Influenza Virus Hemagglutinin Research Articles

Developments and current challenges in the process of cell culture-based seasonal influenza vaccine manufacture in Japan.

Seasonal influenza is an acute respiratory infection primarily caused by influenza A and B viruses, which circulate annually and cause substantial morbidity and mortality worldwide. Annual influenza vaccination is currently the most effective measure for preventing influenza and greatly reduces the risk of disease severity and the incidence of complications and death. Annual seasonal influenza vaccines are traditionally produced in Japan and many other countries using viruses propagated in embryonated chicken eggs. However, at present, the effectiveness of the seasonal influenza vaccines has some significant limitations, partly because of egg-adaptive mutations in the antigenic sites of the influenza virus haemagglutinin, which are caused by the continued evolution of seasonal influenza viruses. To overcome the limitations of egg-based influenza vaccine production, a mammalian cell culture-based influenza vaccine production system has been developed in Japan in the past decade as an alternative to the current production method. In this review, I have summarised the progress in the development of cell-based seasonal influenza vaccines and discussed the technological challenges encountered in the development of influenza vaccines.

The immunodominance of antigenic site Sb on the H1 influenza virus hemagglutinin increases with high immunoglobulin titers of the cohorts and with young age, but not sex

The head domain of the hemagglutinin of influenza viruses plays a dominant role in the antibody response due to the presence of immunodominant antigenic sites that are the main targets of host neutralizing antibodies. For the H1 hemagglutinin, five major antigenic sites defined as Sa, Sb, Ca1, Ca2, and Cb have been described. Although previous studies have focused on defining the hierarchy of the antigenic sites of the hemagglutinin in different human cohorts, it is still unclear if the immunodominance profile of the antigenic sites might change with the antibody levels of individuals or if other demographic factors (such as exposure history, sex, or age) could also influence the importance of the antigenic sites. The major antigenic sites of influenza viruses hemagglutinins are responsible for eliciting most of the hemagglutination inhibition antibodies in the host. To determine the antibody prevalence towards each major antigenic site, we evaluated the hemagglutination inhibition against a panel of mutant H1 viruses, each one lacking one of the “classic” antigenic sites. Our results showed that the individuals from the Stop Flu NYU cohort had an immunodominant response towards the sites Sb and Ca2 of H1 hemagglutinin. A simple logistic regression analysis of the immunodominance profiles and the hemagglutination inhibition titers displayed by each donor revealed that individuals with high hemagglutination inhibition titers against the wild-type influenza virus exhibited higher probabilities of displaying an immunodominance profile dominated by Sb, followed by Ca2 (Sb > Ca2 profile), while individuals with low hemagglutination inhibition titers presented a higher chance of displaying an immunodominance profile in which Sb and Ca2 presented the same level of immunodominance (Sb = Ca2 profile). Finally, while age exhibited an influence on the immunodominance of the antigenic sites, biological sex was not related to displaying a specific immunodominance profile.

C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Prevalence of circulating antibodies against hemagglutinin of influenza viruses in epidemic season 2021/2022 in Poland.

The aim of the study was to determine the level of anti-hemagglutinin antibodies in the serum of patients during the 2021/2022 epidemic season in Poland. A total of 700 sera samples were tested, divided according to the age of the patients into 7 age groups: 0-4years of age, 5-9years of age, 10-14years of age, 15-25years of age, 26-44years of age, 45-64years of age and ≥65years of age, 100 samples were collected from each age group. Anti-hemagglutinin antibody levels was determined using the haemagglutination inhibition assay (OZHA). The results obtained confirm the presence of anti-hemagglutinin antibodies for the antigens A/Victoria/2570/2019 (H1N1) pdm09, A/Cambodia/e0826360/2020 (H3N2), B/Washington/02/2019 and B/Phuket/3073/2013 recommended by World Health Organization (WHO) for the 2021/2022 epidemic season. The analysis of the results shows differences in the levels of individual anti-hemagglutinin antibodies in the considered age groups. In view of very low percentage of the vaccinated population in Poland, which was 6.90% in the 2021/2022 epidemic season, the results obtained in the study would have to be interpreted as the immune system response in patients after a previous influenza virus infection.

