#Genome-informed structural #analysis of #polymerase and glycoprotein #adaptation in #H5N1 clade 2.3.4.4b

 

Abstract

Importance

Understanding the molecular mechanisms driving H5N1 clade 2.3.4.4b is critical for pandemic preparedness.

Objective

To characterize the molecular drivers of viral fitness and mammalian adaptability in recent H5N1 viruses by integrating evolutionary dynamics with structural simulations.

Methods

This study analyzed 2,398 H5Nx genomes (2000–2024) through phylogenetic and selective pressure analyses. HA/NA structures were predicted with AlphaFold 3 and evaluated by AutoDock4 docking, whereas polymerase–ANP32A/B complexes were modeled using template-based methods and their binding free energies were estimated using MM/GBSA. Polymerase–ANP32E complexes were predicted with AlphaFold 3 and similarly evaluated by MM/GBSA. The binding affinities (ΔG) for the sialic acid (SA) receptors and human ANP32 proteins were quantified through molecular mechanics/generalized born surface area calculations.

Results

Clade 2.3.4.4b showed significant antigenic drift in the HA receptor binding site, reducing affinity for α2,3-SA and α2,6-SA receptors. On the other hand, the emergence of a full-length stalk N1 NA with second sialic acid-binding site mutations (e.g., N366S) compensated for reduced HA affinity by enhancing the NA binding stability. In the polymerase complex, both the PB2-627E/631L variant (−144.00 kcal/mol; unadjusted p = 0.0058) and the known mammalian-adaptive 627K/631M variant (−144.67 kcal/mol; unadjusted p = 0.0165) showed more favorable predicted human ANP32B binding free energies than the ancestral 627E/631M state (−136.46 kcal/mol).

Conclusions and Relevance

The co-occurrence of HA, NA, PB1, and PB2 signatures was associated with clade expansion and produced structural predictions consistent with altered receptor or ANP32 interactions; experimental validation is required before inferring effects on fitness or zoonotic risk.

Source: 

Link: https://vetsci.org/DOIx.php?id=10.4142/jvs.26088

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#Influenza A virus #H5N1 genotypes #B3.13 and D1.1 show #temperature-dependent restriction of #replication in primary #human respiratory epithelial cell cultures derived from the upper and lower respiratory tract

 

Abstract

H5N1 clade 2.3.4.4b avian influenza A viruses pose a significant threat to wild animal populations, domesticated animals, and potentially, the human population. For H5N1s to infect and transmit among mammalian species, mutations for improved utilization of mammalian receptors and enhanced replication at the lower temperatures of the upper respiratory tract need to be acquired. A human H1N1pdm09-like virus was compared to H5N1 genotypes B3.13 and D1.1 for replication at 33ºC, 37ºC, and 39ºC – temperatures consistent with the upper and lower respiratory tract in humans, and dairy cow udder tissue. All H5N1 viruses had increased plaque sizes on MDCK cells at 37ºC and 39ºC compared to H1N1pdm09. In primary, differentiated human nasal and bronchial epithelial cultures, all H5N1 viruses show restricted infectious virus production compared to H1N1 at 33ºC. While H5N1 D1.1 also showed restricted replication at 37ºC and 39ºC, the H5N1 B3.13 replicated to nearly equivalent titers as H1N1pdm09. All H5N1 viruses demonstrated similar cell tropism in cells from the upper and lower respiratory tract, infecting more ciliated than non-ciliated cells relative to H1N1pdm09. H1N1, H5N1 B3.13 D1.1 infection induced similar innate immune factors, with nasal epithelial cells producing higher levels compared to bronchial epithelial cells. These data suggest that genotype B3.13 and D1.1 H5N1 viruses show different temperature dependent replication patterns compared to H1N1pdm09.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.08.27.747488v1

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West African Clade C #MERS-CoV Strains BF785 and Mor213 Induce More Robust #Interferon and Inflammatory Signaling Compared to Clade A in #Human Respiratory Cells

 

