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Antigenic drift of influenza virus hemagglutinin (HA) is enabled by facile evolvability. However, HA antigenic site B, which has become immunodominant in recent human H3N2 influenza viruses, is also evolutionarily constrained by its involvement in receptor binding. Here, we employ deep mutational scanning to probe the local fitness landscape of HA antigenic site B in six different human H3N2 strains spanning from 1968 to 2016. We observe that the fitness landscape of HA antigenic site B can be very different between strains. Sequence variants that exhibit high fitness in one strain can be deleterious in another, indicating that the evolutionary constraints of antigenic site B have changed over time. Structural analysis suggests that the local fitness landscape of antigenic site B can be reshaped by natural mutations via modulation of the receptor-binding mode. Overall, these findings elucidate how influenza virus continues to explore new antigenic space despite strong functional constraints.

Background: Influenza is a zoonotic disease that infects millions of people each year resulting in hundreds of thousands of deaths, and in turn devastating pandemics. Influenza is caused by influenza viruses, including influenza A virus (IAV). There are many subtypes of IAV but only a few seem to be able to adapt to humans and to cause disease. In 2013, an H7N9 IAV subtype emerged in China that does not cause clinical symptoms in its chicken host but leads to severe infections when transmitted into humans. Since 2013, there have been six epidemic waves of H7N9 with 1567 laboratory-confirmed human infections and 615 deaths. Pathogenicity of IAV is complex, but a crucial feature contributing to virulence is the activation of the hemagglutinin (HA) fusion protein by host proteases that triggers membrane fusion and leads to subsequent virus propagation., Methods: 293T, VERO, and MDCK cells were used to conduct Western blot analysis, immunofluorescence assays, and pseudoparticle and live virus infections, and to evaluate H7N9 HA cleavage-activation., Results/conclusions: We show that human matriptase/ST 14 is able to cleave H7N9 HA. Cleavage of H7N9 HA expressed in cell culture results in fusogenic HA and syncytia formation. In infection studies with viral pseudoparticles carrying matriptase/ST 14-activated H7N9 HA, we observed a high infectivity of cells. Finally, human matriptase/ST 14 also activated H7N9 live virus which resulted in high infectivity. Our data demonstrate that human matriptase/ST 14 is a likely candidate protease to promote H7N9 infections in humans., (© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

The transmissibility and pandemic potential of influenza viruses depends on their ability to efficiently replicate and be released from an infected host, retain viability as they pass through the environment, and then initiate infection in the next host. There is a significant gap in knowledge about viral properties that enable survival of influenza viruses between hosts, due to a lack of experimental methods to reliably isolate viable virus from the air. Using a novel technique, we isolate and characterise infectious virus from droplets emitted by 2009 pandemic H1N1-infected ferrets. We demonstrate that infectious virus is predominantly released early after infection. A virus containing a mutation destabilising the haemagglutinin (HA) surface protein displayed reduced survival in air. Infectious virus recovered from droplets exhaled by ferrets inoculated with this virus contained mutations that conferred restabilisation of HA, indicating the importance of influenza HA stability for between-host survival. Using this unique approach can improve knowledge about the determinants and mechanisms of influenza transmissibility and ultimately could be applied to studies of airborne virus exhaled from infected people., Competing Interests: The authors have declared that no competing interests exist.

Influenza A viruses (IAV) have been a major public health threat worldwide, and options for antiviral therapy become increasingly limited with the emergence of drug-resisting virus strains. New and effective anti-IAV drugs, especially for highly pathogenic influenza, with different modes of action, are urgently needed. 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. In this study, we show that OA-10, a newly synthesized triterpene out of 11 oleanane-type derivatives, exhibited significant antiviral activity against four different subtypes of IAV (H1N1, H5N1, H9N2 and H3N2) replications in A549 cell cultures with EC 50 ranging from 6.7 to 19.6 μM and a negligible cytotoxicity (CC 50 > 640 μM). It inhibited acid-induced hemolysis in a dose-dependent manner, with an IC 50 of 26 µM, and had a weak inhibition on the adsorption of H5 HA to chicken erythrocytes at higher concentrations (≥40 µM). Surface plasmon resonance (SPR) analysis showed that OA-10 interacted with HA in a dose-dependent manner with the equilibrium dissociation constants (KD) of the interaction of 2.98 × 10 -12 M. Computer-aided molecular docking analysis suggested that OA-10 might bind to the cavity in HA stem region which is known to undergo significant rearrangement during membrane fusion. Our results demonstrate that OA-10 inhibits H5N1 IAV replication mainly by blocking the conformational changes of HA2 subunit required for virus fusion with endosomal membrane. These findings suggest that OA-10 could serve as a lead for further development of novel virus entry inhibitors to prevent and treat IAV infections.

Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low-pathogenicity avian influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortment and emergence in human populations. Since the presence of head region high-mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here, we investigate the activities of two recombinant human SP-D forms against representative LPAIV strains, including H2N1, H5N1, H6N1, H11N9, an avian H3N8, and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPAIV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between the protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite "N165" (H3 numbering) is occupied by high-mannose glycans in H3 HA but by complex glycans in all LPAIV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high-mannose glycan on the head region, our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three-dimensional structural analysis. IMPORTANCE Low-pathogenicity avian influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations, as was seen in the 1957 pandemic, in which an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first-line innate immunity defense, removes influenza type A virus (IAV) through interaction with hemagglutinin (HA) head region high-mannose glycan(s). While it is known that both H1 and H3 HAs have one or more key high-mannose glycosites in the head region, little is known about similar glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high-mannose glycans and do not interact with SP-D, and that sequence analysis can predict glycan subtype, thus predicting the presence or absence of this virulence marker., (Copyright © 2020 American Society for Microbiology.)

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The conserved hemagglutinin (HA) stem has been a focus of universal influenza vaccine efforts. Influenza A group 1 HA stem-nanoparticles have been demonstrated to confer heterosubtypic protection in animals; however, the protection does not extend to group 2 viruses, due in part to differences in glycosylation between group 1 and 2 stems. Here, we show that introducing the group 2 glycan at Asn38 HA1 to a group 1 stem-nanoparticle (gN38 variant) based on A/New Caledonia/20/99 (H1N1) broadens antibody responses to cross-react with group 2 HAs. Immunoglobulins elicited by the gN38 variant provide complete protection against group 2 H7N9 virus infection, while the variant loses protection against a group 1 H5N1 virus. The N38 HA1 glycan thus is pivotal in directing antibody responses by controlling access to group-determining stem epitopes. Precise targeting of stem-directed antibody responses to the site of vulnerability by glycan repositioning may be a step towards achieving cross-group influenza protection.

The influenza A virus (IAV) envelope protein hemagglutinin binds α2,6- or α2,3-linked sialic acid as a host cell receptor. Bat IAV subtypes H17N10 and H18N11 form an exception to this rule and do not bind sialic acid but enter cells via major histocompatibility complex (MHC) class II. Here, we review current knowledge on IAV receptors with a focus on sialoglycan variants, protein coreceptors, and alternative receptors that impact IAV attachment and internalization beyond the well-described sialic acid binding., (Copyright © 2020 American Society for Microbiology.)

Hemagglutinin (HA), a glycoprotein of Influenza A viruses and its proton channel M2 are site-specifically modified with fatty acids. Whereas two cysteines in the short cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to one cysteine located at the cytoplasmic border of the transmembrane region (TMR). M2 is palmitoylated at a cysteine positioned in an amphiphilic helix near the TMR. The enzymes catalyzing acylation of HA and M2 have not been identified, but zinc finger DHHC domain-containing (ZDHHC) palmitoyltransferases are candidates. We used a siRNA library to knockdown expression of each of the 23 human ZDHHCs in HA-expressing HeLa cells. siRNAs against ZDHHC2 and 8 had the strongest effect on acylation of HA as demonstrated by Acyl-RAC and confirmed by 3H-palmitate labeling. CRISPR/Cas9 knockout of ZDHHC2 and 8 in HAP1 cells, but also of the phylogenetically related ZDHHCs 15 and 20 strongly reduced acylation of group 1 and group 2 HAs and of M2, but individual ZDHHCs exhibit slightly different substrate preferences. These ZDHHCs co-localize with HA at membranes of the exocytic pathway in a human lung cell line. ZDHHC2, 8, 15 and 20 are not required for acylation of the HA-esterase-fusion protein of Influenza C virus that contains only stearate at one transmembrane cysteine. Knockout of these ZDHHCs also did not compromise acylation of HA of Influenza B virus that contains two palmitoylated cysteines in its cytoplasmic tail. Results are discussed with respect to the acyl preferences and possible substrate recognition features of the identified ZDHHCs., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses. IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations., (Copyright © 2020 American Society for Microbiology.)

