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Influenza A virus (IAV) is a widespread pathogen that poses a significant threat to human health, causing pandemics with high mortality and pathogenicity. Given the emergence of increasingly drug-resistant strains of IAV, currently available antiviral drugs have been reported to be inadequate to meet clinical demands. Therefore, continuous exploration of safe, effective and broad-spectrum antiviral medications is urgently required. Here, we found that the small molecule compound J1 exhibited low toxicity both in vitro and in vivo. Moreover, J1 exhibits broad-spectrum antiviral activity against enveloped viruses, including IAV, respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human coronavirus OC43 (HCoV-OC43), herpes simplex virus type 1 (HSV-1) and HSV-2. In this study, we explored the inhibitory effects and mechanism of action of J1 on IAV in vivo and in vitro. The results showed that J1 inhibited infection by IAV strains, including H1N1, H7N9, H5N1 and H3N2, as well as by oseltamivir-resistant strains. Mechanistic studies have shown that J1 blocks IAV infection mainly through specific interactions with the influenza virus hemagglutinin HA2 subunit, thereby blocking membrane fusion. BALB/c mice were used to establish a model of acute lung injury (ALI) induced by IAV. Treatment with J1 increased survival rates and reduced viral titers, lung index and lung inflammatory damage in virus-infected mice. In conclusion, J1 possesses significant anti-IAV effects in vitro and in vivo, providing insights into the development of broad-spectrum antivirals against future pandemics., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Influenza A virus (IAV) infection contributes to high annual morbidity and mortality, thus necessitating measures aimed at protecting against the disease. Alcohol-based disinfectants are commonly used to inactivate IAV, but they have several undesirable properties. In search of other means which would inactivate IAV, we focused on the effect of alkaline solutions on IAV. We found the viral infectivity remarkably decreased with treatment of an alkaline solution at pH 12.0 for 1 min, where destruction of the viral spikes was observed using an electron microscope. A more detailed examination revealed that the infectivity of IAV was remarkedly reduced by brief treatment with the alkaline solution at pH 11.75 or above, most likely due to the degradation of viral hemagglutinin protein. These results show that at a high pH, the haemagglutinin protein is degraded, resulting in very rapid inactivation of IAV.

Ferroptosis is a novel form of cell death caused by the accumulation of lipid peroxides in an iron-dependent manner. However, the precise mechanism underlying the exploitation of ferroptosis by influenza A viruses (IAV) remains unclear. The results demonstrate that IAV promotes its own replication through ferritinophagy by sensitizing cells to ferroptosis, with hemagglutinin identified as a key trigger in this process. Hemagglutinin interacts with autophagic receptors nuclear receptor coactivator 4 (NCOA4) and tax1-binding protein 1 (TAX1BP1), facilitating the formation of ferritin-NCOA4 condensates and inducing ferritinophagy. Further investigation shows that hemagglutinin-induced ferritinophagy causes cellular lipid peroxidation, inhibits aggregation of mitochondrial antiviral signaling protein (MAVS), and suppresses the type I interferon response, thereby contributing to viral replication. Collectively, a novel mechanism by which IAV hemagglutinin induces ferritinophagy resulting in cellular lipid peroxidation, consequently impairing MAVS-mediated antiviral immunity, is revealed., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli , and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro . Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents., Importance: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro . We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection., Competing Interests: The authors declare no conflict of interest.

Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry., Competing Interests: Declaration of interests The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories, and Merck outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, and Astrazeneca. A.G.-S. is an 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 filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, influenza virus vaccines, and influenza virus therapeutics, which list F.K. as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. F.K. is a co-founder and scientific advisory board member of Castlevax. F.K. has consulted for Merck, Curevac, Seqirus, and Pfizer and is currently consulting for 3rd Rock Ventures, GSK, Gritstone, and Avimex. The Krammer laboratory is also collaborating with Dynavax on influenza vaccine development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Outbreaks of acute respiratory viral diseases, such as influenza and COVID-19 caused by influenza A virus (IAV) and SARS-CoV-2, pose a serious threat to global public health, economic security, and social stability. This calls for the development of broad-spectrum antivirals to prevent or treat infection or co-infection of IAV and SARS-CoV-2. Hemagglutinin (HA) on IAV and spike (S) protein on SARS-CoV-2, which contain various types of glycans, play crucial roles in mediating viral entry into host cells. Therefore, they are key targets for the development of carbohydrate-binding protein-based antivirals. This study demonstrated that griffithsin (GRFT) and the GRFT-based bivalent entry inhibitor GL25E (GRFT-L25-EK1) showed broad-spectrum antiviral effects against IAV infection in vitro by binding to HA in a carbohydrate-dependent manner and effectively protected mice from lethal IAV infection. Although both GRFT and GL25E could inhibit infection of SARS-CoV-2 Omicron variants, GL25E proved to be significantly more effective than GRFT and EK1 alone. Furthermore, GL25E effectively inhibited in vitro co-infection of IAV and SARS-CoV-2 and demonstrated good druggability, including favorable safety and stability profiles. These findings suggest that GL25E is a promising candidate for further development as a broad-spectrum antiviral drug for the prevention and treatment of infection or co-infection from IAV and SARS-CoV-2.IMPORTANCEInfluenza and COVID-19 are highly contagious respiratory illnesses caused by the influenza A virus (IAV) and SARS-CoV-2, respectively. IAV and SARS-CoV-2 co-infection exacerbates damage to lung tissue and leads to more severe clinical symptoms, thus calling for the development of broad-spectrum antivirals for combating IAV and SARS-CoV-2 infection or co-infection. Here we found that griffithsin (GRFT), a carbohydrate-binding protein, and GL25E, a recombinant protein consisting of GRFT, a 25 amino acid linker, and EK1, a broad-spectrum coronavirus inhibitor, could effectively inhibit IAV and SARS-CoV-2 infection and co-infection by targeting glycans on HA of IAV and spike (S) protein of SARS-CoV-2. GL25E is more effective than GRFT because GL25E can also interact with the HR1 domain in SARS-CoV-2 S protein. Furthermore, GL25E possesses favorable safety and stability profiles, suggesting that it is a promising candidate for development as a drug to prevent and treat IAV and SARS-CoV-2 infection or co-infection., Competing Interests: L.L., S.J., N.C., X.W., and Y.C. are the inventors in the patent or patent application covering the recombinant proteins GRFT and GL25E. Other authors declare no conflict of interest.