Abstract

Clade A Middle East respiratory syndrome coronavirus (MERS-CoV) spilled over from camels into humans in Saudi Arabia in 2012 and caused pneumonia and severe respiratory disease, with a 37% case fatality rate. While clade A and B viruses are closely related phylogenetically, clade B MERS-CoV strains have since outcompeted clade A strains and continue to circulate in camels and humans in the Middle East and cause outbreaks in humans. Clade C strains circulate in camels across the African continent but have not been reported to cause disease in humans. We have shown that a number of East African clade C isolates are less able to utilize the TMPRSS2-mediated pathway for viral entry in both cell lines and primary nasal epithelial cultures, which may underlie the reduced replication of East African clade C strains in humans. However, the reduced replicative capacity of West African strains in human cells appears to be independent of viral entry, suggesting an alternative basis for their attenuation. Here, we report that West African clade C MERS-CoV isolates with deletions in ORF4b encoding a key accessory protein, NS4b, induced significantly more robust interferon and inflammatory responses than clade A MERS in human respiratory cell lines and primary bronchial air–liquid interface cultures. The replication deficit for the West African strain BF785, which has a complete deletion of ORF4b, was partially rescued when RNASEL was knocked out in A549 cells. These findings demonstrate that complete loss of NS4b results in stronger innate immune activation than partial truncation and suggests that differential selection on NS4b may contribute to the varying phenotypes of clade C MERS-CoV strains circulating in African camels.

Source: 

Link: https://www.mdpi.com/1999-4915/18/9/935

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Impaired #Neuraminidase and Polymerase Activities Correspond With Limited Aerosol Infectivity of #B3.13 and #D1.1 #H5N1 Lineages in #Human Respiratory Cultures

 

Abstract

The ongoing panzootic of clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 viruses has reached a critical point, marked by unprecedented mammalian spillover and sustained outbreaks in U.S. dairy cattle. While these viruses remain highly lethal in traditional ferret models, human infections-primarily linked to the B3.13 and D1.1 lineages-have been notably mild, typically presenting as conjunctivitis with minimal respiratory involvement. In this study, we address this disconnect by evaluating the infectivity of recent H5N1 isolates using a physiologically relevant air-liquid interface (ALI) culture system that incorporates an aerosol settling chamber. We demonstrate that while direct liquid inoculation leads to efficient replication, aerosolized H5N1 strains exhibit a significant defect in their ability to infect human respiratory epithelium. In contrast, a prototypic H5N1 virus remains highly pathogenic and lethal in ferrets regardless of the inoculation route, showing systemic dissemination to the brain and other organs. Our findings identify two primary viral determinants driving this respiratory restriction: reduced neuraminidase (NA) enzymatic activity and impaired polymerase activity. Collectively, these results suggest that commonly used mammalian models may overstate current human pandemic risk. This work highlights the critical need for alternative risk-assessment platforms to identify the specific genetic shifts required for these viruses to overcome existing barriers to human adaptation.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.08.18.745466v1

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A mouse #monoclonal #antibody against #H7N9 #influenza virus cross-react with #human #platelets

 

Highlights

    • H7N9–98 mAb specifically binds human platelets and targets a ~60 kDa platelet protein.

    • H7N9 virus induces ITP via molecular mimicry with platelet antigens.

    • H7N9 virus-related ITP correlates with disease severity.

Abstract

Objective

To investigate the association and potential mechanisms between H7N9 influenza virus infection or vaccination and immune thrombocytopenia (ITP), providing foundational data for the prevention and treatment of related ITP.

Methods

Using laboratory-prepared anti-H7N9 influenza virus monoclonal antibodies (mAbs) (H7N9-98 and H7N9-120) as research subjects, the interactions between these antibodies and human platelets were analyzed through Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) techniques.

Results

The mAb H7N9-98 exhibited specific binding to human platelets, showing positive results in both IHC and IF assays. Western blotting results demonstrated that this antibody could specifically recognize approximately 60 kDa human platelet proteins. The isotype control mAb H7N9-120 did not exhibit the aforementioned binding reactions, with all test results being negative.

Conclusion

These findings suggest that the specific antibodies induced by the H7N9 virus may mediate platelet damage through cross-reactivity with platelet autoantigens. This mechanism warrants further investigation to provide experimental evidence for the pathogenesis of secondary ITP associated with H7N9 infection.

