Influenza Virus Entry Research Articles

Influenza Viral Hemagglutinin Peptide Inhibits Influenza Viral Entry by Shielding the Host Receptor.

Influenza viral infection of the host begins by the attachment of viral hemagglutinin to a cell surface receptor. In the current study, a hemagglutinin fragment peptide library was screened using an H5N1 recombinant pseudotyped viral system. One peptide, designated HA-pep25, showed effective antiviral activity against both human and avian influenza viral strains (IC50 = 12.0-51.0 μM). A mechanistic study demonstrated direct binding between HA-pep25 and sialyllactose, which mimics the host receptor for the influenza virus. This binding was independent of the presence of sialic acid on the cell membrane. By generating alanine substitutions in HA-pep25, eight residues were identified as essential for the peptide's anti-influenza activity. HA-pep25 derived from hemagglutinin blocked influenza viral entry by shielding the host receptor on the cell membrane. This peptide might be a candidate drug for influenza virus entry inhibition and may be combined with other antivirals targeting different steps of the influenza viral life cycle.

Design, synthesis and biological evaluation of novel l-ascorbic acid-conjugated pentacyclic triterpene derivatives as potential influenza virus entry inhibitors

Since the influenza viruses can rapidly evolve, it is urgently required to develop novel anti-influenza agents possessing a novel mechanism of action. In our previous study, two pentacyclic triterpene derivatives (Q8 and Y3) have been found to have anti-influenza virus entry activities. Keeping the potential synergy of biological activity of pentacyclic triterpenes and l-ascorbic acid in mind, we synthesized a series of novel l-ascorbic acid-conjugated pentacyclic triterpene derivatives (18–26, 29–31, 35–40 and 42–43). Moreover, we evaluated these novel compounds for their anti-influenza activities against A/WSN/33 virus in MDCK cells. Among all evaluated compounds, the 2,3-O,O-dibenzyl-6-deoxy-l-ascorbic acid-betulinic acid conjugate (30) showed the most significant anti-influenza activity with an EC50 of 8.7 μM, and no cytotoxic effects on MDCK cells were observed. Time-of-addition assay indicated that compound 30 acted at an early stage of the influenza life cycle. Further analyses revealed that influenza virus-induced hemagglutination of chicken red blood cells was inhibited by treatment of compound 30, and the interaction between the influenza hemagglutinin (HA) and compound 30 was determined by surface plasmon resonance (SPR) with a dissociation constant of KD = 3.76 μM. Finally, silico docking studies indicated that compound 30 and its derivative 31 were able to occupy the binding pocket of HA for sialic acid receptor. Collectively, these results suggested that l-ascorbic acid-conjugated pentacyclic triterpenes were promising anti-influenza entry inhibitors, and HA protein associated with viral entry was a promising drug target.

Pentacyclic triterpenes grafted on CD cores to interfere with influenza virus entry: A dramatic multivalent effect

Multivalent effect plays an important role in biological processes, particularly in the specific recognition of virus with its host cell during the first step of infection. Here we report the synthesis of multivalent pentacyclic triterpene grafted on cyclodextrin core and potency of against influenza entry activity. Nine star-shaped compounds containing six, seven and eight pentacyclic triterpene pharmacophore on cyclodextrin scaffold were prepared by way of copper-catalyzed azide-alkyl cycloaddition reaction under microwave activation. Some of the multimers exhibited much potent antiviral activity against H1N1 virus (A/WSN/33), even equivalent or superior to oseltamivir. The most active compound 31, a heptavalent oleanolic acid-β-cyclodextrin conjugate, shows an up to 125-fold potency enhancement by its IC50 value over the corresponding monovalent conjugate and oleanolic acid, disclosing a clear multivalent effect. Further studies show that three compounds 31–33 exhibited broad spectrum inhibitory activity against other two human influenza A/JX/312 (H3N2) and A/HN/1222 (H3N2) viruses with the IC50 values at 2.47–14.90 μM. Most importantly, we found that compound 31, one of the best representative conjugate, binds tightly to the viral envelope hemagglutinin with a dissociation constant of KD = 2.08 μM, disrupting the interaction of hemagglutinin with the sialic acid receptor and thus the attachment of viruses to host cells. Our study might establish a strategy for the design of new pharmaceutical agents based on multivalency so as to block influenza virus entry into host cells.

