolite baloxavir acid (baloxavir [MedChem Express]) was determined by Influenza Replication Inhibition NA-based Assay (IRINA) in MDCK cells\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Virus inoculum was standardized to 1.9 nM/well of 4-MU and incubated with baloxavir (6 pM \u0026minus;\u0026thinsp;111 nM) on cell monolayer (96-well microplates, 8 h at 37\u0026deg;C) without TPCK-treated trypsin to achieve a single cycle of virus replication. NA activity of the infected cells was measured, and baloxavir half-maximal effective concentrations (EC\u003csub\u003e50s\u003c/sub\u003e) were calculated as described for NAI assay.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSerologic testing and virus antigenicity\u003c/h2\u003e \u003cp\u003eAntigenic cross-reactivity and/or seroconversion of post-challenge animal (mouse, chicken, ferret) sera was assessed using the cattle-derived viral antigens with post-infection ferret antisera (Supplementary Table\u0026nbsp;3) in an HI assay. Briefly, sera samples were treated with receptor-destroying enzyme II (Denka Seiken Co.) and were serially diluted and incubated with 4 HA units of respective virus for 45 min at RT before addition of 0.5% chicken erythrocytes (Rockland Immunochemicals). HI titers were recorded after 30 min incubation at RT as the reciprocal of the highest serum dilution with complete inhibition of hemagglutination.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked lectin assay\u003c/h2\u003e \u003cp\u003eThe presence of NA-specific antibodies in human was determined in enzyme-linked lectin assays (ELLAs) as previously described\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The influenza A(H6N1) viruses used in ELLA assay were generated by reverse genetics, all having the same HA gene from A/Teal/Hong Kong/w312/1997 (H6N1) virus, six internal segments from A/Puerto Rico/8/1934 (H1N1) virus and the NA gene of bovine/OH/439 (H5N1) virus, eagle/FL/W22 (H5N1), or CA/09 (H1N1)pdm09 viruses. The flat-bottom, 96-well plates (Thermo Scientific) were coated with fetuin (Sigma-Aldrich) at 25 g/mL in 0.1 M PBS at 4\u0026ordm;C for 48 h. Heat-inactivated human sera (56\u0026ordm;C for 1 h) that were commercially obtained from BioIVT were titrated by serially diluted in Dulbecco's phosphate-buffered saline (DPBS), 1% BSA, 0.5% Tween-20 and added to plates. An optimized amount of rg\u0026ndash;derived H6N1 virus was added, and the plates were incubated for 18 h at 37\u0026ordm;C. After three washing, horseradish peroxidase\u0026ndash;conjugated peanut agglutinin (Sigma-Aldrich) was added, and the plates were incubated for 2 h at RT. After washing, 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) substrate was added. Reactions were stopped after 10 min by adding 1 N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and the plates were read at 490 nM using a Synergy H1 microplate reader. The NI titers were defined as the reciprocal of the last dilution that resulted in at least 50% inhibition using nonlinear regression analysis of GraphPad Prism software (version 10.1.2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using two-way Anova with multiple comparisons in GraphPad Prism v.10.1.2. Replicates, group comparisons, and P values are listed in each figure legend.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData generated in this study are provided in the main manuscript, figures, supplemental figures, and in source data files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRichard J. Webby\u003c/p\u003e\n\u003cp\u003eDepartment of Host\u0026ndash;Microbe Interactions, St. Jude Children\u0026rsquo;s Research Hospital,\u003c/p\u003e\n\u003cp\u003e262 Danny Thomas Place MS330, Memphis, Tennessee 38105-3678, USA\u003c/p\u003e\n\u003cp\u003ePhone: +1 (901) 595-3014\u003c/p\u003e\n\u003cp\u003eE-mail: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u0026amp; Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded in whole or in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract 75N93021C00016 and by St. Jude ALSAC. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We gratefully acknowledge the efforts of the GISAID Data Science Initiative and associated public databases that contributed to this study, and the US CDC for reagents including Astrakhan/3212 (H5N8) and wigeon/SC/345 (H5N1), and recombinant 6+2 ck/Ghana/21 (H5N1) AVL-763.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGlobal Influenza Program, W. H. O. (https://www.who.int/publications/m/item/cumulative-number-of-confirmed-human-cases-for-avian-influenza-a(h5n1)-reported-to-who-2003-2024, 2024).