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects "self" cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AN declared a shared affiliation with the author AP at the time of review. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Rabeeah, Billington, Nal, Sadeyen, Pathan, Iqbal, Temperton, Zipfel, Skerka, Kishore and Shelton.)

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N -acetylneuraminic acid (NeuAc) or N -glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo . We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo , the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N -glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N -glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N -acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N -glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N -glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection., Competing Interests: The authors declare no conflict of interest.

The transmembrane serine protease 2 (TMPRSS2) activates the outer structural proteins of a number of respiratory viruses including influenza A virus (IAV), parainfluenza viruses, and various coronaviruses for membrane fusion. Previous studies showed that TMPRSS2 interacts with the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), a cell surface protein that serves as an entry receptor for some coronaviruses. Here, by using protease activity assays, we determine that ACE2 increases the enzymatic activity of TMPRSS2 in a non-catalytic manner. Furthermore, we demonstrate that ACE2 knockdown inhibits TMPRSS2-mediated cleavage of IAV hemagglutinin (HA) in Calu-3 human airway cells and suppresses virus titers 100- to 1.000-fold. Transient expression of ACE2 in ACE2-deficient cells increased TMPRSS2-mediated HA cleavage and IAV replication. ACE2 knockdown also reduced titers of MERS-CoV and prevented S cleavage by TMPRSS2 in Calu-3 cells. By contrast, proteolytic activation and multicycle replication of IAV with multibasic HA cleavage site typically cleaved by furin were not affected by ACE2 knockdown. Co-immunoprecipitation analysis revealed that ACE2-TMPRSS2 interaction requires the enzymatic activity of TMPRSS2 and the carboxypeptidase domain of ACE2. Together, our data identify ACE2 as a new co-factor or stabilizer of TMPRSS2 activity and as a novel host cell factor involved in proteolytic activation and spread of IAV in human airway cells. Furthermore, our data indicate that ACE2 is involved in the TMPRSS2-catalyzed activation of additional respiratory viruses including MERS-CoV.IMPORTANCEProteolytic cleavage of viral envelope proteins by host cell proteases is essential for the infectivity of many viruses and relevant proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of several respiratory viruses, including influenza A virus. TMPRSS2 was previously shown to interact with angiotensin-converting enzyme 2 (ACE2). Here, we report the mechanistic details of this interaction. We demonstrate that ACE2 increases or stabilizes the enzymatic activity of TMPRSS2. Furthermore, we describe ACE2 involvement in TMPRSS2-catalyzed cleavage of the influenza A virus hemagglutinin and MERS-CoV spike protein in human airway cells. These findings expand our knowledge of the activation of respiratory viruses by TMPRSS2 and the host cell factors involved. In addition, our results could help to elucidate a physiological role for TMPRSS2., Competing Interests: The authors declare no conflict of interest.

Influenza, a pervasive viral respiratory illness, remains a significant global health concern. The influenza A virus, capable of causing pandemics, necessitates timely identification of specific subtypes for effective prevention and control, as highlighted by the World Health Organization. The genetic diversity of influenza A virus, especially in the hemagglutinin protein, presents challenges for accurate subtype prediction. This study introduces PreIS as a novel pipeline utilizing advanced protein language models and supervised data augmentation to discern subtle differences in hemagglutinin protein sequences. PreIS demonstrates two key contributions: leveraging pre-trained protein language models for influenza subtype classification and utilizing supervised data augmentation to generate additional training data without extensive annotations. The effectiveness of the pipeline has been rigorously assessed through extensive experiments, demonstrating a superior performance with an impressive accuracy of 94.54% compared to the current state-of-the-art model, the MC-NN model, which achieves an accuracy of 89.6%. PreIS also exhibits proficiency in handling unknown subtypes, emphasizing the importance of early detection. Pioneering the classification of HxNy subtypes solely based on the hemagglutinin protein chain, this research sets a benchmark for future studies. These findings promise more precise and timely influenza subtype prediction, enhancing public health preparedness against influenza outbreaks and pandemics. The data and code underlying this article are available in https://github.com/CBRC-lab/PreIS., Competing Interests: Declaration of competing interest We declare that there is no conflict of interest regarding the research presented in this paper. No financial or personal relationships have influenced the design, execution, or interpretation of the study. This work is solely based on our objective investigation and analysis of the subject matter, and we have no affiliations with any organizations or individuals that could be perceived as affecting the integrity of our findings., (Copyright © 2024. Published by Elsevier Ltd.)