Source: 

Link: https://www.sciencedirect.com/science/article/pii/S1567134826001346?via%3Dihub

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Emerging Haemagglutinin #Mutations in #Bovine-origin #H5N1 Influenza Viruses from #Humans and #Cattle Retain Avian #Receptor Binding with Increased Stability

 

Abstract

The current H5N1 panzootic has seen an unprecedented host range expansion, including sustained circulation in US dairy cattle, detected in March 2024. By July 2026, infections had been reported on more than 1,150 dairy farms across 19 states. Although the outbreak initially centred in Texas, California has emerged as the principal focus of transmission and accounts for most human infections associated with exposure to infected dairy cattle. Continued transmission in cattle and repeated spillover into humans increase opportunities for acquisition of mammalian-adaptive mutations that could elevate zoonotic and pandemic risk. The haemagglutinin (HA) protein plays a central role in modulating virus receptor binding and airborne transmission. Here, we characterised the receptor-binding and stability phenotypes of HA mutations identified in viruses circulating in Californian dairy cattle. Receptor-binding specificity was assessed using bio-layer interferometry and pseudotype virus entry assays. All tested HA variants maintained a preference for avian-type α2,3-linked sialic acid receptors. We evaluated HA stability using fusion and thermostability assays. All mutants exhibited fusion pH values >5.5, outside the range associated with efficient airborne transmission in humans (pH 5.0-5.5). However, mutations D88G and S94N increased pH stability, with fusion pH values of 5.6 and 5.7, respectively, compared with 5.9 for wild-type HA. Viruses harbouring both mutations displayed increased thermostability. These findings demonstrate that cattle-origin H5N1 viruses retain avian-like receptor specificity despite acquiring mutations that modestly enhance HA stability. Evolution of H5N1 viruses in dairy cattle underscores the importance of genomic and phenotypic surveillance to identify mutations that may increase zoonotic risk.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Biotechnology and Biological Sciences Research Council, BB/X006166/1, BB/Y007298/1, BB/X006204/1 BB/Y007271/1, APP104179,, BBS/E/PI/230001B, BBS/E/PI/230001C, BBS/E/PI/230002B, BBS/E/PI/230002C), BBS/E/PI/23NB0004, BBS/E/PI/23NB0003, BB/S011269/1

Medical Research Council, https://ror.org/03x94j517, MR/Y03368X/1,

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.08.18.745528v1

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#Maraviroc Inhibits #SARS-CoV-2 Through Variant-Dependent Effects on Viral Entry and #Mpro Activity Using Single-Round Infectious Particle and Virus-like Particle Models

 

Abstract

Maraviroc (MVC), a CCR5 antagonist, has been proposed as a potential antiviral agent against SARS-CoV-2; however, its mechanism of action across viral variants remains unclear. Here, we evaluated the antiviral activity of MVC against SARS-CoV-2 wild-type (WT) and Omicron BA.1 variants using single-round infectious particles (SRIPs), virus-like particles (VLPs), and cell-based assays, with a focus on its impact on viral entry and Mpro function. MVC potently inhibited infection of both WT and BA.1 SRIPs in Vero E6 cells, exhibiting EC50 values of 0.0065 μM and 0.016 μM, respectively. Time-of-addition assays revealed that MVC primarily targets the early phase of infection, with the strongest inhibition observed at the viral entry stage, while moderate effects were detected during attachment and post-entry stages. Fluorescence-labeled VLP imaging demonstrated distinct entry pathways, with WT predominantly entering via plasma membrane fusion and BA.1 via endocytosis, independent of cell type. MVC altered WT-VLP trafficking by promoting internalization and lysosomal localization, whereas it had minimal impact on BA.1 internalization. In spike-mediated cell–cell fusion assays, MVC preferentially inhibited WT spike-driven syncytium formation but showed limited effects on BA.1 or BA.4 fusion, while more effectively reducing Omicron spike-mediated binding. At the post-entry stage, MVC inhibited SARS-CoV-2 main protease (Mpro) activity, with BA.1 Mpro (P132H) exhibiting greater sensitivity (IC50 = 0.496 µM) than WT (1.869 µM). Collectively, these findings demonstrate that MVC exerts variant-dependent antiviral effects by targeting viral entry, modulating trafficking pathways, and inhibiting Mpro activity. This study highlights MVC as a multi-stage inhibitor with differential efficacy against SARS-CoV-2 variants, providing insights into its potential therapeutic application.