A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus.

BackgroundGenome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening.MethodsTwo genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits.ResultsThe parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study.ConclusionsThis strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.

Reduction of Influenza Virus Envelope's Fusogenicity by Viral Fusion Inhibitors.

During cell entry of an enveloped virus, the viral membrane must be fused with the cellular membrane. The virus envelope has a unique structure consisting of viral proteins and a virus-specific lipid composition, whereas the host membrane has its own structure with host membrane proteins. Compound 136 was previously found to bind in close proximity to the viral envelope and inhibit influenza virus entry. We showed here that the 136-treated influenza virus still caused hemolysis. When liposomes were used as the target membrane for 136-treated viruses, aberrant fusion occurred; few liposomes fused per virion, and glycoproteins were not distributed evenly across fusion complexes. Additionally, large fusion aggregates did not form, and in some instances, neck-like structures were found. Based on previous results and hemolysis, fusion inhibition by 136 occurs post-scission but prior to lipid mixing.

The serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells

BackgroundSerine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear. MethodsPrimary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2). ResultsCamostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat. ConclusionsThese findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.

Pseudotype-based neutralization assays for influenza: a systematic analysis.

The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI)-competent antibodies that target the globular head of hemagglutinin (HA) thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D toward the production of a “universal vaccine” has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1–H16). The accurate and sensitive measurement of antibody responses elicited by these “next-generation” influenza vaccines is, however, hampered by the lack of sensitivity of the traditional influenza serological assays HI, single radial hemolysis, and microneutralization. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies.

Oral intake of Lactobacillus rhamnosus M21 enhances the survival rate of mice lethally infected with influenza virus.

Influenza viruses cause acute respiratory disease. Because of the high genetic variability of viruses, effective vaccines and antiviral agents are limited. Considering the fact that the site of influenza virus entry is the mucosa of the upper respiratory tract, probiotics that can enhance mucosal immunity as well as systemic immunity could be an important source of treatment against influenza infection. Mice were fed with Lactobacillus rhamnosus M21 or skim milk and were challenged with influenza virus. The resulting survival rate, lung inflammation, and changes in the cytokine and secretory immunoglobulin A (sIgA) levels were examined. Because of infection (influenza virus), all the mice in the control group and 60% of the mice in the L. rhamnosus M21 group died; however, the remaining 40% of the mice fed with L. rhamnosus M21 survived the infection. Pneumonia was severe in the control group but moderate in the group treated with L. rhamnosus M21. Although there were no significant changes in the proinflammatory cytokines in the lung lysates of mice collected from both groups, levels of interferon-γ and interleukin-2, which are representative cytokines of type I helper T cells, were significantly increased in the L. rhamnosus M21-treated group. An increase in sIgA as well as the diminution of inflammatory cells in bronchoalveolar lavage fluid was also observed in the L. rhamnosus M21-treated group. These results demonstrate that orally administered L. rhamnosus M21 activates humoral as well as cellular immune responses, conferring increased resistance to the host against influenza virus infection.

Phospholipase D Facilitates Efficient Entry of Influenza Virus, Allowing Escape from Innate Immune Inhibition

Lipid metabolism plays a fundamental role during influenza virus replication, although key regulators of lipid-dependent trafficking and virus production remain inadequately defined. This report demonstrates that infection by influenza virus stimulates phospholipase D (PLD) activity and that PLD co-localizes with influenza during infection. Both chemical inhibition and RNA interference of PLD delayed viral entry and reduced viral titers in vitro. Although there may be contributions by both major isoenzymes, the effects on viral infectivity appear to be more dependent on the PLD2 isoenzyme. In vivo, PLD2 inhibition reduced virus titer and correlated with significant increases in transcription of innate antiviral effectors. The reduction in viral titer downstream of PLD2 inhibition was dependent on Rig-I (retinoic acid-inducible gene-1), IRF3, and MxA (myxovirus resistance gene A) but not IRF7. Inhibition of PLD2 accelerated the accumulation of MxA in foci as early as 30 min postinfection. Together these data suggest that PLD facilitates the rapid endocytosis of influenza virus, permitting viral escape from innate immune detection and effectors that are capable of limiting lethal infection.