Computationally optimized broadly reactive Ags (COBRA) targeting H1 elicit a broad cross-reactive and cross-neutralizing Ab response against multiple H1N1 viral strains. To assess B cell breadth, Mus musculus (BALB/c) Ab-secreting cells elicited by a candidate COBRA hemagglutinin (HA) (termed P1) were compared with Ab-secreting cells elicited by historical H1N1 vaccine strains. In addition, to evaluate the Ab response elicited by P1 HA at increased resolution, a panel of P1 HA-specific B cell hybridomas was generated following immunization of mice with COBRA P1 and the corresponding purified mAbs were characterized for Ag specificity and neutralization activity. Both head- and stem-directed mAbs were elicited by the P1 HA Ag, with some mAbs endowed with Ab-dependent cell-mediated cytotoxicity activity. P1 HA-elicited mAbs exhibited a wide breadth of HA recognition, ranging from narrowly reactive to broadly reactive mAbs. Interestingly, we identified a P1 HA-elicited mAb (1F8) exhibiting broad hemagglutination inhibition activity against both seasonal and pandemic H1N1 influenza strains. Furthermore, mAb 1F8 recognized an overlapping, but distinct, epitope compared with other narrowly hemagglutination inhibition-positive mAbs elicited by the P1 or wild-type HA Ags. Finally, P1 HA-elicited mAbs were encoded by distinct H chain variable and L chain variable gene segment rearrangements and possessed unique CDR3 sequences. Collectively, the functional characterization of P1 HA-elicited mAbs sheds further insights into the underlying mechanism(s) of expanded Ab breadth elicited by a COBRA HA-based immunogen and advances efforts toward design and implementation of a more broadly protective influenza vaccine., (Copyright © 2020 by The American Association of Immunologists, Inc.)

An aptamer is a synthetic oligonucleotide with a unique spatial structure that provides specific binding to a target. To date, several aptamers to hemagglutinin of the influenza A virus have been described, which vary in affinity and strain specificity. Among them, the DNA aptamer RHA0385 is able to recognize influenza hemagglutinins with highly variable sequences. In this paper, the structure of RHA0385 was studied by circular dichroism spectroscopy, nuclear magnetic resonance, and size-exclusion chromatography, demonstrating the formation of a parallel G-quadruplex structure. Three derivatives of RHA0385 were designed in order to determine the contribution of the major loop to affinity. Shortening of the major loop from seven to three nucleotides led to stabilization of the scaffold. The affinities of the derivatives were studied by surface plasmon resonance and an enzyme-linked aptamer assay on recombinant hemagglutinins and viral particles, respectively. The alterations in the loop affected the binding to influenza hemagglutinin, but did not abolish it. Contrary to aptamer RHA0385, two of the designed aptamers were shown to be conformationally homogeneous, retaining high affinities and broad binding abilities for both recombinant hemagglutinins and whole influenza A viruses., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Low pathogenic avian influenza (LPAI) H7N9 viruses have recently evolved to gain a polybasic cleavage site in the hemagglutinin (HA) protein, resulting in variants with increased lethality in poultry that meet the criteria for highly pathogenic avian influenza (HPAI) viruses. Both LPAI and HPAI variants can cause severe disease in humans (case fatality rate of ~40%). Here, we investigated the virulence of HPAI H7N9 viruses containing a polybasic HA cleavage site (H7N9-PBC) in mice. Inoculation of mice with H7N9-PBC did not result in observable disease; however, mice inoculated with a mouse-adapted version of this virus, generated by a single passage in mice, caused uniformly lethal disease. In addition to the PBC site, we identified three other mutations that are important for host-adaptation and virulence in mice: HA (A452T), PA (D347G), and PB2 (M483K). Using reverse genetics, we confirmed that the HA mutation was the most critical for increased virulence in mice. Our study identifies additional disease determinants in a mammalian model for HPAI H7N9 virus. Furthermore, the ease displayed by the virus to adapt to a new host highlights the potential for H7N9-PBC viruses to rapidly acquire mutations that may enhance their risk to humans or other animal species., Competing Interests: The authors declare no conflicts of interest.

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Native and recombinant collectins are purified by using mannan-agarose and an anti-collectin antibody column. The use of sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies against human mannan-binding lectin (MBL) enables elucidation of the collectin concentration in the blood, serum, and plasma. The collectin sugar specificity is demonstrated by determining the concentration of saccharide required to inhibit sugar binding by 50% in a saccharide-binding assay. Biological analyses including the complement-dependent hemolysis test and several other methods are used to evaluate collectin.