\u003c/li\u003e\n\u003cli\u003eCDC. \u003cem\u003eH5N1 Bird Flu Detections across the United States in Backyard and Commercial Poultry\u003c/em\u003e, \u0026lt;https://www.cdc.gov/bird-flu/situation-summary/data-map-commercial.html\u0026gt; (2024).\u003c/li\u003e\n\u003cli\u003eCaliendo, V.\u003cem\u003e et al.\u003c/em\u003e Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 11729 (2022). https://doi.org:10.1038/s41598-022-13447-z\u003c/li\u003e\n\u003cli\u003ePohlmann, A.\u003cem\u003e et al.\u003c/em\u003e Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e0060922 (2022). https://doi.org:10.1128/mbio.00609-22\u003c/li\u003e\n\u003cli\u003eKandeil, A.\u003cem\u003e et al.\u003c/em\u003e Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 3082 (2023). https://doi.org:10.1038/s41467-023-38415-7\u003c/li\u003e\n\u003cli\u003eYouk, S.\u003cem\u003e et al.\u003c/em\u003e H5N1 highly pathogenic avian influenza clade 2.3.4.4b in wild and domestic birds: Introductions into the United States and reassortments, December 2021-April 2022. \u003cem\u003eVirology\u003c/em\u003e \u003cstrong\u003e587\u003c/strong\u003e, 109860 (2023). https://doi.org:10.1016/j.virol.2023.109860\u003c/li\u003e\n\u003cli\u003ePlaza, P. I., Gamarra-Toledo, V., Eugui, J. R. \u0026amp; Lambertucci, S. A. Recent Changes in Patterns of Mammal Infection with Highly Pathogenic Avian Influenza A(H5N1) Virus Worldwide. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 444-452 (2024). https://doi.org:10.3201/eid3003.231098\u003c/li\u003e\n\u003cli\u003eNational Wildlife Health Center, U. Highly pathogenic avian influenza H5N1 virus (HPAIv) has been present in North America since its first detection in wild birds in November 2021 (Newfoundland and Labrador, Canada), and has subsequently spread across the Americas., (2023).\u003c/li\u003e\n\u003cli\u003ePuryear, W.\u003cem\u003e et al.\u003c/em\u003e Highly Pathogenic Avian Influenza A(H5N1) Virus Outbreak in New England Seals, United States. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 786-791 (2023). https://doi.org:10.3201/eid2904.221538\u003c/li\u003e\n\u003cli\u003eHarvey, J. A., Mullinax, J. M., Runge, M. C. \u0026amp; Prosser, D. J. The changing dynamics of highly pathogenic avian influenza H5N1: Next steps for management \u0026amp; science in North America. \u003cem\u003eBiol Conserv\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e (2023). https://doi.org:ARTN 110041 10.1016/j.biocon.2023.110041\u003c/li\u003e\n\u003cli\u003eMurawski, A.\u003cem\u003e et al.\u003c/em\u003e Highly pathogenic avian influenza A(H5N1) virus in a common bottlenose dolphin (Tursiops truncatus) in Florida. \u003cem\u003eCommun Biol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e (2024). https://doi.org:ARTN 476 10.1038/s42003-024-06173-x\u003c/li\u003e\n\u003cli\u003eBurrough, E. R.\u003cem\u003e et al.\u003c/em\u003e Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1335-1343 (2024). https://doi.org:10.3201/eid3007.240508\u003c/li\u003e\n\u003cli\u003eHu, X.\u003cem\u003e et al.\u003c/em\u003e Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2380421 (2024). https://doi.org:10.1080/22221751.2024.2380421\u003c/li\u003e\n\u003cli\u003eSpackman, E.\u003cem\u003e et al.\u003c/em\u003e Characterization of highly pathogenic avian influenza virus in retail dairy products in the US. \u003cem\u003eJ Virol\u003c/em\u003e (2024). https://doi.org:10.1128/jvi.00881-24\u003c/li\u003e\n\u003cli\u003eFDA. \u003cem\u003eUpdates on Highly Pathogenic Avian Influenza (HPAI)\u003c/em\u003e, \u0026lt;https://www.fda.gov/food/alerts-advisories-safety-information/updates-highly-pathogenic-avian-influenza-hpai#secondtesting\u0026gt; (2024).\u003c/li\u003e\n\u003cli\u003eUyeki, T. M.\u003cem\u003e et al.\u003c/em\u003e Highly Pathogenic Avian Influenza A(H5N1) Virus Infection in a Dairy Farm Worker. \u003cem\u003eNew Engl J Med\u003c/em\u003e (2024). https://doi.org:10.1056/NEJMc2405371\u003c/li\u003e\n\u003cli\u003eCDC. \u003cem\u003eCDC Confirms Second Human H5 Bird Flu Case in Michigan; Third Case Tied to Dairy Outbreak\u003c/em\u003e, \u0026lt;https://www.cdc.gov/media/releases/2024/p0530-h5-human-case-michigan.html\u0026gt; (2024).\u003c/li\u003e\n\u003cli\u003eCDC. \u003cem\u003eCDC Reports Fourth Human Case of H5 Bird Flu Tied to Dairy Cow Outbreak\u003c/em\u003e, \u0026lt;https://www.cdc.gov/media/releases/2024/p-0703-4th-human-case-h5.html#:~:text=This%20is%20the%20fourth%20case,for%20A(H5N1)%20virus.