Inhibition of Akt kinase activity suppresses entry and replication of influenza virus

The possibility of the pandemic spread of influenza viruses highlights the need for an effective cure for this life-threatening disease. Influenza A virus, belonging to a family of orthomyxoviruses, is a negative-strand RNA virus which encodes 11 viral proteins. A numbers of intracellular signaling pathways in the host cells interact with influenza the viral proteins, which affect various stages of viral infection and replication.In this study, we investigated how inhibition of Akt kinase activity impacts on influenza virus infection by using “Akt-in”, a peptide Akt inhibitor. In PR8 influenza-infected A549 cells, Akt interacted with the NS1 (Non structural protein 1), and hence increased phosphorylation of Akt kinase activity and NS1. Treatment of cells with either “TCL1- or TCL1b-based Akt-in” efficiently suppressed Akt kinase activity while decreasing the levels of phosphorylated NS1; this, in turn, inhibited viral replication in a dose- and time-dependent manner. The inhibitory effect on viral replication appears to not be due to inhibition of the production of inflammatory cytokines, including IL-6 and IL-8, in the host cells. Inhibition of Akt kinase activity in the host cells inhibited the efficiency of viral entry, which is associated with decreased levels of phosphorylated glycogen synthase kinase 3, a substrate of Akt. Thus inhibition of Akt kinase activity in host cells may have therapeutic advantages for influenza virus infection by inhibiting viral entry and replication.

Influenza A virus encoding secreted Gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins.

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.

Novel Roles of Focal Adhesion Kinase in Cytoplasmic Entry and Replication of Influenza A Viruses

Viruses modulate cellular signaling pathways at almost every step of the infection cycle. Cellular signaling pathways activated at later times of influenza infection have previously been investigated; however, early influenza virus-host cell interactions remain understudied. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates phosphatidylinositol 3-kinase (PI3K) activation and actin reorganization, two critical processes during influenza A virus (IAV) infection in most cell types. Using 6 influenza A virus strains (A/Puerto Rico/8/1934, A/Aichi/2/1968 × A/Puerto Rico/8/1934 reassortant [X-31], A/California/04/2009, mouse-adapted A/California/04/2009, A/WSN/1933, and A/New Caledonia/20/1999), we examined the role of FAK during IAV entry. We found that influenza virus attachment induced PI3K-dependent FAK-Y397 phosphorylation. Pharmacological FAK inhibition or expression of a kinase-dead mutant of FAK led to disruption of the actin meshwork that resulted in sequestration of IAV at the cell periphery and reduced virion localization to early endosomes. Additionally, FAK inhibition impeded viral RNA replication at later times of infection and ultimately resulted in significantly reduced viral titers in both A549 and differentiated normal human bronchial epithelial (NHBE) cells. Although not all tested strains activated FAK, all of them exhibited a reduction in viral replication in response to inhibition of FAK signaling. These findings highlight novel biphasic roles of FAK activation during IAV infection and indicate that FAK serves as a central link between receptor-mediated PI3K activation and actin reorganization during IAV infection. We found that FAK links early activation of PI3K and actin reorganization, thereby regulating influenza virus entry. Surprisingly, we also found that FAK can regulate viral RNA replication independently of its role in entry. Our study addresses a knowledge gap in the understanding of signaling events triggered by influenza virus that mediate its internalization and initiation of the infection cycle. Understanding of these fundamental molecular events will be necessary to identify novel host targets, such as FAK, and development of future anti-influenza virus therapeutics.