Source: 

Link: https://www.mdpi.com/1999-4915/18/8/911

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Enhanced #Pathogenicity and Contact #Transmissibility of #Human-origin Avian #Influenza #H5N1 Clade 2.3.4.4b Genotype B3.13 Compared to D1.1 in #Ferrets

 

Abstract

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness.

Competing Interest Statement

The A.G.-S. laboratory has received research support from Avimex, Dynavax, Pharmamar, and Accurius, outside of the reported work within the last three years. A.G.-S. has consulting agreements for the following companies involving cash and/or stock within the last three years: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, Avimex, Pagoda, Accurius, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Virofend, Prosetta and A.A.C.T., outside of the reported work. A.G.-S. has been an invited speaker in meeting events within the last three years organized by Seqirus, Novavax and Hipra. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The Icahn School of Medicine at Mount Sinai has licensed some of these inventions to Medimmune, Avimex, Leinco Technologies, Castlevax, Virofend, Kerafast, Cell Signaling, EMD Millipore, Genentech, Paratus and Nura Bio, and as a result receives financial compensation. Subject to Mount Sinai receiving such financial consideration, AG-S will receive a portion of that consideration pursuant to the terms of the Mount Sinai Intellectual Property Policy. All other authors declare no commercial or financial conflict of interest.

Funder Information Declared

NIH/NIAID, 75N93021C00014

Horizon Europe Program, KAPPA-FLU no. 101084171

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.08.10.744032v1

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Identification and characterization of #PB2 #mutations associated with #mammalian #adaptation of highly pathogenic #H5N1 avian #influenza viruses

 

Abstract

The highly pathogenic avian influenza virus (HPAIV) subtype H5N1 has been continuously circulating among wild bird populations and domestic poultry. It’s ongoing circulation has led to outbreaks in poultry and U.S. dairy cattle populations, as well as sporadic severe infections in individuals engaged in poultry and dairy farming. These occurrences have raised concerns about the potential evolution of this virus into a pandemic strain. To elucidate the molecular determinants facilitating H5N1 cross-species adaptation and to evaluate its implications for public health, we conducted serials of sequence analysis and specific-site mutations on the viral polymerase subunit PB2 to determine its effect on polymerase activity and viral infectivity. The results showed that three mutations in the PB2 protein (E362G, D441N and M631L) were presented cooperative effects associated with enhanced viral replication in mammalian cells. Compared to the original isolated strain of the 2.3.4.4b clade, A/chicken/NL/FAV-0033/2021, these three mutations were predominantly identified in isolates obtained from cattle and other mammalian hosts between 2021 and 2024. The M631L mutation, identified as the primary determinant of increased polymerase activity in mammalian cells, significantly enhanced the binding affinity of PB2 to ANP32A. The mutation E362G and D441N did not increased polymerase activity and viral replication significantly but enhanced binding affinity of PB2 to ANP32A. The combined mutations with E362G, D441N and M631L resulted in a significantly increased polymerase activity and viral replication in H5N1 virus, and significantly elevated viral loads and aggravated pulmonary pathology in lungs of mice with H5N1 infection. These findings indicate that the PB2-M631L mutation constitutes a crucial molecular marker for the adaptation of H5N1 to mammalian hosts, whereas the E362G and D441N mutations likely function as supportive modulatory factors that optimize this host-adaptation process.

Source: 

Link: https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2026.1867604/full

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Loss-of-function #mutation in #Omicron #variants reduces #spike protein expression and attenuates #SARS-CoV-2 infection

 

Abstract

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel mutations in the spike alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. Here, we examine three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we find that the mutant increases spike processing, consistent with prior reports for H655Y/P681H. In addition, the YKH mutant induces attenuated disease, but augments viral loads in male golden Syrian hamsters. Next, we generate a single N679K mutant, finding it reduces viral replication in Calu3 human respiratory cells and induces less disease in male golden Syrian hamsters. Mechanistically, the N679K mutant has increased spike processing but also reduces spike in purified virions; spike decreases are further exacerbated in infected Calu3 cell lysates. Importantly, exogenous spike expression reveals that N679K reduces overall spike protein in the context of the epidemic strain. Although a loss-of-function mutation, transmission competition demonstrates that N679K confers a replication advantage in the upper airway, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein during Omicron infection, which has implications for infection, immunity, and transmission.