We describe a method to detect influenza virus using an evanescent-field-activated fluorescence scanner type glycan array and ELISA system. Neoglycoprotein was prepared by combination of organic chemistry and biomaterial preparation. These ligands were spotted on a glass plate or plastic well to make a glycan array and ELISA plate. We detected cultured influenza virus using glycan array and ELISA. Then, we showed that the neoglycoprotein binds to Cy3-labeled hemagglutinins (H1 and H5), a NeuAcα2,6LacNAc or NeuAcα2,3LacNAc recognized protein, as detected.

Antibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identify several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that are sensitive to substitutions in conventional antigenic sites and are therefore not broadly reactive. However, we also identify an H3N2 HA RBS-targeting mAb that is exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We show that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that HA RBS-targeting antibodies can be effective against variable viral strains even when they are somewhat sensitive to substitutions in HA residues adjacent to the RBS., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I, and K58 2 I (K58I in the HA2 subunit), which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1 virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells. IMPORTANCE Receptor specificity of the HA of IAVs is known to be a critical determinant of viral cell tropism. Here, we show that fusion properties of the HA may also play a key role in the tropism. Thus, we demonstrate that IAVs having a relatively low pH optimum of fusion cannot efficiently infect human endothelial cells owing to their relatively high endosomal pH and increased expression of fusion-inhibiting IFITM3 protein. These restrictions can be overcome by IAVs with elevated pH of fusion, such as zoonotic H5N1 and H7N9. Our results illustrate that the infectivity of IAVs depends on an interplay between HA conformational stability, endosomal acidification and IFITM3 expression in target cells, and the extracellular pH. Given significant variation of levels of HA stability among animal, human, and zoonotic IAVs, our findings prompt further studies on the fusion-dependent tropism of IAVs to different cell types in humans and its role in viral host range and pathogenicity., (Copyright © 2019 American Society for Microbiology.)

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population. IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses., (Copyright © 2019 American Society for Microbiology.)

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC 50 values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,600 human infections, posing a threat to public health. An emerging concern is whether H7N9 AIVs will cause pandemics among humans. Molecular analysis of hemagglutinin (HA), which is a critical determinant of interspecies transmission, shows that the current H7N9 AIVs are still dual-receptor tropic, indicating limited human-to-human transmission potency. Mutagenesis and structural studies reveal that a G186V substitution is sufficient for H7N9 AIVs to acquire human receptor-binding capacity, and a Q226L substitution would favor binding to both avian and human receptors only when paired with A138/V186/P221 hydrophobic residues. These data suggest a different evolutionary route of H7N9 viruses compared to other AIV-subtype HAs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we demonstrate a novel mechanism for hemagglutinin (HA) activation in a naturally occurring H7N6 avian influenza A virus strain, A/mallard duck/Korea/6L/2007 (A/Mdk/6L/07). This novel mechanism allows for systemic infection of chickens, ducks, and mice, and A/Mdk/6L/07 can replicate in vitro without exogenous trypsin and exhibits broad tissue tropism in animals despite the presence of a monobasic HA cleavage motif (PEIPKGR/G). The trypsin-independent growth phenotype requires the N6 neuraminidase and the specific recognition of glycine at the P2 position of the HA cleavage motif by a thrombin-like protease. Correspondingly, viral growth is significantly attenuated by the addition of a thrombin-like protease inhibitor (argatroban). These data provide evidence for a previously unrecognized virus replication mechanism and support the hypothesis that thrombin-mediated HA cleavage is an important virulence marker and potential therapeutic target for H7 influenza viruses. IMPORTANCE The identification of virulence markers in influenza viruses underpins risk assessment programs and the development of novel therapeutics. The cleavage of the influenza virus HA is a required step in the viral life cycle, and phenotypic differences in viruses can be caused by changes in this process. Here, we describe a novel mechanism for HA cleavage in an H7N6 influenza virus isolated from a mallard duck. The mechanism requires the N6 protein and full activity of thrombin-like proteases and allows the virus to cause systemic infection in chickens, ducks, and mice. The thrombin-mediated cleavage of HA is thus a novel virulence determinant of avian influenza viruses., (Copyright © 2019 Kwon et al.)