Influenza A virus (IAV) binds sialic acid receptors on the cell surface to enter the host cells, which is the key step in initiating infection, transmission and pathogenesis. Understanding the factors that contribute to the highly efficient entry of IAV into human cells will help elucidate the mechanism of viral entry and pathogenicity, and provide new targets for intervention. In the present study, we reported a novel membrane protein, C1QTNF5, which binds to the hemagglutinin protein of IAV and promotes IAV infection in vitro and in vivo. We found that the HA1 region of IAV hemagglutinin is critical for the interaction with C1QTNF5 protein, and C1QTNF5 interacts with hemagglutinin mainly through its N-terminus (1-103 aa). In addition, we further demonstrated that overexpression of C1QTNF5 promotes IAV entry, while blocking the interaction between C1QTNF5 and IAV hemagglutinin greatly inhibits viral entry. However, C1QTNF5 does not function as a receptor to mediate IAV infection in sialic acid-deficient CHO-Lec2 cells, but promotes IAV to attach to these cells, suggesting that C1QTNF5 is an important attachment factor for IAV. This work reveals C1QTNF5 as a novel IAV attachment factor and provides a new perspective for antiviral strategies., Competing Interests: Conflict of interest Professor Ke Lan is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. We declare that we have no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Influenza A virus (IAV) is a negative-sense RNA virus that causes seasonal infections and periodic pandemics, inflicting huge economic and human costs on society. The current production of influenza virus for vaccines is initiated by generating a seed virus through the transfection of multiple plasmids in HEK293 cells followed by the infection of seed viruses into embryonated chicken eggs or cultured mammalian cells. We took a system design and synthetic biology approach to engineer cell lines that can be induced to produce all viral components except hemagglutinin (HA) and neuraminidase (NA), which are the antigens that specify the variants of IAV. Upon the transfection of HA and NA, the cell line can produce infectious IAV particles. RNA-Seq transcriptome analysis revealed inefficient synthesis of viral RNA and upregulated expression of genes involved in host response to viral infection as potential limiting factors and offered possible targets for enhancing the productivity of the synthetic cell line. Overall, we showed for the first time that it was possible to create packaging cell lines for the production of a cytopathic negative-sense RNA virus. The approach allows for the exploitation of altered kinetics of the synthesis of viral components and offers a new method for manufacturing viral vaccines.

Background: Highly pathogenic H5Nx viruses cause avian influenza, a zoonotic disease that can infect humans. The vaccine can facilitate the prevention of human infections from infected poultry. Our previous study showed that an H5 cleavage-site peptide vaccine containing the polybasic amino acid RRRK could protect chickens from lethal infections of the highly pathogenic H5N6 avian influenza virus., Methods: Chickens immunized with the various polybasic amino combinations (RRRK, RRR, RR, R, RK, and K) of H5 cleavage-site peptides were challenged with highly pathogenic H5N6 avian influenza viruses. The challenged chickens were monitored for survival rate, and viral titers in swabs and tissue samples were measured in Madin-Darby canine kidney (MDCK) cells using the median tissue culture infectious dose 50 (log10 TCID50/mL)., Results: Most H5 cleavage-site vaccines containing various combinations of polybasic amino acids protected chickens from lethal infection. Chickens immunized with the RK-containing peptide combination of the H5 cleavage site were not protected., Conclusions: The polybasic amino acids (RRRK) of H5 cleavage cleavage-site peptide vaccines are important for protecting chickens against HP H5N6 avian influenza virus. The H5 cleavage cleavage-site peptide containing RK did not protect chickens against the virus., Competing Interests: The author declares no conflict of interest. Given his role as Guest Editor, SHS had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Giuseppe Murdaca., (© 2024 The Author(s). Published by IMR Press.)

Aim: Mineralo-organic particles, naturally present in human body fluids, participate in ectopic calcification and inflammatory diseases. These particles coexist with influenza A virus (IAV) in the same microenvironment during viral infection. Our objective was to investigate the functional consequences of the potential interactions between these particles and the virions. Materials & methods: We used in vitro models, including electron microscopy, fluorescence microscopy, hemagglutination assay and viral infection assays to examine the interactions. Results: Mineralo-organic particles bind to IAV virions through interactions involving particle-bound fetuin-A and mineral content, effectively engaging viral hemagglutinin. These interactions result in hindered viral infection. Conclusion: These findings uncover the novel interactions between mineralo-organic particles and IAV, highlighting the impact of virus microenvironment complexity.

A synthetic multivalent hemagglutinin and neuraminidase inhibitor was developed by the conjugation of a septa-valent triazolylsialoside to bovine serum albumin using di-(N-succinimidyl) adipate. Matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) confirmed the attachment of five septa-valent sialyl lactosides to the protein backbone, resulting in a pentatrideca-valent sialyl conjugate. This pseudo-glycoprotein demonstrated a high affinity for hemagglutinin/neuraminidase as well as for the drug-resistant NA mutation on the influenza virus surface due to the cluster effect. The conjugate also exhibited potent antiviral activity against a wide range of virus strains without cytotoxicity at high concentrations. Mechanistic studies revealed that the pentatrideca-valent sialyl conjugate bound strongly to the influenza virion particles through interactions with HA/NA on the virion surfaces. The KD of the interaction was approximately 1 μM, as determined by isothermal calorimetric titration, allowing the capture and trapping of the influenza virions and preventing their further infection of host cells. These findings provide insight into the development of new antiviral agents using multivalent sialic acid clusters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Importance: Influenza A viruses (IAVs) contain hemagglutinin (HA) proteins involved in sialoglycan receptor binding and neuraminidase (NA) proteins that cleave sialic acids. While the importance of the NA protein in virion egress is well established, its role in virus entry remains to be fully elucidated. NA activity is needed for the release of virions from mucus decoy receptors, but conflicting results have been reported on the importance of NA activity in virus entry in the absence of decoy receptors. We now show that inhibition of NA activity affects virus entry depending on the receptor-binding properties of HA and the receptor repertoire present on cells. Inhibition of entry by the presence of mucus correlated with the importance of NA activity for virus entry, with the strongest inhibition being observed when mucus and OsC were combined. These results shed light on the importance in virus entry of the NA protein, an important antiviral drug target., Competing Interests: The authors declare no conflict of interest.