Exploring sialic acid derivative inhibitors targeting influenza virus hemagglutinin (HA) protein

Exploring sialic acid derivative inhibitors targeting influenza virus hemagglutinin (HA) protein

Identification of novel inhibitor phytoconstituents for Influenza A H3N2: an in silico approach.

Influenza A virus subtype H3N2 is a highly infectious respiratory virus that is responsible for global seasonal flu epidemics. The current study was designed to investigate the antiviral activity of 150 phytocompounds of North Western Himalayas medicinal plants by molecular docking. Two target proteins of hemagglutinin of influenza virus A (PDB ID 4WE8) and Influenza virus H3N2 nucleoprotein - R416A mutant (PDB ID 7NT8) are selected for this study. Molecular docking was done by AutoDock vina tool, toxicity and drug-likeness prediction was done by protox II and Moleinspiration. MD simulation of best protein-ligand complexes was done by using Gromacs, version 2021.5. Molecular docking and toxicity data revealed that clicoemodin and rumexocide showed the best binding with both target proteins 4WEB & 7NT8. Clicoemodin showed the -7.5 KJ/mol binding energy with 4WE8 and 7NT8. Similarly, rumexoside showed the -7.6 KJ/mol binding energy with 4WE8 and -7.6 KJ/mol with 7NT8. Furthermore, Molecular dynamic simulation and MMPBSA binding free energy validated the stability of protein-ligand complexes. The current study suggested that clicoemodin and rumexocide are the promising inhibitors of H3N2 proteins hemagglutinin of influenza virus A and Influenza virus H3N2 nucleoprotein - R416A mutant, though there is further invitro and invivo validation is required.Communicated by Ramaswamy H. Sarma.

Design, synthesis, and biological evaluation of covalent inhibitors targeting influenza virus hemagglutinin

Design, synthesis, and biological evaluation of covalent inhibitors targeting influenza virus hemagglutinin

Problems and Collisions of Vaccinology

The article discusses the limitations of the protective potential of the immune system associated with the peculiarities of the evolutionary mechanisms of the emergence of protein diversity and the late emergence in the evolution of the adaptive immune system, as well as problems associated with the formation of immunity to viral infections and immune collisions during vaccination. Using the example of hemagglutinin of the H1N1 influenza virus and S protein of the SARS-Cov-2 coronavirus, the features of the amino acid composition of their immunodominant (NA1 and S1) and subdominant (NA2 and S2) subunits are illustrated and the possibility of creating a universal vaccine against influenza viruses is analyzed. The principle of a new method for detecting linear peptide immunoepitopes recognized by MHC I and II and biomarkers of long-term immunity in surface viral proteins used as vaccines is described. The model of proteolysis of vaccine proteins in immunoprotesomes and lysosomes, features of the amino acid composition of surface proteins of viruses to which vaccines cause long-term immunity, and viruses to which vaccines have not yet been developed, as well as possible collisions with mRNA vaccines are examined. Possible collisions with mRNA vaccines are also being considered in connection with the identification of gene encoding limitations.

C1QTNF5 is a novel attachment factor that facilitates the entry of influenza A virus

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.

Engineering of novel hemagglutinin biosensors for rapid detection and drug screening of Influenza A H7N9 virus

New pathogenic influenza virus strains are constantly emerging, posing a serious risk to both human health and economic growth. To effectively control the spread of this virus, there is an urgent need for early, rapid, sensitive, simple, and cost-effective detection technologies, as well as new and effective antiviral drugs. In this study, we have successfully achieved a significant milestone by successfully fusing the H7N9 influenza virus hemagglutinin (HA) protein with the nano-luciferase component, resulting in the development of a novel set of biosensors. This remarkable achievement marks the first instance of utilizing this biosensor technology for influenza antibody detection. Our biosensor technology also has the potential to facilitate the development of antiviral drugs targeting specific epitopes of the HA protein, providing a promising avenue for the treatment of H7N9 influenza virus infections. Furthermore, our biosensors have broad applications beyond H7N9 influenza virus detection, as they can be expanded for the detection of other pathogens and drug screening applications in the future. By providing a novel and effective solution to the detection and treatment of influenza viruses, our biosensors have the potential to revolutionize the field of infectious disease control.

Roles and functions of IAV proteins in host immune evasion.