Source: 

Link: https://www.nature.com/articles/s41467-026-76680-4

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#Mutations in severe #human #H5N1 cases facilitate #evasion from human mucus and #antivirals

 

Abstract

In late 2024, two individuals in Canada and the United States were treated in intensive care for acute respiratory distress caused by infection with the avian Influenza A Virus H5N1 2.3.4.4b genotype D1.1. Viral sequence data obtained from sampling these patients indicated mixed alleles at haemagglutinin (HA) positions 190 and 226. Mutations at these positions are key determinants of HA usage of α2,6-linked sialic acids (SA), the most abundant influenza receptors in human upper respiratory tracts. Thus, these mutations raised concerns about human adaptation and pandemic potential of the H5N1 virus. In this study, we investigated the impact of the mutations at residues 190 and 226 in H5 HA. We studied the receptor binding properties, cell entry phenotypes and fitness impacts of the mutations using recombinant proteins, pseudotyped lentiviruses, and in the context of influenza viruses using reverse genetics. The mutations did not confer any detectable α2,6-linked sialic acid receptor usage either alone or in combination. Rather, viruses carrying these mutations exhibit weakened binding towards α2,3-linked sialic acid receptors. This correlated with an enhanced capacity to evade human airway mucus, and a reduced susceptibility to oseltamivir and zanamivir. This research underscores that in addition to the way HA interacts with SA as entry receptors, other factors that impact the HA/NA balance might influence the evolutionary trajectory of a zoonotic virus in the human respiratory tract. This study presents a new paradigm for the evolutionary drivers of HA, where reduced sialic acid binding can serve as an advantage for escape from host barriers and antivirals.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Medical Research Council, https://ror.org/03x94j517, MR/Y03368X/1, MR/Y015061/1, CC2127

Biotechnology and Biological Sciences Research Council, BB/Y007298/1, APP104179, BBS/E/PI/23NB000, BBS/E/PI/23NB0003

Wellcome Trust, https://ror.org/029chgv08, CC2127, 218304/Z/19/Z

Department for Environment Food and Rural Affairs, BB/Y007298/1

The Pirbright Institute, BBS/E/PI/230002A, BBS/E/PI/230001C, BBS/E/PI/230002B

Cancer Research UK, CC2127

UK Research and Innovation, https://ror.org/001aqnf71, UKRI3602

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.28.740943v1

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#H5N1 #influenza binding and cell entry via #human class II #MHC, and blocking by cross-reactive #antibodies

 

Abstract

Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses are currently responsible for a multi-species outbreak affecting wild birds, poultry, numerous mammalian species, and humans. Influenza A viruses typically initiate infection through binding to sialic acid, although select bat and human influenza viruses can also exploit class II major histocompatibility complex (MHC–II) molecules for cell entry. Here we show that emerging H5N1 clade 2.3.4.4b viruses, but not historical H5 lineages, bind human MHC–II HLA-DR and mediate sialic acid-independent cell entry. Hemagglutinin binding to primary human immune cells varies with MHC–II expression and is further shaped by HLA–DR allelic variation, identifying host genetic determinants that may influence susceptibility to infection. Mammalian-adaptive substitutions within the hemagglutinin sialic acid receptor-binding domain reduce MHC–II binding, suggesting this interaction is remodeled during clade 2.3.4.4b H5 adaptation to a human host. Lastly, cross-reactive monoclonal antibodies isolated from clade 2.3.4.4b H5–naive humans can block the hemagglutinin–MHC–II interaction. These findings identify a previously unrecognized receptor pathway in contemporary H5N1 viruses and reveal that both human genetic variation and pre-existing humoral immunity can modulate this interaction, with implications for host range, cellular tropism, spillover risk, and therapeutic intervention.