Influenza A virus (IAV) is one of the most common infectious pathogen and associated with significant morbidity and mortality. Although processing the IAV hemagglutinin (HA) envelope glycoprotein precursor is a pre-requisite for viral membrane fusion activity, viral entry and transmission, HA-processing protease is not encoded in the IAV genome and thus the cellular trypsin-type serine HA-processing proteases determine viral infectious tropism and viral pathogenicity. The initial process of IAV infection of the airway is followed by marked upregulation of ectopic trypsin in various organs and endothelial cells through the induction of various proinflammatory cytokines, and this process has been termed the "influenza virus-cytokine-trypsin" cycle. In the advanced stage of IAV infection, the cytokine storm induces disorders of glucose and lipid metabolism and the "metabolic disorders-cytokine" cycle is then linked with the "influenza virus-cytokine-trypsin" cycle, to advance the pathogenic process into energy crisis and multiple organ failure. Application of protease inhibitors and treatment of metabolic disorders that break these cycles and their interconnection is therefore a promising therapeutic approach against influenza. This review discusses IAV pathogenicity on trypsin type serine HA-processing proteases, cytokines, metabolites and therapeutic options., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: A pandemic influenza viral strain, influenza A/California/07/2009 (pdmH1N1), has been considered to be a potential issue that needs to be controlled to avoid the seasonal emergence of mutated strains., Materials and Methods: In this study, aptamer-antibody complementation was implemented on a multiwalled carbon nanotube-gold conjugated sensing surface with a dielectrode to detect pandemic pdmH1N1. Preliminary biomolecular and dielectrode surface analyses were performed by molecular and microscopic methods. A stable anti-pdmH1N1 aptamer sequence interacted with hemagglutinin (HA) and was compared with the antibody interaction. Both aptamer and antibody attachments on the surface as the basic molecule attained the saturation at nanomolar levels., Results: Aptamers were found to have higher affinity and electric response than antibodies against HA of pdmH1N1. Linear regression with aptamer-HA interaction displays sensitivity in the range of 10 fM, whereas antibody-HA interaction shows a 100-fold lower level (1 pM). When sandwich-based detection of aptamer-HA-antibody and antibody-HA-aptamer was performed, a higher response of current was observed in both cases. Moreover, the detection strategy with aptamer clearly discriminated the closely related HA of influenza B/Tokyo/53/99 and influenza A/Panama/2007/1999 (H3N2)., Conclusion: The high performance of the abovementioned detection methods was supported by the apparent specificity and reproducibility by the demonstrated sensing system., Competing Interests: The authors have no conflicts of interest to declare in this work., (© 2019 Wang et al.)

Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections. IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment., (Copyright © 2019 American Society for Microbiology.)

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal., Competing Interests: The authors have declared that no competing interests exist.