\u0026gt; (2024).\u003c/li\u003e\n\u003cli\u003eCDC. \u003cem\u003eH5 Bird Flu: Current Situation\u003c/em\u003e, \u0026lt;https://www.cdc.gov/bird-flu/situation-summary/index.html\u0026gt; (2024).\u003c/li\u003e\n\u003cli\u003eRogers, G. N. \u0026amp; Paulson, J. C. Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin. \u003cem\u003eVirology\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 361-373 (1983). https://doi.org:10.1016/0042-6822(83)90150-2\u003c/li\u003e\n\u003cli\u003eLin, T. H.\u003cem\u003e et al.\u003c/em\u003e A single mutation in bovine influenza H5N1 hemagglutinin switches specificity to human receptors. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e386\u003c/strong\u003e, 1128-1134 (2024). https://doi.org:10.1126/science.adt0180\u003c/li\u003e\n\u003cli\u003eSwayne, D. E. \u0026amp; Suarez, D. L. Highly pathogenic avian influenza. \u003cem\u003eRev Sci Tech\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 463-482 (2000). https://doi.org:10.20506/rst.19.2.1230\u003c/li\u003e\n\u003cli\u003eAndreev, K.\u003cem\u003e et al.\u003c/em\u003e Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Circulating Globally in 2022-2023. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e229\u003c/strong\u003e, 1830-1835 (2023). https://doi.org:10.1093/infdis/jiad418\u003c/li\u003e\n\u003cli\u003eOmoto, S.\u003cem\u003e et al.\u003c/em\u003e Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 9633 (2018). https://doi.org:10.1038/s41598-018-27890-4\u003c/li\u003e\n\u003cli\u003eAndreev, K.\u003cem\u003e et al.\u003c/em\u003e Genotypic and phenotypic susceptibility of emerging avian influenza A viruses to neuraminidase and cap-dependent endonuclease inhibitors. \u003cem\u003eAntiviral Res\u003c/em\u003e \u003cstrong\u003e229\u003c/strong\u003e, 105959 (2024). https://doi.org:10.1016/j.antiviral.2024.105959\u003c/li\u003e\n\u003cli\u003eNeuzil, K. M.\u003cem\u003e et al.\u003c/em\u003e Safety and Immunogenicity of Influenza A/H5N8 Virus Vaccine in Healthy Adults: Durability and Cross-reactivity of Antibody Responses. \u003cem\u003eClin Infect Dis\u003c/em\u003e (2023). https://doi.org:10.1093/cid/ciac982\u003c/li\u003e\n\u003cli\u003eHobson, D., Curry, R. L., Beare, A. S. \u0026amp; Ward-Gardner, A. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. \u003cem\u003eJ Hyg (Lond)\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 767-777 (1972). https://doi.org:10.1017/s0022172400022610\u003c/li\u003e\n\u003cli\u003eCoudeville, L.\u003cem\u003e et al.\u003c/em\u003e Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model. \u003cem\u003eBMC Med Res Methodol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 18 (2010). https://doi.org:10.1186/1471-2288-10-18\u003c/li\u003e\n\u003cli\u003eKozlov, M. \u0026amp; Mallapaty, S. Bird flu outbreak in US cows: why scientists are concerned. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e628\u003c/strong\u003e, 484-485 (2024). https://doi.org:10.1038/d41586-024-01036-1\u003c/li\u003e\n\u003cli\u003eNguyen, T.-Q.\u003cem\u003e et al.\u003c/em\u003e Emergence and interstate spread of highly pathogenic avian influenza A(H5N1) in dairy cattle. \u003cem\u003ebioRxiv\u003c/em\u003e, 2024.2005.2001.591751 (2024). https://doi.org:10.1101/2024.05.01.591751\u003c/li\u003e\n\u003cli\u003eGu, C.\u003cem\u003e et al.\u003c/em\u003e A human isolate of bovine H5N1 is transmissible and lethal in animal models. \u003cem\u003eNature\u003c/em\u003e (2024). https://doi.org:10.1038/s41586-024-08254-7\u003c/li\u003e\n\u003cli\u003ePulit-Penaloza, J. A.\u003cem\u003e et al.\u003c/em\u003e Transmission of a human isolate of clade 2.3.4.4b A(H5N1) virus in ferrets. \u003cem\u003eNature\u003c/em\u003e (2024). https://doi.org:10.1038/s41586-024-08246-7\u003c/li\u003e\n\u003cli\u003eSantos, J. J. S.\u003cem\u003e et al.\u003c/em\u003e Bovine H5N1 influenza virus binds poorly to human-type sialic acid receptors. \u003cem\u003ebioRxiv\u003c/em\u003e, 2024.2008.2001.606177 (2024). https://doi.org:10.1101/2024.08.01.606177\u003c/li\u003e\n\u003cli\u003eChopra, P.\u003cem\u003e et al.\u003c/em\u003e Receptor Binding Specificity of a Bovine A(H5N1) Influenza Virus. \u003cem\u003ebioRxiv\u003c/em\u003e, 2024.2007.2030.605893 (2024). https://doi.org:10.1101/2024.07.30.605893\u003c/li\u003e\n\u003cli\u003eHalwe, N. J.\u003cem\u003e et al.\u003c/em\u003e H5N1 clade 2.3.4.4b dynamics in experimentally infected calves and cows. \u003cem\u003eNature\u003c/em\u003e (2024). https://doi.org:10.1038/s41586-024-08063-y\u003c/li\u003e\n\u003cli\u003eNelli, R. K.