Competing Interest Statement

S.D.B. has consulted for Regeneron, Sanofi, Novartis, Genentech, Pfizer, Visterra, and Otsuka on topics unrelated to the research presented here; owns stock in AbCellera Biologics; and is a scientific cofounder of Immunera, Inc.; S.E.H reports receiving consulting fees from Sanofi, Pfizer, Lumen, Novavax, and Merck.

Funder Information Declared

NIH/NIAID CEIRR contract, 75N93021C00015

NIH, 1U54CA260517

HIPC, U19AI057266

P01 grant, 5P01AI153559

David Crown Foundation endowment

Early Postdoc Mobility Fellowship Stipend from the Swiss

National Institutes of Health NRSA T32, T32OD011121

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.22.739677v1

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#MHC-II acts as a #fusion-triggering #receptor for #bat #influenza virus

 

Abstract

Influenza A virus hemagglutinin is a prototypical class I viral fusion protein that binds sialylated glycans and is activated by low pH in endosomes. In contrast, bat-derived IAV subtypes H17N10 and H18N11 use major histocompatibility complex class II (MHC-II) as an entry receptor, but how this receptor contributes to membrane fusion remains unknown. We find that MHC-II-dependent hemagglutinin subtypes H17, H18, and H19 possess an increased negative net charge relative to canonical HAs. Using cryo-electron tomography, we demonstrate that H18N11 morphology remains stable and H18 is in prefusion conformation at strongly acidic pH. Remarkably, H18 undergoes fusion-relevant conformational changes only when both MHC-II binding and low pH are present. By reconstitution of H18N11 fusion with liposomes and purified MHC-II, we show that receptor engagement is required to trigger the fusion activity of H18. These findings identify MHC-II as a receptor that directly triggers membrane fusion and reveal a previously unrecognized receptor-dependent mechanism of influenza virus entry.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 240245660 – P19, 537227910

European Research Council, 882631—Bat Flu

Excellence Initiative of the German Research Foundation, GSC-4, Spemann Graduate School

Hans A. Krebs Medical Scientist Programme

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.08.737388v1

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Lactylation of #Influenza Virus #Polymerase Acidic Protein Promotes Viral #Replication and #Pathogenicity

 

Abstract

Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

Competing Interest Statement

The authors have declared no competing interest.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.10.737663v1

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Isolation and characterization of a clade 2.3.4.4b genotype #D1.1 #H5N1 virus from dairy #cattle in #Wisconsin

 

ABSTRACT

Highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses of clade 2.3.4.4b have recently been detected in U.S. dairy cattle following multiple spillover events from avian reservoirs. In December 2025, HPAI H5N1 virus was identified in a dairy herd in Wisconsin through the National Milk Testing Strategy. Here, we report the isolation of a clade 2.3.4.4b, genotype D1.1 H5N1 virus, A/dairy cow/Wisconsin/25G05743-001/2025 (WI5743-H5N1), from bulk milk associated with the affected herd, describe its phylogenetic relationships, and assess its pathogenicity in mice. Infectious virus was recovered following blind passage in embryonated chicken eggs. Phylogenetic analysis demonstrated that WI5743-H5N1 is distinct from previously reported D1.1 viruses detected in dairy cattle in Nevada and Arizona, supporting an independent introduction into cattle, and indicating a likely local avian source. Compared with closely related avian viruses, WI5743-H5N1 encoded the mammalian-adapting substitution PB2-E627K and additional amino acid differences in HA, PB1-F2, and NS1. In mice, WI5743-H5N1 replicated efficiently in respiratory tissues and was detectable in the brain but exhibited lower lethality relative to other recent clade 2.3.4.4b, genotype B3.13 viruses. Together, these findings highlight the genetic and phenotypic diversity of HPAI H5N1 viruses infecting dairy cattle and underscore the importance of continued surveillance and functional characterization of emerging strains.