Novel highly pathogenic avian influenza (HPAI) H5Nx viruses are predominantly circulating worldwide, with an increasing potential threat of an outbreak in humans. It remains largely unknown how the stably maintained HPAI H5N1 suddenly altered its neuraminidase (NA) to other NA subtypes, which resulted in the emergence and evolution of H5Nx viruses. Here, we found that a combination of four specific amino acid (AA) substitutions (S123P-T156A-D183N- S223 R) in the hemagglutinin (HA) protein consistently observed in the H5Nx markedly altered the NA preference of H5N1 viruses. These molecular changes in H5N1 impaired its fitness, particularly viral growth and the functional activities of the HA and NA proteins. Among the AA substitutions identified, the T156A substitution, which contributed to the NA shift, also dramatically altered the antigenicity of H5N1 viruses, suggesting an occurrence of antigenic drift triggered by selective pressure. Our study shows the importance of how HA and NA complement each other and that antigenic drift in HA can potentially cause a shift in the NA protein in influenza A virus evolution.

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.

Amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large branched polysaccharide molecules on proteins, can modulate the function of HA and shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to quantitatively measure glycosylation changes in variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages, and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 (H3N2): WT and mutant, each expressed in embryonated chicken eggs and Madin-Darby canine kidney cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence variations and changing viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster genomic surveillance already being done, for a more complete understanding of IAV evolution., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Isoquercitrin (IQC) is a component abundantly present in many plants and is known to have an anti-viral effect against various viruses. In this study, we demonstrate that IQC exhibits strong anti-influenza A virus infection, and its effect is closely related to the suppression of hemagglutinin (HA) and neuraminidase (NA) activities. We used green fluorescent protein-tagged Influenza A/PR/8/34 (H1N1), A/PR/8/34 (H1N1), and HBPV-VR-32 (H3N2) to evaluate the anti-IAV effect of IQC. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that IQC significantly decreases the levels of GFP expressed by IAV infection, dose-dependently. Consistent with that, IQC inhibited cytopathic effects by H1N1 or H3N2 IAV infection. Immunofluorescence analysis confirmed that IQC represses the IAV protein expression. Time-of-addition assay showed that IQC inhibits viral attachment and entry and exerts a strong virucidal effect during IAV infection. Hemagglutination assay confirmed that IQC affects IAV HA. Further, IQC potently reduced the NA activities of H1N1 and H3N2 IAV. Collectively, IQC prevents IAV infection at multi-stages via virucidal effects, inhibiting attachment, entry and viral release. Our results indicate that IQC could be developed as a potent antiviral drug to protect against influenza viral infection.

The influenza A virus (IAV) is a highly contagious virus that causes pandemics and seasonal epidemics, which are major public health issues. Current anti-influenza therapeutics are limited partly due to the continuous emergence of drug-resistant IAV strains; thus, there is an unmet need to develop novel anti-influenza therapies. Here, we present a novel imidazo[1,2- a ]pyrimidine scaffold that targets group 2 IAV entry. We have explored three different regions of the lead compound, and we have developed a series of small molecules that have nanomolar activity against oseltamivir-sensitive and -resistant forms of group 2 IAVs. These small molecules target hemagglutinin (HA), which mediates the viral entry process. Mapping a known small-molecule-binding cavity of the HA structure with resistant mutants suggests that these molecules bind to that cavity and block HA-mediated membrane fusion.

Antigenic evolution of the influenza A virus (IAV) hemagglutinin (HA) gene limits efforts to effectively control the spread of the virus in the population. Efforts to understand the mechanisms governing HA antigenic evolution typically examine the HA gene in isolation. This can ignore the importance of balancing HA receptor binding activities with the receptor-destroying activities of the viral neuraminidase (NA) to maintain viral fitness. We hypothesize that the need to maintain functional balance with NA significantly constrains the evolutionary potential of the HA. We use deep mutational scanning and show that variation in NA activity significantly reshapes the HA fitness landscape by modulating the overall mutational robustness of HA. Consistent with this, we observe that different NA backgrounds support the emergence of distinct repertoires of HA escape variants under neutralizing antibody pressure. Our results reveal a critical role for intersegment epistasis in influencing the evolutionary potential of the HA gene., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Influenza A viruses pose a serious pandemic risk, while generation of efficient vaccines against seasonal variants remains challenging. There is thus a pressing need for new treatment options. We report here a set of macrocyclic peptides that inhibit influenza A virus infection at low nanomolar concentrations by binding to hemagglutinin, selected using ultrahigh-throughput screening of a diverse peptide library. The peptides are active against both H1 and H5 variants, with no detectable cytotoxicity. Despite the high sequence diversity across hits, all tested peptides were found to bind to the same region in the hemagglutinin stem by HDX-MS epitope mapping. A mutation in this region identified in an escape variant confirmed the binding site. This stands in contrast to the immunodominance of the head region for antibody binding and suggests that macrocyclic peptides from in vitro display may be well suited for finding new druggable sites not revealed by antibodies. Functional analysis indicates that these peptides stabilize the prefusion conformation of the protein and thereby prevent virus-cell fusion. High-throughput screening of macrocyclic peptides is thus shown here to be a powerful method for the discovery of novel broadly acting viral fusion inhibitors with therapeutic potential.