Influenza A viruses (IAVs) evade the immune system of the host by several regulatory mechanisms. Their genomes consist of eight single-stranded segments, including nonstructural proteins (NS), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), acidic polymerase (PA), matrix (M), neuraminidase (NA), and nucleoprotein (NP). Some of these proteins are known to suppress host immune responses. In this review, we discuss the roles, functions and underlying strategies adopted by IAV proteins to escape the host immune system by targeting different proteins in the interferon (IFN) signaling pathway, such as tripartite motif containing 25 (TRIM25), inhibitor of nuclear factor κB kinase (IKK), mitochondrial antiviral signaling protein (MAVS), Janus kinase 1 (JAK1), type I interferon receptor (IFNAR1), interferon regulatory factor 3 (IRF3), IRF7, and nuclear factor-κB (NF-κB). To date, the IAV proteins NS1, NS2, PB1, PB1-F2, PB2, HA, and PA have been well studied in terms of their roles in evading the host immune system. However, the detailed mechanisms of NS3, PB1-N40, PA-N155, PA-N182, PA-X, M42, NA, and NP have not been well studied with respect to their roles in immune evasion. Moreover, we also highlight the future perspectives of research on IAV proteins.

Antibodies elicited in humans upon chimeric hemagglutinin-based influenza virus vaccination confer FcγR-dependent protection in vivo.

Antibody responses against highly conserved epitopes on the stalk domain of influenza virus hemagglutinin (HA) confer broad protection; however, such responses are limited. To effectively induce stalk-specific immunity against conserved HA epitopes, sequential immunization strategies have been developed based on chimeric HA (cHA) constructs featuring different head domains but the same stalk regions. Immunogenicity studies in small animal models, as well as in humans, revealed that cHA immunogens elicit stalk-specific IgG responses with broad specificity against heterologous influenza virus strains. However, the mechanisms by which these antibodies confer in vivo protection and the contribution of their Fc effector function remain unclear. To characterize the role of Fc-FcγR (Fcγ receptor) interactions to the in vivo protective activity of IgG antibodies elicited in participants in a phase I trial of a cHA vaccine candidate, we performed passive transfer studies of vaccine-elicited IgG antibodies in mice humanized for all classes of FcγRs, as well as in mice deficient for FcγRs. IgG antibodies elicited upon cHA vaccination completely protected FcγR humanized mice against lethal influenza virus challenge, while no protection was evident in FcγR-deficient mice, suggesting a major role for FcγR pathways in the protective function of vaccine-elicited IgG antibodies. These findings have important implications for influenza vaccine development, guiding the design of vaccination approaches with the capacity to elicit IgG responses with optimal Fc effector function.

Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides.

Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types, where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to preparing well-defined KS oligosaccharides by exploiting the known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfate terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify the internal galactosides of a poly-LacNAc chain; however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over the other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides, including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins, and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also the presentation of epitopes as part of an O- or N-glycan determines binding properties.

Sequencing methods for HA and NA genes of avian influenza viruses from wild bird feces using Oxford Nanopore sequencing

We developed a method to determine the sequences of hemagglutinin (HA) and neuraminidase (NA) from RNA extracted directly from wild bird fecal samples, using Nanopore Flongle. We determined the nucleotide sequences and subtypes of HA and NA in 16 and 15 samples respectively, using Flongle. The results of HA and NA subtyping determined by the conventional method were consistent with their subtypes determined by our method, thereby the applicability of this method in the identification of HA and NA subtypes. In addition, the homology between the HA fragments in this and the Sanger methods ranged from 98.5 % to 100 %. Compared with conventional PCR with the Sanger method, this method can easily determine HA and NA subtypes and sequences directly from the fecal samples. It is easier to implement and has lower running costs (USD100$) than other NGS-based methods, making it a useful tool for avian influenza surveillance in wild birds.

A novel in vitro system of supported planar endosomal membranes (SPEMs) reveals an enhancing role for cathepsin B in the final stage of Ebola virus fusion and entry.