Inhibition of influenza virus infection with chitosan–sialyloligosaccharides ionic complex

With the recent emergence of drug-resistant influenza viruses, effective means of preventing and treating these contagious pathogens have become imperative. The binding receptors of influenza virus are sialyloligosaccharides (SOS), which are present on the surfaces of host cells, and are therefore attractive targets for antiviral development. We report the preparation and identification of a novel influenza virus entry inhibitor, designated chitosan–SOS complex (CS complex). The CS complex was formed through noncovalent adsorption between cationic chitosan and anionic SOS, the latter derived from bovine colostrum. The preparation was accomplished in gram quantities from chitosan and bovine colostrum oligosaccharides by a one-step dialysis process. The inhibitory activity of the complex against influenza virus infection was determined by cytotoxicity inhibition assay (IC50=42μM). This simple preparation, combined with efficient anti-infective activity and the rich natural availability of chitosan and SOS, highlights the potential of the CS complex as a safe, practical agent for influenza prevention and control.

Evidence for Influenza Virus CNS Invasion Along the Olfactory Route in an Immunocompromised Infant

Central nervous system (CNS) disease is the most common extrarespiratory complication of influenza in humans. However, the pathogenesis, including the route of virus entry, is largely unknown. Here we present, for the first time, evidence of influenza virus entry into the CNS via the olfactory route in an immune-compromised infant. Since the nasal cavity is a primary site of influenza virus replication and is directly connected to the CNS via the olfactory nerve, these results imply that influenza virus invasion of the CNS may occur more often than previously believed.

Entry of influenza A virus: host factors and antiviral targets

Influenza virus is a major human pathogen that causes annual epidemics and occasional pandemics. Moreover, the virus causes outbreaks in poultry and other animals, such as pigs, requiring costly and laborious countermeasures. Therefore, influenza virus has a substantial impact on health and the global economy. Here, we review entry of this important pathogen into target cells, an essential process by which viral genomes are delivered from extracellular virions to sites of transcription/replication in the cell nucleus. We summarize current knowledge on the interaction of influenza viruses with their receptor, sialic acid, and highlight the ongoing search for additional receptors. We describe receptor-mediated endocytosis and the recently discovered macropinocytosis as alternative virus uptake pathways, and illustrate the subsequent endosomal trafficking of the virus with advanced live microscopy techniques. Release of virus from the endosome and import of the viral ribonucleoproteins into the host cell nucleus are also outlined. Although a focus has been on viral protein function during entry, recent studies have revealed exciting information on cellular factors required for influenza virus entry. We highlight these, and discuss established entry inhibitors targeting viral and host factors, as well as the latest prospects for designing novel 'anti-entry' compounds. New entry inhibitors are of particular importance for current efforts to develop the next generation of anti-influenza drugs - entry is the first essential step of virus replication and is an ideal target to block infection efficiently.

New Small Molecule Entry Inhibitors Targeting Hemagglutinin-Mediated Influenza A Virus Fusion

Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC(50)] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM(2) % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.

Structure–activity relationship analysis of curcumin analogues on anti‐influenza virus activity

Curcumin (Cur) is a commonly used colouring agent and spice in food. Previously, we reported that Cur inhibits type A influenza virus (IAV) infection by interfering with viral haemagglutination (HA) activity. To search for a stable Cur analogue with potent anti-IAV activity and to investigate the structure contributing to its anti-IAV activity, a comparative analysis of structural and functional analogues of Cur, such as tetrahydrocurcumin (THC) and petasiphenol (Pet), was performed. The result of time-of-drug addition tests indicated that these curcuminoids were able to inhibit IAV production in cell cultures. Noticeably, Pet and THC inhibit IAV to a lesser extent than Cur, which is in line with their effect on reducing plaque formation when IAV was treated with Cur analogues before infection. Unexpectedly, both THC and Pet did not harbour any HA inhibitory effect. It should be noted that the structure of Pet and THC differs from Cur with respect to the number of double bonds present in the central seven-carbon chain, and structure modelling of Cur analogues indicates that the conformations of THC and Pet are distinct from that of Cur. Moreover, simulation docking of Cur with the HA structure revealed that Cur binds to the region constituting sialic acid anchoring residues, supporting the results obtained by the inhibition of HA activity assay. Collectively, structure-activity relationship analyses indicate that the presence of the double bonds in the central seven-carbon chain enhanced the Cur -dependent anti-IAV activity and also that Cur might interfere with IAV entry by its interaction with the receptor binding region of viral HA protein.