Background/aim: Influenza A virus (IAV) infection causes an inflammatory response to the respiratory mucosa. The viral glycoprotein hemagglutinin (HA) binds to the sialylated voltage-dependent Ca 2+ channel (Cav1.2) in ciliated epithelium. The binding of HA and sialylated Cav1.2 is considered essential to IAV infection, entry, and IAV-induced Ca 2+ oscillation. The epipharynx comprises the ciliated epithelium, which is the initial target for viruses that cause upper respiratory tract infections. Previously, we showed that epipharyngeal abrasive therapy (EAT), a treatment for chronic epipharyngitis in Japan, which scratches the epipharyngeal mucosa with a cotton swab containing zinc chloride, induces squamous metaplasia. In this study, we evaluated whether squamous metaplasia by EAT affects the expression patterns of Cav1.2., Patients and Methods: The study subjects were seven patients who had not been treated with EAT and 11 patients who had. For the immunohistochemical assessment of the epipharyngeal mucosa, the staining intensity of Cav1.2 was described using the immunohistochemical score (IHC score)., Results: The IHC scores for Cav1.2 in the EAT-treated group was 4.19-fold lower than those in the non-treated group (p=0.0034)., Conclusion: EAT down-regulates the expression of Cav1.2, a key cell surface molecule in influenza virus entry via squamous metaplasia. Thus, EAT may be a simple method for preventing influenza infection., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Understanding mechanisms of resistance to antiviral inhibitors can reveal nuanced features of targeted viral mechanisms and, in turn, lead to improved strategies for inhibitor design. Arbidol is a broad-spectrum antiviral that binds to and prevents the fusion-associated conformational changes in the trimeric influenza A virus (IAV) hemagglutinin (HA). The rate-limiting step during the HA-mediated membrane fusion is the release of the hydrophobic fusion peptides from a conserved pocket on HA. Here, we investigated how destabilizing or stabilizing mutations in or near the fusion peptide affect viral sensitivity to Arbidol. The degree of sensitivity was proportional to the extent of fusion-peptide stability on the prefusion HA: stabilized mutants were more sensitive, and destabilized ones were resistant to Arbidol. Single-virion membrane fusion experiments for representative wild-type (WT) and mutant viruses demonstrated that resistance is a direct consequence of fusion-peptide destabilization not requiring reduced Arbidol binding to HA. Our results support the model whereby the probability of individual HAs extending to engage the target membrane is determined by the composite of two critical forces: a "tug" on the fusion peptide by HA rearrangements near the Arbidol binding site and the key interactions stabilizing the fusion peptide in the prefusion pocket. Arbidol increases and destabilizing mutations decrease the free-energy cost for fusion-peptide release, accounting for the observed resistance. Our findings have broad implications for fusion inhibitor design, viral mechanisms of resistance, and our basic understanding of HA-mediated membrane fusion.

Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. In this study, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. IMPORTANCE The acquisition of mutations that allow avian influenza A virus hemagglutinins to recognize human-type receptors is mandatory for the transmission of avian viruses to humans, which could lead to a pandemic. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study contributes to elucidation of the relationship between the physical binding of virus and receptor and viral infectivity. Furthermore, the system using sialic acid knockout cells could provide a useful tool to explore the sialic acid-independent entry mechanism. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism.

Establishment of zoonotic viruses, causing pandemics like the Spanish flu and Covid-19, requires adaptation to human receptors. Pandemic influenza A viruses (IAV) that crossed the avian-human species barrier switched from binding avian-type α2-3-linked sialic acid (2-3Sia) to human-type 2-6Sia receptors. Here, we show that this specificity switch is however less dichotomous as generally assumed. Binding and entry specificity were compared using mixed synthetic glycan gradients of 2-3Sia and 2-6Sia and by employing a genetically remodeled Sia repertoire on the surface of a Sia-free cell line and on a sialoglycoprotein secreted from these cells. Expression of a range of (mixed) 2-3Sia and 2-6Sia densities shows that non-binding human-type receptors efficiently enhanced avian IAV binding and entry provided the presence of a low density of high affinity avian-type receptors, and vice versa. Considering the heterogeneity of sialoglycan receptors encountered in vivo, hetero-multivalent binding is physiologically relevant and will impact evolutionary pathways leading to host adaptation., (© 2022. The Author(s).)

Mucus in the lung plays an essential role as a barrier to infection by viral pathogens such as influenza A virus (IAV). Previous work determined mucin-associated sialic acid acts as a decoy receptor for IAV hemagglutinin (HA) binding and the sialic-acid cleaving enzyme, neuraminidase (NA), facilitates virus passage through mucus. However, it has yet to be fully addressed how the physical structure of the mucus gel influences its barrier function and its ability to trap viruses via glycan mediated interactions to prevent infection. To address this, IAV and nanoparticle diffusion in human airway mucus and mucin-based hydrogels is quantified using fluorescence video microscopy. We find the mobility of IAV in mucus is significantly influenced by the mesh structure of the gel and in contrast to prior reports, these effects likely influence virus passage through mucus gels to a greater extent than HA and NA activity. In addition, an analytical approach is developed to estimate the binding affinity of IAV to the mucus meshwork, yielding dissociation constants in the mM range, indicative of weak IAV-mucus binding. Our results provide important insights on how the adhesive and physical barrier properties of mucus influence the dissemination of IAV within the lung microenvironment., (© 2022. The Author(s).)