Antibody-based molecular recognition plays a central role in today's life sciences, ranging from immunoassays to molecular imaging and antibody-based therapeutics. Control over antibody activity by using external triggers such as light could further increase the specificity of antibody-based targeting. Here we present bivalent peptide-DNA ligands containing photocleavable linkers as a noncovalent approach by which to allow photoactivation of antibody activity. Light-triggered cleavage of the 3-amino-3-(2-nitrophenyl)propionic acid peptide linker converted the high-affinity bivalent peptide-DNA lock into weakly binding monovalent ligands, effectively restoring antibody targeting of cell-surface receptors. In this work, a proof of principle was provided with an anti-hemagglutinin antibody, but the molecular design of the lock is generic and applicable to any monoclonal antibody for which an epitope or mimotope of sufficient affinity is available., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Influenza A viruses enter host cells through the endocytic pathway, where acidification triggers conformational changes of the viral hemagglutinin (HA) to drive membrane fusion. During this process, the HA fusion peptide is extruded from its buried position in the neutral pH structure and targeted to the endosomal membrane. Conserved ionizable residues near the fusion peptide may play a role in initiating these structural rearrangements. We targeted highly conserved histidine residues in this region, at HA1 position 17 of Group-2 HA subtypes and HA2 position 111 of Group-1 HA subtypes, to determine their role in fusion activity. WT and mutant HA proteins representing several subtypes were expressed and characterized, revealing that His 111 is essential for HA functional activity of Group-1 subtypes, supporting continued efforts to target this region of the HA structure for vaccination strategies and the design of antiviral compounds., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The glycoprotein hemagglutinin of influenza virus plays a key role in the initial stage of virus infection, making it a potential target for novel influenza viruses entry inhibitors. Two "privileged fragments", 2-(piperidin-1-yl)ethan-1-amine and 2-(1,3-oxazinan-3-yl)ethan-1-amine were integrated into 3-O-β-chacotriosyl ursolic acid producing new derivatives 5 and 6 with improved activity against IAVs in vitro. Mechanistically, compound 6 was effective in inhibiting infection of H1-, H3-, and H5-typed influenza A viruses by interfering with the viral hemagglutinin. Furthermore, the docking studies were in agreement with the antiviral data. These results showed that the title compound 6 as a new lead compound was meriting further optimization and development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The potential avian influenza pandemic remains a threat to public health, as the avian-origin influenza A(H7N9) virus has caused more than 1,560 laboratory-confirmed human infections since 2013, with nearly 40% mortality. Development of low-pathogenic candidate vaccine viruses (CVVs) for vaccine production is essential for pandemic preparedness. However, the suboptimal growth of CVVs in mammalian cells and chicken eggs is often a challenge. By introducing a single adaptive substitution, G218E, into the hemagglutinin (HA), we generated reassortant A(H7N9)-G218E CVVs that were characterized by significantly enhanced growth in both cells and eggs. These G218E CVVs retained the original antigenicity, as determined by a hemagglutination inhibition assay, and effectively protected ferrets from lethal challenge with the highly pathogenic parental virus. We found that the suboptimal replication of the parental H7 CVVs was associated with impeded progeny virus release as a result of strong HA receptor binding relative to weak neuraminidase (NA) cleavage of receptors. In contrast, the G218E-mediated growth improvement was attributed to relatively balanced HA and NA functions, resulted from reduced HA binding to both human- and avian-type receptors, and thus facilitated NA-mediated virus release. Our findings revealed that a single amino acid mutation at residue 218 of the HA improved the growth of A(H7N9) influenza virus by balancing HA and NA functions, shedding light on an alternative approach for optimizing certain influenza CVVs. IMPORTANCE The circulating avian influenza A(H7N9) has caused recurrent epidemic waves with high mortality in China since 2013, in which the alarming fifth wave crossing 2016 and 2017 was highlighted by a large number of human infections and the emergence of highly pathogenic avian influenza (HPAI) A(H7N9) strains in human cases. We generated low-pathogenic reassortant CVVs derived from the emerging A(H7N9) with improved virus replication and protein yield in both MDCK cells and eggs by introducing a single substitution, G218E, into HA, which was associated with reducing HA receptor binding and subsequently balancing HA-NA functions. The in vitro and in vivo experiments demonstrated comparable antigenicity of the G218E CVVs with that of their wild-type (WT) counterparts, and both the WT and the G218E CVVs fully protected ferrets from parental HPAI virus challenge. With high yield traits and the anticipated antigenicity, the G218E CVVs should benefit preparedness against the threat of an A(H7N9) influenza pandemic., (Copyright © 2019 Li et al.)

Vaccines that elicit broadly cross-neutralizing antibodies, including antibodies that target the conserved stem of hemagglutinin (HA), are being developed as a strategy for next-generation influenza vaccines that protect against influenza across multiple years. However, efficient induction of cross-neutralizing antibodies remains a challenge, and potential escape mutations have not been well characterized. Here we elicited cross-neutralizing antibodies by immunizing animals with the hemagglutinins from H5 and H9 subtype influenza A viruses that are sensitive to neutralization by stem antibodies. We further isolated and characterized an HA stem monoclonal antibody 4C2 that broadly neutralizes group 1 influenza viruses and identified HA mutations that reduced sensitivity to stem antibodies. Our results offer insights for next-generation influenza vaccine strategies for inducing cross-neutralizing antibodies., Competing Interests: The authors have declared that no competing interests exist.