\u003cem\u003e et al.\u003c/em\u003e Sialic Acid Receptor Specificity in Mammary Gland of Dairy Cattle Infected with Highly Pathogenic Avian Influenza A(H5N1) Virus. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1361-1373 (2024). https://doi.org:10.3201/eid3007.240689\u003c/li\u003e\n\u003cli\u003eRios Carrasco, M., Grone, A., van den Brand, J. M. A. \u0026amp; de Vries, R. P. The mammary glands of cows abundantly display receptors for circulating avian H5 viruses. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, e0105224 (2024). https://doi.org:10.1128/jvi.01052-24\u003c/li\u003e\n\u003cli\u003eEisfeld, A. J.\u003cem\u003e et al.\u003c/em\u003e Pathogenicity and transmissibility of bovine H5N1 influenza virus. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e633\u003c/strong\u003e, 426-432 (2024). https://doi.org:10.1038/s41586-024-07766-6\u003c/li\u003e\n\u003cli\u003eJassem, A. N.\u003cem\u003e et al.\u003c/em\u003e Critical Illness in an Adolescent with Influenza A(H5N1) Virus Infection. \u003cem\u003eN Engl J Med\u003c/em\u003e (2024). https://doi.org:10.1056/NEJMc2415890\u003c/li\u003e\n\u003cli\u003eMcCullers, J. A. Preparing for the next influenza pandemic. \u003cem\u003ePediatr Infect Dis J\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, S57-59 (2008). https://doi.org:10.1097/INF.0b013e3181684d41\u003c/li\u003e\n\u003cli\u003eKhurana, S.\u003cem\u003e et al.\u003c/em\u003e Licensed H5N1 vaccines generate cross-neutralizing antibodies against highly pathogenic H5N1 clade 2.3.4.4b influenza virus. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 2771-2776 (2024). https://doi.org:10.1038/s41591-024-03189-y\u003c/li\u003e\n\u003cli\u003eZhang, X. \u0026amp; Ross, T. M. Anti-neuraminidase immunity in the combat against influenza. \u003cem\u003eExpert Rev Vaccines\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 474-484 (2024). https://doi.org:10.1080/14760584.2024.2343689\u003c/li\u003e\n\u003cli\u003eNguyen, H. T.\u003cem\u003e et al.\u003c/em\u003e Antiviral susceptibility of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses isolated from birds and mammals in the United States, 2022. \u003cem\u003eAntiviral Res\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 105679 (2023). https://doi.org:10.1016/j.antiviral.2023.105679\u003c/li\u003e\n\u003cli\u003eBaker, A. L.\u003cem\u003e et al.\u003c/em\u003e Dairy cows inoculated with highly pathogenic avian influenza virus H5N1. \u003cem\u003eNature\u003c/em\u003e (2024). https://doi.org:10.1038/s41586-024-08166-6\u003c/li\u003e\n\u003cli\u003eLDH. \u003cem\u003eLDH reports first U.S. H5N1-related human death\u003c/em\u003e, \u0026lt;https://ldh.la.gov/news/H5N1-death\u0026gt; (2025).\u003c/li\u003e\n\u003cli\u003eScott Krauss, D. W., Robert G. Webster. in \u003cem\u003eInfluenza Virus\u003c/em\u003e Vol. 865 \u003cem\u003eMethods in Molecular Biology\u003c/em\u003e (ed Gabriele Neumann Yoshihiro Kawaoka) Ch. 2, 234 (Humana Totowa, 2012).\u003c/li\u003e\n\u003cli\u003eReed, L. J., Muench, H. A Simple Method of Estimating Fifty Per Cent Endpoints. \u003cem\u003eAmerican Journal of Epidemiology\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 493-497 (1938). https://doi.org:10.1093/oxfordjournals.aje.a118408\u003c/li\u003e\n\u003cli\u003eREED, L. J. \u0026amp; MUENCH, H. A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS12. \u003cem\u003eAmerican Journal of Epidemiology\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 493-497 (1938). https://doi.org:10.1093/oxfordjournals.aje.a118408\u003c/li\u003e\n\u003cli\u003eMaher, J. A. \u0026amp; DeStefano, J. The ferret: An animal model to study influenza virus. \u003cem\u003eLab Animal\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 50-+ (2004). https://doi.org:DOI 10.1038/laban1004-50\u003c/li\u003e\n\u003cli\u003eWHO. \u003cem\u003eSummary of polymerase acidic (PA) protein amino acid substitutions analysed for their effects on baloxavir susceptibility\u003c/em\u003e, \u0026lt;https://www.who.int/publications/m/item/summary-of-polymerase-acidic-(pa)-protein-amino-acid-substitutions-analysed-for-their-effects-on-baloxavir-susceptibility\u0026gt; (2024).\u003c/li\u003e\n\u003cli\u003eWHO. \u003cem\u003eSummary of neuraminidase (NA) amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors (NAIs) among avian influenza viruses of Group 1 and Group 2 NAs\u003c/em\u003e, \u0026lt;https://www.who.int/publications/m/item/summary-of-neuraminidase-(na)-amino-acid-substitutions-associated-with-reduced-inhibition-by-neuraminidase-inhibitors-(nais)-among-avian-influenza-viruses-of-group-1-and-group-2-nas\u0026gt; (2024).