Ebola virus (EBOV) causes a hemorrhagic fever with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP) is processed to a 19-kDa form, which binds to the EBOV intracellular receptor Niemann-Pick type C1. We previously showed that the cathepsin protease inhibitor, E-64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs), and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy. We validated the system by demonstrating the pH dependencies of influenza virus hemagglutinin (HA)-mediated and Lassa virus (LASV) GP-mediated fusion. Using SPEMs, we showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH, enhanced by Ca2+, and augmented by the addition of cathepsins. Subsequently, we found that E-64d inhibits full fusion, but not lipid mixing, mediated by 19-kDa GP, which we corroborated with the reversible cathepsin inhibitor VBY-825. Hence, we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of 19-kDa Ebola GP. In addition to providing new insights into how Ebola GP mediates fusion, the approach we developed employing SPEMs can now be broadly used for studies of virus and toxin entry through endosomes. IMPORTANCE Ebola virus is the causative agent of Ebola virus disease, which is severe and frequently lethal. EBOV gains entry into cells via late endosomes/lysosomes. The events immediately preceding fusion of the viral and endosomal membranes are incompletely understood. In this study, we report a novel in vitro system for studying virus fusion with endosomal membranes. We validated the system by demonstrating the low pH dependencies of influenza and Lassa virus fusion. Moreover, we show that further cathepsin B action enhances the fusion activity of the primed Ebola virus glycoprotein. Finally, this model endosomal membrane system should be useful in studying the mechanisms of bilayer breaching by other enveloped viruses, by non-enveloped viruses, and by acid-activated bacterial toxins.

Rational Design of Novel Peptidomimetics against Influenza A Virus: Biological and Computational Studies.

Effective therapy against the influenza virus is still an unmet goal. Drugs with antiviral effects exist, but the appearance of resistant viruses pushes towards the discovery of drugs with different mechanisms of action. New anti-influenza molecules should target a good candidate, as a new anti-influenza molecule could be an inhibitor of the influenza A virus hemagglutinin (HA), which plays a key role during the early phases of infection. In previous work, we identified two tetrapeptide sequences, SLDC (1) and SKHS (2), derived from bovine lactoferrin (bLf) C-lobe fragment 418-429, which were able to bind HA and inhibit cell infection at picomolar concentration. Considering the above, the aim of this study was to synthesize a new library of peptidomimetics active against the influenza virus. In order to test their ability to bind HA, we carried out a preliminary screening using biophysical assays such as surface plasmon resonance (SPR) and orthogonal immobilization-free microscale thermophoresis (MST). Biological and computational studies on the most interesting compounds were carried out. The methods applied allowed for the identification of a N-methyl peptide, S(N-Me)LDC, which, through high affinity binding of influenza virus hemagglutinin, was able to inhibit virus-induced hemagglutination and cell infection at picomolar concentration. This small sequence, with high activity, represents a good starting point for the design of new peptidomimetics and small molecules.

Forecasting influenza hemagglutinin mutations through the lens of anomaly detection

The influenza virus hemagglutinin is an important part of the virus attachment to the host cells. The hemagglutinin proteins are one of the genetic regions of the virus with a high potential for mutations. Due to the importance of predicting mutations in producing effective and low-cost vaccines, solutions that attempt to approach this problem have recently gained significant attention. A historical record of mutations has been used to train predictive models in such solutions. However, the imbalance between mutations and preserved proteins is a big challenge for the development of such models that need to be addressed. Here, we propose to tackle this challenge through anomaly detection (AD). AD is a well-established field in Machine Learning (ML) that tries to distinguish unseen anomalies from normal patterns using only normal training samples. By considering mutations as anomalous behavior, we could benefit existing rich solutions in this field that have emerged recently. Such methods also fit the problem setup of extreme imbalance between the number of unmutated vs. mutated training samples. Motivated by this formulation, our method tries to find a compact representation for unmutated samples while forcing anomalies to be separated from the normal ones. This helps the model to learn a shared unique representation between normal training samples as much as possible, which improves the discernibility and detectability of mutated samples from the unmutated ones at the test time. We conduct a large number of experiments on four publicly available datasets, consisting of three different hemagglutinin protein datasets, and one SARS-CoV-2 dataset, and show the effectiveness of our method through different standard criteria.

Designed nanoparticles elicit cross-reactive antibody responses to conserved influenza virus hemagglutinin stem epitopes.

Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that tandem copies of an alpha-helical fragment from the conserved stem region (helix-A) can be displayed on the protruding spikes structures of a capsid scaffold. The stem region of HA on these designed chimeric nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, mice vaccinated with H1-nanoparticles were protected from lethal challenge with H1N1 influenza virus. By using a nanoparticle library approach with this helix-A nanoparticle design, we show that this vaccine nanoparticle construct design could be applicable to different influenza HA subtypes. Importantly, antibodies elicited by H1, H5, and H7 nanoparticles demonstrated homosubtypic and heterosubtypic cross-reactivity binding to different HA subtypes. Also, helix-A nanoparticle immunizations were used to isolate mouse monoclonal antibodies that demonstrated heterosubtypic cross-reactivity and provided protection to mice from viral challenge via passive-transfer. This tandem helix-A nanoparticle construct represents a novel design to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes.

Novel mono- and multivalent N-acetylneuraminic acid glycoclusters as potential broad-spectrum entry inhibitors for influenza and coronavirus infection

N-acetylneuraminic acid (Neu5Ac) is a glycan receptor of viruses spread in many eukaryotic cells. The present work aimed to design, synthesis and biological evaluation of a panel of Neu5Ac derivatives based on a cyclodextrin (CD) scaffold for targeting influenza and coronavirus membrane proteins. The multivalent Neu5Ac glycoclusters efficiently inhibited chicken erythrocyte agglutination induced by intact influenza virus in a Neu5Ac density–dependent fashion. Compared with inhibition by Neu5Ac, the multivalent inhibitor with 21 Neu5Ac residues on the primary face of the β-CD scaffold afforded 1788-fold higher binding affinity inhibition for influenza virus hemagglutinin with a dissociation constant (KD) of 3.87 × 10−7 M. It showed moderate binding affinity to influenza virus neuraminidase, but with only about one-thirtieth the potency of that with the HA protein. It also exhibited strong binding affinity to the spike protein of three human coronaviruses (severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus 2), with KD values in the low micromolar range, which is about 10-time weaker than that of HA. Therefore, these multivalent sialylated CD derivatives have possible therapeutic application as broad-spectrum antiviral entry inhibitors for many viruses by targeting the Neu5Ac of host cells.

The M2 proteins of bat influenza A viruses reveal atypical features compared to conventional M2 proteins.

The influenza A virus (IAV) M2 protein has proton channel activity, which plays a role in virus uncoating and may help to preserve the metastable conformation of the IAV hemagglutinin (HA). In contrast to the highly conserved M2 proteins of conventional IAV, the primary sequences of bat IAV H17N10 and H18N11 M2 proteins show remarkable divergence, suggesting that these proteins may differ in their biological function. We, therefore, assessed the proton channel activity of bat IAV M2 proteins and investigated its role in virus replication. Here, we show that the M2 proteins of bat IAV did not fully protect acid-sensitive HA of classical IAV from low pH-induced conformational change, indicating low proton channel activity. Interestingly, the N31S substitution not only rendered bat IAV M2 proteins sensitive to inhibition by amantadine but also preserved the metastable conformation of acid-sensitive HA to a greater extent. In contrast, the acid-stable HA of H18N11 did not rely on such support by M2 protein. When mutant M2(N31S) protein was expressed in the context of chimeric H18N11/H5N1(6:2) encoding HA and NA of avian IAV H5N1, amantadine significantly inhibited virus entry, suggesting that ion channel activity supported virus uncoating. Finally, the cytoplasmic domain of the H18N11 M2 protein mediated rapid internalization of the protein from the plasma membrane leading to low-level expression at the cell surface. However, cell surface levels of H18N11 M2 protein were significantly enhanced in cells infected with the chimeric H18N11/H5N1(6:2) virus. The potential role of the N1 sialidase in arresting M2 internalization is discussed. IMPORTANCE Bat IAV M2 proteins not only differ from the homologous proteins of classical IAV by their divergent primary sequence but are also unable to preserve the metastable conformation of acid-sensitive HA, indicating low proton channel activity. This unusual feature may help to avoid M2-mediated cytotoxic effects and inflammation in bats infected with H17N10 or H18N11. Unlike classical M2 proteins, bat IAV M2 proteins with the N31S substitution mediated increased protection of HA from acid-induced conformational change. This remarkable gain of function may help to understand how single point mutations can modulate proton channel activity. In addition, the cytoplasmic domain was found to be responsible for the low cell surface expression level of bat IAV M2 proteins. Given that the M2 cytoplasmic domain of conventional IAV is well known to participate in virus assembly at the plasma membrane, this atypical feature might have consequences for bat IAV budding and egress.