IMPORTANCE

Highly pathogenic avian influenza A(H5N1) viruses have recently entered U.S. dairy cattle through multiple spillover events from avian reservoirs, creating new opportunities for viral adaptation in mammals. Here, we describe the isolation and characterization of a clade 2.3.4.4b, genotype D1.1 H5N1 virus from bulk milk collected during a spillover event in Wisconsin in December 2025. Phylogenetic analyses demonstrated that this virus represents an independent introduction into dairy cattle distinct from previously reported D1.1 viruses identified in Nevada and Arizona. Although the virus encoded the mammalian-adapting PB2-E627K substitution, it exhibited comparatively low lethality in mice, highlighting the complexity of mammalian adaptation and pathogenicity in H5N1 viruses. These findings expand current understanding of the genetic and phenotypic diversity of H5N1 viruses infecting dairy cattle and emphasize the importance of continued surveillance and functional characterization of emerging strains.

Source: 

Link: https://journals.asm.org/doi/10.1128/jvi.00761-26

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#Cattle and #human #organoids reveal 2.3.4.4b #H5N1 cross-species #transmission potential and #neuraminidase-specific neutralizing #antibodies in humans

 

Abstract

The unexpected circulation of clade 2.3.4.4b H5N1 influenza viruses in dairy cattle and the transmission to diverse mammalian species poses a pandemic risk. We sought to explore cattle and human respiratory susceptibility to the 2.3.4.4b H5N1 virus. We establish long-term expandable cattle airway and mammary organoids. The 2.3.4.4b H5N1 virus exhibits high replicative fitness in cattle mammary organoids, recapitulating its remarkable mammary tropism. The virus also replicates robustly in cattle airway organoids, suggesting an underrecognized respiratory component in ongoing outbreaks. Interestingly, human airway and nasal organoids are highly susceptible to the 2.3.4.4b H5N1 virus. Yet, a novel organoid-based neutralization assay reveals that N1 antibodies in human sera had cross-neutralizing activity against the 2.3.4.4b H5N1 and ancestral H5N1-VN1194 viruses. The cross-neutralization, exclusively manifested in the organoid-based assay, is enhanced after seasonal influenza vaccination and diminished after depleting N1-specific antibodies. Therefore, cross-neutralizing N1 antibodies are likely limiting zoonotic infection by H5N1 viruses in humans.

Source: 

Link: https://www.nature.com/articles/s41467-026-74345-w

____

#Assessment of #influenza virus and #coronavirus #tropism, #replication competence and disease severity in ex vivo and in vitro cultures of the #human respiratory tract

ABSTRACT

The emergence of animal influenza viruses circulating in poultry and human populations poses a significant public health threat, yet current risk assessment tools that connect surveillance data to human transmission risk and disease severity are lacking. To address this, we employed a semi-quantitative approach to analyze virus tropism and replication competence, conducting risk assessments of influenza and coronavirus adaptation to human transmission in an ex vivo model, and evaluating virus-induced impairment of alveolar fluid clearance (AFC) in vitro as a correlation of disease severity. Our results showed that seasonal influenza A H1N1, H3N2, influenza B, MERS-CoV, and SARS-CoV exhibited productive viral replication and tissue infection in bronchial tissues, whereas wild bird surveillance isolates such as H5N3 and H7N1 showed minimal replication when compared to pandemic H1N1 and highly pathogenic avian influenza (HPAI) H5N1. Notably, differential lung viral replication and tissue tropism were detected for H5N6 and H9N2. HPAI H5N1, H7N9, MERS-CoV, and SARS-CoV caused more severe AFC impairment than seasonal H1N1, H3N2, and influenza B viruses, correlating with their clinical severity. Overall, these findings revealed an important association between viral tropism and human transmissibility in ex vivo explants, as well as the impairment of AFC in vitro, which aligns with the clinical manifestations of disease severity across different viral strains.