The highly pathogenic avian influenza H5N6 viruses are widely circulating in poultry and wild birds, and have caused 38 human infections including 21 deaths; however, the key genetic determinants of the pathogenicity of these viruses have yet to be fully investigated. Here, we characterized two H5N6 avian influenza viruses - A/duck/Guangdong/S1330/2016 (GD/330) and A/environment/Fujian/S1160/2016 (FJ/160) - that have similar viral genomes but differ markedly in their lethality in mice. GD/330 is highly pathogenic with a 50% mouse lethal dose (MLD 50 ) of 2.5 log 10 50% egg infectious doses (EID 50 ), whereas FJ/160 exhibits low pathogenicity with an MLD 50 of 7.4 log 10 EID 50 . We explored the molecular basis for the difference in virulence between these two viruses. By using reverse genetics, we created a series of reassortants and mutants in the GD/330 background and assessed their virulence in mice. We found that the HA gene of FJ/160 substantially attenuated the virulence of GD/330 and that the mutation of glycine (G) to tryptophan (W) at position 225 (H3 numbering) in HA played a key role in this function. We further found that the amino acid mutation G225W in HA decreased the acid and thermal stability and increased the pH of HA activation, thereby attenuating the H5N6 virus in mice. Our study thus identifies a novel molecular determinant in the HA protein and provides a new target for the development of live attenuated vaccines and antiviral drugs against H5 influenza viruses.

Influenza (flu) is a serious global health threat. The Hemagglutinin (HA) protein binds the flu virus to the sialic acids at the surface of the host cells' membrane which allows the endocytosis of the virus. Therefore, potential inhibitors can attach to the active site of HA and block the virus life-cycle. In this study, the antiviral drug arbidol (ARB) and 16 HA-subtypes were docked and analyzed to represent different approaches in predicting the conformation of protein-ligand, protein-protein, and protein-glycan complex and its binding energy. Our findings show that ARB interacts with all HA subtypes, and H 7 possesses the best affinity. The next influenza pandemic could be caused by H 4 , H 5 , H 6 , and H 14 subtypes, which prompts further studies in investigating the interaction between these particular HA subtypes and other antiviral drugs to obtain higher efficacy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.

The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding hemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype specific, varying significantly in sequence and length at both the 3' and 5' termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3'-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable, and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multisegment environment. These results provide novel insights into the role of the 3'-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3'-end H1-ssNCR and observed a synergistic effect between the 3'-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of the HA segment on template preference in a multisegment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.

Influenza viruses cause approximately half a million deaths every year worldwide. Vaccines are available but partially effective, and the number of antiviral medications is limited. Thus, it is crucial to develop therapeutic strategies to counteract this major pathogen. Influenza viruses enter the host cell via their hemagglutinin (HA) proteins. The HA subtypes of influenza A virus are phylogenetically classified into groups 1 and 2. Here, we identified an inhibitor of the HA protein, a tertiary aryl sulfonamide, that prevents influenza virus entry and replication. This compound shows potent antiviral activity against diverse H1N1, H5N1, and H3N2 influenza viruses encoding HA proteins from both groups 1 and 2. Synthesis of derivatives of this aryl sulfonamide identified moieties important for antiviral activity. This compound may be considered as a lead for drug development with the intent to be used alone or in combination with other influenza A virus antivirals to enhance pan-subtype efficacy.

Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N-glycosylation sites, and alteration of N-glycan micro- and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N-glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin-Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N-glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N-glycan microheterogeneity showed an increasing variability and a higher complexity for N-glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N-glycosylated at site N73. Almost all N-glycan structures were fucosylated. Furthermore, HA and NA N-glycan structures were exclusively hybrid- and complex-type structures, to some extent terminated with alpha-linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose-type, alpha-linked galactose, and multiantennary complex-type N-glycans., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5'- and 3'-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385-hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.

Dabbling and diving ducks partly occupy shared habitats but have been reported to play different roles in wildlife infectious disease dynamics. Influenza A virus (IAV) epidemiology in wild birds has been based primarily on surveillance programs focused on dabbling duck species, particularly mallard ( Anas platyrhynchos ). Surveillance in Eurasia has shown that in mallards, some subtypes are commonly (H1 to H7 and H10), intermediately (H8, H9, H11, and H12), or rarely (H13 to H16) detected, contributing to discussions on virus host range and reservoir competence. An alternative to surveillance in determining IAV host range is to study virus attachment as a determinant for infection. Here, we investigated the attachment patterns of all avian IAV subtypes (H1 to H16) to the respiratory and intestinal tracts of four dabbling duck species ( Mareca and Anas spp.), two diving duck species ( Aythya spp.), and chicken, as well as to a panel of 65 synthetic glycan structures. We found that IAV subtypes generally showed abundant attachment to colon of the Anas duck species, mallard, and Eurasian teal ( Anas crecca ), supporting the fecal-oral transmission route in these species. The reported glycan attachment profile did not explain the virus attachment patterns to tissues but showed significant attachment of duck-originated viruses to fucosylated glycan structures and H7 virus tropism for Neu5Gc-LN. Our results suggest that Anas ducks play an important role in the ecology and epidemiology of IAV. Further knowledge on virus tissue attachment, receptor distribution, and receptor binding specificity is necessary to understand the mechanisms underlying host range and epidemiology of IAV. IMPORTANCE Influenza A viruses (IAVs) circulate in wild birds worldwide. From wild birds, the viruses can cause outbreaks in poultry and sporadically and indirectly infect humans. A high IAV diversity has been found in mallards ( Anas platyrhynchos ), which are most often sampled as part of surveillance programs; meanwhile, little is known about the role of other duck species in IAV ecology and epidemiology. In this study, we investigated the attachment of all avian IAV hemagglutinin (HA) subtypes (H1 to H16) to tissues of six different duck species and chicken as an indicator of virus host range. We demonstrated that the observed virus attachment patterns partially explained reported field prevalence. This study demonstrates that dabbling ducks of the Anas genus are potential hosts for most IAV subtypes, including those infecting poultry. This knowledge is useful to target the sampling of wild birds in nature and to further study the interaction between IAVs and birds., (Copyright © 2021 Verhagen et al.)

The presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multibasic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points., (© 2021 Gultyaev et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among V H 3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of V H 3-30 antibodies and reveals that 3I14 represents a novel structural solution within the V H 3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knockout significantly inhibited the budding of NA. Further studies showed that the NLG knockout caused attenuation of virus in vitro and in vivo Notably, the NLG at 219 position plays an important role in the budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, the unfolded protein response (UPR) was determined in NLG knockout NA overexpressed cells, which showed that the mutant NA was mainly located in the endoplasmic reticulum (ER), the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knockout NA was stacked in the ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding. IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. In particular, the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo , which suggested that NLG of NA protein is a novel virulence marker for influenza viruses., (Copyright © 2021 American Society for Microbiology.)

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

Through annual epidemics and global pandemics, influenza A viruses (IAVs) remain a significant threat to human health as the leading cause of severe respiratory disease. Within the last century, four global pandemics have resulted from the introduction of novel IAVs into humans, with components of each originating from avian viruses. IAVs infect many avian species wherein they maintain a diverse natural reservoir, posing a risk to humans through the occasional emergence of novel strains with enhanced zoonotic potential. One natural barrier for transmission of avian IAVs into humans is the specificity of the receptor-binding protein, hemagglutinin (HA), which recognizes sialic-acid-containing glycans on host cells. HAs from human IAVs exhibit "human-type" receptor specificity, binding exclusively to glycans on cells lining the human airway where terminal sialic acids are attached in the α2-6 configuration (NeuAcα2-6Gal). In contrast, HAs from avian viruses exhibit specificity for "avian-type" α2-3-linked (NeuAcα2-3Gal) receptors and thus require adaptive mutations to bind human-type receptors. Since all human IAV pandemics can be traced to avian origins, there remains ever-present concern over emerging IAVs with human-adaptive potential that might lead to the next pandemic. This concern has been brought into focus through emergence of SARS-CoV-2, aligning both scientific and public attention to the threat of novel respiratory viruses from animal sources. In this review, we summarize receptor-binding adaptations underlying the emergence of all prior IAV pandemics in humans, maintenance and evolution of human-type receptor specificity in subsequent seasonal IAVs, and potential for future human-type receptor adaptation in novel avian HAs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Protective immunity against influenza A viruses (IAVs) generally depends on antibodies to the major envelope glycoprotein, hemagglutinin (HA), whose antigenicity is distinctive among IAV subtypes. On the other hand, the matrix 2 (M2) protein is antigenically highly conserved and has been studied as an attractive vaccine antigen to confer cross-protective immunity against multiple subtypes of IAVs. However, antiviral mechanisms of M2-specific antibodies are not fully understood. Here, we report the molecular basis of antiviral activity of an M2-specific monoclonal antibody (MAb), rM2ss23. We first found that rM2ss23 inhibited A/Aichi/2/1968 (H3N2) (Aichi) but not A/PR/8/1934 (H1N1) (PR8) replication. rM2ss23 altered the cell surface distribution of M2, likely by cross-linking the molecules, and interfered with the colocalization of HA and M2, resulting in reduced budding of progeny viruses. However, these effects were not observed for another strain, PR8, despite the binding capacity of rM2ss23 to PR8 M2. Interestingly, HA was also involved in the resistance of PR8 to rM2ss23. We also found that two amino acid residues at positions 54 and 57 in the M2 cytoplasmic tail were critical for the insensitivity of PR8 to rM2ss2. These findings suggest that the disruption of the M2-HA colocalization on infected cells and subsequent reduction of virus budding is one of the principal mechanisms of antiviral activity of M2-specific antibodies and that anti-M2 antibody-sensitive and -resistant IAVs have different properties in the interaction between M2 and HA. IMPORTANCE Although the IAV HA is the major target of neutralizing antibodies, most of the antibodies are HA subtype specific, restricting the potential of HA-based vaccines. On the contrary, the IAV M2 protein has been studied as a vaccine antigen to confer cross-protective immunity against IAVs with multiple HA subtypes, since M2 is antigenically conserved. Although a number of studies highlight the protective role of anti-HA neutralizing and nonneutralizing antibodies, precise information on the molecular mechanism of action of M2-specific antibodies is still obscure. In this study, we found that an anti-M2 antibody interfered with the HA-M2 association, which is important for efficient budding of progeny virus particles from infected cells. The antiviral activity was IAV strain dependent despite the similar binding capacity of the antibody to M2, and, interestingly, HA was involved in susceptibility to the antibody. Our data provide a novel mechanism underlying antiviral activity of M2-specific antibodies., (Copyright © 2020 American Society for Microbiology.)

N-linked glycans commonly contribute to secretory protein folding, sorting, and signaling. For enveloped viruses, such as the influenza A virus (IAV), large N-linked glycans can also be added to prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs, three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased over time and then decreased with and after the introduction of the 2009 pandemic H1N1 IAV of Eurasian swine origin. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells and eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions. IMPORTANCE N-linked glycans are transferred to secretory proteins upon entry into the endoplasmic reticulum lumen. In addition to promoting secretory protein maturation, enveloped viruses also utilize these large oligosaccharide structures to prevent access to surface antigen epitopes. Sequence analyses of the influenza A virus (IAV) surface antigen neuraminidase (NA or N) showed that the conservation of N-linked glycosylation sites on the NA enzymatic head domain differs by IAV subtype (H1N1 versus H3N2) and species of origin, with human-derived IAVs possessing the most variability. Experimental analyses verified that the N-linked glycosylation sites on the NA head domain contribute to virion incorporation and replication. It also revealed that the head domain glycans affect N1 stability more than N2, suggesting N2 is more accommodating to glycan additions. These results demonstrate that in addition to antigenicity, changes in N-linked glycosylation sites can alter other properties of viral surface antigens and virions.