\u003c/li\u003e\n\u003cli\u003eKatoh, K., Misawa, K., Kuma, K. \u0026amp; Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 3059-3066 (2002). https://doi.org:10.1093/nar/gkf436\u003c/li\u003e\n\u003cli\u003eLeang, S. K. \u0026amp; Hurt, A. C. Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals. \u003cem\u003eJove-J Vis Exp\u003c/em\u003e (2017). https://doi.org:ARTN e55570 10.3791/55570\u003c/li\u003e\n\u003cli\u003eMarathe, B. M., L\u0026eacute;v\u0026ecirc;que, V., Klumpp, K., Webster, R. G. \u0026amp; Govorkova, E. A. Determination of Neuraminidase Kinetic Constants Using Whole Influenza Virus Preparations and Correction for Spectroscopic Interference by a Fluorogenic Substrate. \u003cem\u003ePlos One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2013). https://doi.org:ARTN e71401 10.1371/journal.pone.0071401\u003c/li\u003e\n\u003cli\u003ePatel, M. C.\u003cem\u003e et al.\u003c/em\u003e An optimized cell-based assay to assess influenza virus replication by measuring neuraminidase activity and its applications for virological surveillance. \u003cem\u003eAntivir Res\u003c/em\u003e \u003cstrong\u003e208\u003c/strong\u003e (2022). https://doi.org:ARTN 105457 10.1016/j.antiviral.2022.105457\u003c/li\u003e\n\u003cli\u003eCouzens, L.\u003cem\u003e et al.\u003c/em\u003e An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. \u003cem\u003eJ Virol Methods\u003c/em\u003e \u003cstrong\u003e210\u003c/strong\u003e, 7-14 (2014). https://doi.org:10.1016/j.jviromet.2014.09.003\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInfluenza viruses, including bovine A(H5N1) clade 2.3.4.4b viruses used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfluenza A virus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubtype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHA clade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbbreviation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenotypes\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eReference CVV\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/Astrakhan/3212/2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAstrakhan/3212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Ghana/AVL-76321VIR7050-39/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eck/Ghana/39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/American wigeon/South Carolina/22-000345-001/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ewigeon/SC/345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bald eagle/Florida/W22-134-OP/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eeagle/FL/W22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/gyrfalcon/Washington/41088-6/2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003egyrfalcon/41088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBovine and avian influenza viruses\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-302/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-342/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-358/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-368/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-432/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-439/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-497/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Ohio/B24OSU-541/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/OH/541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Texas/3301/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/TX/38301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Texas/38161/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/TX/38161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Texas/40106/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/TX/40106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Texas/42041/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/TX/42041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Texas/43134/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/TX/43134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/bovine/Texas/97794/2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebovine/TX/97794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/Ross\u0026rsquo;s goose/Kansas/W23-930F/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003egoose/KS/930F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinor60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/snow goose/Louisiana/W23-957/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003egoose/LA/957\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB3.