Emerging antiviral strategies to interfere with influenza virus entry.

Influenza A and B viruses are highly contagious respiratory pathogens with a considerable medical and socioeconomical burden and known pandemic potential. Current influenza vaccines require annual updating and provide only partial protection in some risk groups. Due to the global spread of viruses with resistance to the M2 proton channel inhibitor amantadine or the neuraminidase inhibitor oseltamivir, novel antiviral agents with an original mode of action are urgently needed. We here focus on emerging options to interfere with the influenza virus entry process, which consists of the following steps: attachment of the viral hemagglutinin to the sialylated host cell receptors, endocytosis, M2‐mediated uncoating, low pH‐induced membrane fusion, and, finally, import of the viral ribonucleoprotein into the nucleus. We review the current functional and structural insights in the viral and cellular components of this entry process, and the diverse antiviral strategies that are being explored. This encompasses small molecule inhibitors as well as macromolecules such as therapeutic antibodies. There is optimism that at least some of these innovative concepts to block influenza virus entry will proceed from the proof of concept to a more advanced stage. Special attention is therefore given to the challenging issues of influenza virus (sub)type‐dependent activity or potential drug resistance.

The pH of Activation of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Replication and Pathogenesis in Mice

After receptor binding and internalization during influenza virus entry, the hemagglutinin (HA) protein is triggered by low pH to undergo irreversible conformational changes that mediate membrane fusion. To investigate how mutations that alter the activation pH of the HA protein influence the fitness of an avian H5N1 influenza virus in a mammalian model, we infected C57BL/6J or DBA/2J mice and compared the replication and virulence of recombinant A/chicken/Vietnam/C58/04 (H5N1) HA-Y231H mutant, wild-type, and HA-H241Q and HA-K582I mutant viruses that have HA activation pH values of 6.3, 5.9, 5.6, and 5.4, respectively. The HA-Y231H mutant virus was highly susceptible to acid inactivation in vitro and was attenuated for growth and virulence in mice, suggesting that an H5N1 HA protein triggered at pH 6.3 is too unstable for the virus to remain fit. Wild-type and HA-H241Q viruses were similar in pathogenicity and grew to similar levels in mice, ducks, and cell cultures derived from both avian and mammalian tissues, suggesting that H5N1 HA proteins triggered at pH values in the range of 5.9 to 5.6 broadly support replication. The HA-K582I mutant virus had greater growth and virulence in DBA/2J mice than the wild type did, although the mutant virus was highly attenuated in ducks. The data suggest that adaptation of avian H5N1 influenza virus for infection in mammals is supported by a decrease in the HA activation pH to 5.4. Identification of the HA activation pH as a host-specific infectivity factor is expected to aid in the surveillance and risk assessment of currently circulating H5N1 influenza viruses.

Bat-Derived Influenza Hemagglutinin H17 Does Not Bind Canonical Avian or Human Receptors and Most Likely Uses a Unique Entry Mechanism

A new influenza-like virus genome (H17N10) was recently discovered in bats and offers a new perspective about the origin and evolution of influenza viruses. The viral envelope glycoprotein hemagglutinin (HA) is responsible for influenza virus receptor binding, fusion, and entry into the cell; therefore, the structure and function of HA H17 was characterized. The 2.70 Å resolution crystal structure revealed that H17 has a typical influenza A virus HA fold, but with some special features, including a distorted putative sialic acid (SA) binding site and low thermostability. No binding to either the canonical human α2,6 SA-linkage or avian α2,3 SA-linkage receptor was observed. Furthermore, H17 glycan binding was not detected using a chip covering more than 600 glycans. Our results demonstrate that H17 is unique among characterized HAs and that the bat-derived influenza virus may use a different entry mechanism compared to canonical influenza viruses.