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential., Competing Interests: The authors have declared that no competing interests exist.

Background: Type A influenza viruses circulate and spread among wild birds and mostly consist of low pathogenic strains. However, fast genome variation timely results in the insurgence of high pathogenic strains, which when infecting poultry birds may cause a million deaths and strong commercial damage. More importantly, the host shift may concern these viruses and sustained human-to-human transmission may result in a dangerous pandemic outbreak. Therefore, fingerprints specific to either low or high pathogenic strains may represent a very important tool for global surveillance., Results: We combined Normal Modes Analysis and surface electrostatic analysis of a mixed strain dataset of influenza A virus haemagglutinins from high and low pathogenic strains in order to infer specific fingerprints. Normal Modes Analysis sorted the strains in two different, homogeneous clusters; sorting was independent of clades and specific instead to high vs low pathogenicity. A deeper analysis of fluctuations and flexibility regions unveiled a special role for the 110-helix region. Specific sorting was confirmed by surface electrostatics analysis, which further allowed to focus on regions and mechanisms possibly crucial to the low-to-high transition., Conclusions: Evidence from previous work demonstrated that changes in surface electrostatics are associated with the evolution and spreading of avian influenza A virus clades, and seemingly involved also in the avian to mammalian host shift. This work shows that a combination of electrostatics and Normal Modes Analysis can also identify fingerprints specific to high and low pathogenicity. The possibility to predict which specific mutations may result in a shift to high pathogenicity may help in surveillance and vaccine development.

Owing to the emergence of drug resistance and high morbidity and mortality, the need for novel anti-influenza A virus (IAV) drugs with divergent targets is highly sought after. Herein, we reveal the discovery of an anti-IAV agent as a dual inhibitor to block hemagglutinin-mediated adsorption and membrane fusion using a chemoreactive ortho -quinone methide ( o -QM) equivalent. Based on the o -QM equivalent nonenzymatically multipotent behavior, we created a series of clavatol-derived pseudo-natural products and found that penindolone (PND), a new diclavatol indole adduct, exhibited potent and broad-spectrum anti-IAV activities with low risk of inducing drug resistance. Distinct from current anti-IAV drugs, PND possesses a novel scaffold and is the first IAV inhibitor targeting both HA1 and HA2 subunits of virus hemagglutinin to dually block the IAV adsorption and membrane fusion process. More importantly, intranasal and oral administration of PND can protect mice against IAV-induced death and weight loss, superior to the effects of the clinical drug oseltamivir. Thus, the use of chemoreactive intermediates could expand our understanding of chemical diversity and aid in the development of anti-IAV drugs with novel targets.

Hemagglutinin (HA) glycosylation of avian influenza virus (AIV) effects differently depending on the variation of glycosylation position and numbers. The natural mutation on the glycosylation sites of the AIV HA head occurs frequently. Our previous study shows that deletion of 158 or 169 glycosylation site on the HA head of the H5 subtype AIV strain rS-144-/158+/169+ increases the viral virulence in mammals; however, the mechanism remains unknown. In this study, several AIVs with different deletions at HA head glycosylation sites 144, 158 or 169 were tested for their biological characteristics to clarify the possible mechanism. We found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of inflammatory cytokines than rS-144-/158+/169+ did in the infected cells, but the TCID 50 , EID 50 and MDT of the viruses showed no difference. Moreover, we found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of endoplasmic reticulum (ER) stress in the cells. Inhibition of inositol-requiring enzyme 1α (IRE1α) phosphorylation reduced the inflammation induced by AIV infection. Furthermore, we found that rS-144-/158-/169+ virus activated the c-Jun N-terminal kinase (JNK), X-box binding protein 1 (XBP1), and nuclear factor-κB pathways by activating IRE1α phosphorylation under ER stress, whereas the rS-144-/158+/169- virus activated only the JNK pathway by altering IRE1α phosphorylation. In vivo analysis of Kira6 intervention further confirmed that ER stress played a key role in higher virulence for HA head 158 or 169 site de-glycosylation AIV. Our findings reveal that deletion of additional HA head glycosylation sites 158 or 169 enhanced the AIV virulence via activating of strong ER stress and inflammation., (© 2020 Blackwell Verlag GmbH.)

Influenza continues to be a significant public health challenge. Two glycoproteins on the surface of influenza virus, hemagglutinin and neuraminidase, play a prominent role in the process of influenza virus infection and release. Monoclonal antibodies targeting glycoproteins can effectively prevent the spread of the virus. In this review, we summarized currently reported human monoclonal antibodies targeting glycoproteins of influenza A and B viruses., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion 1-4 . Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies 5 and peptides 6 or that address late steps of the viral replication cycle 7 .

The host cell protease TMPRSS2 cleaves the influenza A virus (IAV) hemagglutinin (HA). Several reports have described resistance of Tmprss2 -/- knock-out (KO) mice to IAV infection but IAV of the H2 subtype have not been examined yet. Here, we demonstrate that TMPRSS2 is able to cleave H2-HA in cell culture and that Tmprss2 -/- mice are resistant to infection with a re-assorted PR8_HA(H2) virus. Infection of KO mice did not cause major body weight loss or death. Furthermore, no significant increase in lung weights and no virus replication were observed in Tmprss2 -/- mice. Finally, only minor tissue damage and infiltration of immune cells were detected and no virus-positive cells were found in histological sections of Tmprss2 -/- mice. In summary, our studies indicate that TMPRSS2 is required for H2 IAV spread and pathogenesis in mice. These findings extend previous results pointing towards a central role of TMPRSS2 in IAV infection and validate host proteases as a potential target for antiviral therapy.