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHuman influenza virus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/California/04/2009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH1N1pdm09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCA/09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eGenotype of A(H5N1) virus was conducted using GenoFLU\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003csup\u003eb\u003c/sup\u003eNA, not assigned.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhenotypic NAI and CENI susceptibility of bovine HPAI A(H5N1) 2.3.4.4b viruses\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInfluenza A virus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCENI baloxavir EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;SD [nM] (fold change)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eNAI, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;SD [nM] (fold change)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOseltamivir\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZanamivir\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePeramivir\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/TX/38161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/TX/97794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/TX/43134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/TX/42041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/OH/302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/OH/358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/OH/439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebovine/OH/541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMean values from two independent experiments are provided \u0026plusmn;\u0026thinsp;the standard deviation (SD).\u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eRelative to the baseline susceptibility of North American HPAI A(H5N1) clade 2.3.4.4b viruses\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5806806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5806806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn March 2024, clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses were first detected in U.S. dairy cattle. Similar viruses have since caused 66 zoonotic human infections. To assess changes to zoonotic potential, we characterized A(H5N1) clade 2.3.4.4b viruses isolated from cows\u0026rsquo; milk and birds. Bovine-derived viruses were lethal in mice and ferrets and transmitted to direct but not airborne contact ferrets. All viruses replicated in human bronchial epithelial cells despite preferentially binding avian virus-like receptors. The bovine-derived viruses remained susceptible to FDA-approved antivirals and were neutralized by sera from ferrets vaccinated with WHO CVVs or humans vaccinated with clade 2.3.4.4c vaccine. While 2.3.4.4b viruses induce severe disease in mammalian models, they retain many avian virus-like characteristics. Combined, we conclude that the risk of contemporary bovine-derived viruses to humans not in contact with affected animals is low. However, heightened vigilance remains essential to promptly detect and respond to any changes.\u003c/p\u003e","manuscriptTitle":"Influenza A(H5N1) Viruses Isolated From Dairy Cattle Demonstrate High Virulence in Laboratory Models, but Retain Avian Virus-like Properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 06:05:08","doi":"10.21203/rs.3.rs-5806806/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fba0b8b9-19f7-4f01-a6bb-a5180e23d88b","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43860486,"name":"Biological sciences/Microbiology/Virology/Influenza virus"},{"id":43860487,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2025-07-24T08:01:56+00:00","versionOfRecord":{"articleIdentity":"rs-5806806","link":"https://doi.org/10.1038/s41467-025-61757-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-23 04:00:00","publishedOnDateReadable":"July 23rd, 2025"},"versionCreatedAt":"2025-02-05 06:05:08","video":"","vorDoi":"10.1038/s41467-025-61757-3","vorDoiUrl":"https://doi.org/10.1038/s41467-025-61757-3","workflowStages":[]},"version":"v1","identity":"rs-5806806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5806806","identity":"rs-5806806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}