Source: 

Link: https://www.microbiologyresearch.org/content/journal/jgv/10.1099/jgv.0.002281

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A #bovine #H5N1 virus efficiently replicates in differentiated #human #nasal epithelial #cells

 

Abstract

Highly pathogenic avian influenza (H5N1) viruses of clade 2.3.4.4b have caused significant losses in bird populations worldwide and repeatedly infected mammals, including humans, without sustained human to human transmission. Here we show that an H5N1 virus (H5N1Tex/24) isolated from bovine milk in Texas in 2024 replicates just as efficiently in differentiated human nasal epithelial cells as a pandemic H1N1 virus strain from 2009 (H1N1HH4/09), at both 37 °C and 33 °C. The adaptive mutations PB2 M631L and PA K497R promoted replication at 33 °C but had no effect on replication at 37 °C. An H5N1 virus (H5N1BE/22) isolated from a pelican in 2022, which lacked these mutations, replicated efficiently at 37 °C but poorly at 33 °C, and this limitation was not overcome by the introduction of the PB2 M631L and PA K497R mutations. The differentiated nasal epithelial cell cultures expressed receptors for both human and avian influenza viruses. Accordingly, no HA mutations associated with altered receptor specificity were detected. H5N1Tex/24 was able to effectively suppress the production of interferon-λ, yet remained sensitive to the antiviral effects of this cytokine. These findings suggest that H5N1Tex/24 possesses intrinsic traits supporting efficient replication in differentiated human upper airway cell cultures.

Source: npj Viruses, https://www.nature.com/npjviruses/

Link: https://www.nature.com/articles/s44298-026-00208-2

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Mechanistic and #antigenic boundaries of #Henipavirus and Parahenipavirus #glycoproteins

 

Abstract

Henipaviruses, in the Paramyxoviridae family, includes the highly virulent Nipah virus that causes reoccurring outbreaks of deadly disease. Recent discoveries of Henipavirus-like species, including the zoonotic Langya virus, have revealed much higher antigenic diversity than currently characterized and prompted the reorganization of these viruses into the Henipavirus and Parahenipavirus genera. Here, to explore the limits of structural and antigenic variation in both genera, collectively referred to as HNVs, we construct an expanded, diverse panel of HNV fusion and attachment glycoproteins from non-redundant HNV strains that better reflect global HNV diversity. We express and purify the fusion protein ectodomains and the attachment protein head domains and study their biochemical and biophysical properties. We perform immunization experiments in mice, eliciting antibodies reactive to multiple HNV fusion proteins. Cryo-electron microscopy structures elucidate molecular determinants of differential pre-fusion state stability and higher order contacts. A crystal structure of the Gamak virus attachment head domain reveals an additional domain appended to the conserved 6-bladed, β-propeller fold. Taken together, these studies expand the known structural and antigenic limits of the HNVs, reveal cross-reactive epitopes within both genera and provide foundational data for the development of broadly reactive countermeasures.

Source: 

Link: https://www.nature.com/articles/s41467-026-74212-8

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#Antibodies to #influenza A virus #hemagglutinin and #neuraminidase limit egress and alter the physical properties of released virus particles

 

Abstract

Influenza A virus (IAV)-specific antibodies neutralize mature virions by inhibiting functional sites or, in some cases, by promoting virion aggregation. Many antibodies also act on infected cells to reduce virion yields, but the underlying mechanisms and effects on the physical properties and function of released virus particles remain incompletely characterized. Here we use flow virometry to acquire high-sensitivity yield and size measurements of virus particles released in the presence of antibodies. Combined with digital-droplet PCR and electron microscopy, this approach enables comprehensive characterization of antibody-induced changes in particle genome-content and morphology. We show that antibodies rapidly and dynamically alter released particle distributions, reducing yields and inducing the production of larger particles. Both effects result in part from aggregation induced by crosslinking of viral antigen on the infected-cell surface and inhibition of viral NA. However, yield reduction is not fully explained by aggregation, and a subset of induced larger particles are elongated virions. Finally, particles formed in the presence of HA stem-binding antibody, which does not inhibit attachment of mature virions, show reduced attachment in subsequent rounds of infection. Altogether, we uncover an unappreciated mechanism by which antibodies interfere with viral infection that occurs only during budding and release. Our work highlights the necessity of studying how the immune response shapes virus populations in the context of active infection processes.

Competing Interest Statement

T.I. is involved on a patent related to the flow virometry methodology: PCT/US2022/042125, status pending. The authors declare no other competing interests.

Funder Information Declared

Intramural Research Program of the National Institutes of Health (NIH), ZIAAI001385

G. Harold & Leila Y. Mathers Foundation, https://ror.org/02a7hjv13

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.06.15.729605v1

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