The highly pathogenic (HP) avian influenza virus (AIV), H5N1 and reassortant H5-subtype HPAIVs, H5N2, H5N6, and H5N8, cause high mortality in domestic birds, resulting in economic losses in the poultry industry. H5N1 and H5N6 also pose significant public health risks and H5N1 viruses are a permanent pandemic threat. To control HPAIVs, eukaryotic expression systems have traditionally been exploited to produce vaccines based on hemagglutinin (HA), a protective viral antigen. In contrast, we used a bacterial expression system to produce vaccine targeting the HA protein. A fragment of the HA ectodomain from H5N1, with a multibasic cleavage site deletion, was expressed in Escherichia coli , refolded, and chromatographically purified from inclusion bodies. The resulting antigen, rH5- E. coli , was validated in terms of conformational integrity and oligomerization status. Previously, the protective efficacy of rH5- E. coli adjuvanted with aluminum hydroxide, has been positively verified by challenging the specific pathogen-free layer chickens with homologous and heterologous H5N1 HPAIVs. Protection was provided primarily by the H5 subtype-specific antibodies, as detected in the FluAC H5 test. The present studies were conducted to assess the performance of alum-adjuvanted rH5- E. coli in commercial birds. Broiler chickens were vaccinated twice with 25 μg of rH5- E. coli at 2- and 4-week intervals, while the layer chickens were vaccinated with 5- to 25-μg antigen doses at 4- and 6-week intervals. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition (HI) tests. Prime-boost immunizations with rH5- E. coli elicited H5 HA-specific antibodies in all the chickens tested. Two antigen doses administered at 4- or 6-week intervals were sufficient to induce neutralizing antibodies against H5-subtype HAs; however, they were ineffective when applied with a 2-week delay. In the layers, 80% to 100% of individuals developed antibodies that were active in the FluAC H5 and/or HI tests. A dose-sparing effect was seen when using the longer prime-boost interval. In the broiler chickens, 62.5% positivity was achieved in the FluAC H5 and/or HI tests. The trials confirmed the vaccine potential of rH5- E. coli and provided indications for anti-influenza vaccination with respect to the chicken type and immunization scheme.

We previously demonstrated that despite no airborne transmissibility increase compared to low pathogenic avian influenza viruses, select human isolates of highly pathogenic avian influenza A(H7N9) virus exhibit greater virulence in animal models and a lower threshold pH for fusion. In the current study, we utilized both in vitro and in vivo approaches to identify key residues responsible for hemagglutinin (HA) intracellular cleavage, acid stability, and virulence in mice. We found that the four amino acid insertion (-KRTA-) at the HA cleavage site of A/Taiwan/1/2017 virus is essential for HA intracellular cleavage and contributes to disease in mice. Furthermore, a lysine to glutamic acid mutation at position HA2-64 increased the threshold pH for HA activation, reduced virus stability, and replication in mice. Identification of a key residue responsible for enhanced acid stability of A(H7N9) viruses is of great significance for future surveillance activities and improvements in vaccine stability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

An influenza A(H1N1)pdm09 and an influenza B virus were passaged in 3-fluoro(eq)-4-guanidino difluoro sialic acid (3Feq4Gu DFSA), an inhibitor of the influenza neuraminidase (NA) to determine whether resistant variants could be selected. 3Feq4Gu DFSA is a mechanism-based inhibitor, forming a covalent link to Y406 in the NA active site. Given its similarity to the natural substrate, sialic acid, we predicted resistant variants would be difficult to select. Yields of both viruses decreased with passaging, so that after 12 passages both viruses were only growing to low titers. Drug concentrations were decreased for another three passages. There was no difference in NA sensitivity in the MUNANA fluorescence-based assay, nor in plaque assays for the passaged virus stocks. All influenza B plaques were still wild type in all assays. There were isolated small diffuse plaques in the P15 pdm09 stock, which after purification had barely detectable NA or hemagglutinin (HA) activity. These had a novel non-active site I106M substitution in the NA gene, but unexpectedly no HA changes. The I106M may impact NA function through steric effects on the movement of the 150 and 430-loops. The I106M viruses had similar replication kinetics in MDCK cells as wild type viruses, but their ability to bind to and infect CHO-K1 cells expressing high levels of cell-bound mucin was compromised. The I106M substitution was unstable, with progeny rapidly reverting to wild type by three different mechanisms. Some had reverted to I106, some had V106, both with wild type NA and HA properties. A third group retained the I106M, but had a compensating R363K substitution, which regained almost wild type NA properties. These viruses now agglutinated chicken red blood cells (CRBCs) but unlike the I/V106, they rebound after elution at 37 °C. There were no mutations in the HA, but each phenotype correlated with the NA sequence. We propose that the activity in the I106M mutant is insufficient to remove carbohydrates from the virion HA and NA, sterically limiting HA access to CRBC receptors, thus resulting in poor HA binding., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug. IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug., (Copyright © 2019 American Society for Microbiology.)

Avian influenza viruses (AIV) are classified into 16 hemagglutinin (HA; H1-H16) and 9 neuraminidase (NA; N1-N9) subtypes. All AIV are low pathogenic (LP) in birds, but subtypes H5 and H7 AIV can evolve into highly pathogenic (HP) forms. In the last two decades evolution of HPAIV H7 from LPAIV has been frequently reported. However, little is known about the pathogenesis and evolution of HP H7 from LP ancestors particularly, in non-chicken hosts. In 2015, both LP and HP H7N7 AIV were isolated from chickens in two neighbouring farms in Germany. Here, the virulence of these isogenic H7N7 LP, HP and LP virus carrying a polybasic HA cleavage site (HACS) from HP (designated LP-Poly) was studied in chickens, turkeys and different duck breeds. The LP precursor was avirulent in all birds. In contrast, all inoculated and contact chickens and turkeys died after infection with HP. HP infected Pekin and Mallard ducks remained clinically healthy, while Muscovy ducks exhibited moderate depression and excreted viruses at significantly higher amounts. The polybasic HACS increased virulence in a species-specific manner with intravenous pathogenicity indices of 3.0, 1.9 and 0.2 in chickens, turkeys and Muscovy ducks, respectively. Infection of endothelial cells was only observed in chickens. In summary, Pekin and Mallard were more resistant to HPAIV H7N7 than chickens, turkeys and Muscovy ducks. The polybasic HACS was the main determinant for virulence and endotheliotropism of HPAIV H7N7 in chickens, whereas other viral and/or host factors play an essential role in virulence and pathogenesis in turkeys and ducks.

Hemagglutinin (HA) of influenza virus is incorporated into cholesterol-enriched nanodomains of the plasma membrane. Phylogenetic group 2 HAs contain the conserved cholesterol consensus motif (CCM) YKLW in the transmembrane region. We previously reported that mutations in the CCM retarded intracellular transport of HA and decreased its nanodomain association. Here, we analyzed whether cholesterol interacts with the CCM. Incorporation of photocholesterol into HA was significantly reduced if the whole CCM is replaced by alanine, both using immunoprecipitated HA and when HA is embedded in the membrane. We next used reverse genetics to investigate the significance of the CCM for virus replication. No virus was rescued if the whole motif is exchanged (YKLW4A); singly (LA) or doubly (YK2A and LW2A) mutated virus showed decreased titers and a comparative fitness disadvantage. In polarized cells, transport of HA mutants to the apical membrane was not disturbed. Reduced amounts of HA and cholesterol were incorporated into the viral membrane. Mutant viruses exhibit a decrease in hemolysis, which is only partially corrected if the membrane is replenished with cholesterol. More specifically, viruses have a defect in hemifusion, as demonstrated by fluorescence dequenching. Cells expressing HA YKLW4A fuse with erythrocytes, but the number of events is reduced. Even after acidification unfused erythrocytes remain cell bound, a phenomenon not observed with wild-type HA. We conclude that cholesterol binding to a group 2 HA is essential for virus replication. It has pleiotropic effects on virus assembly and membrane fusion, mainly on lipid mixing and possibly a preceding step. IMPORTANCE The glycoprotein HA is a major pathogenicity factor of influenza viruses. Whereas the structure and function of HA's ectodomain is known in great detail, similar data for the membrane-anchoring part of the protein are missing. Here, we demonstrate that the transmembrane region of a group 2 HA interacts with cholesterol, the major lipid of the plasma membrane and the defining element of the viral budding site nanodomains of the plasma membrane. The cholesterol binding motif is essential for virus replication. Its partial removal affects various steps of the viral life cycle, such as assembly of new virus particles and their subsequent cell entry via membrane fusion. A cholesterol binding pocket in group 2 HAs might be a promising target for a small lipophilic drug that inactivates the virus., (Copyright © 2019 American Society for Microbiology.)

Multivalent carbohydrate-based ligands were synthesized and evaluated as inhibitors of the adhesion protein HA of the influenza A virus (IAV). HA relies on multivalency for strong viral adhesion. While viral adhesion inhibition by large polymeric molecules has proven viable, limited success was reached for smaller multivalent compounds. By linking of sialylated LAcNAc units to di- and trivalent scaffolds, inhibitors were obtained with an up to 428-fold enhanced inhibition in various assays.

Candidate vaccine viruses (CVVs) for seasonal influenza A virus are made by reassortment of the antigenic virus with an egg-adapted strain, typically A/Puerto Rico/8/34 (PR8). Many 2009 A(H1N1) pandemic (pdm09) high-growth reassortants (HGRs) selected this way contain pdm09 segment 2 in addition to the antigenic genes. To investigate this, we made CVV mimics by reverse genetics (RG) that were either 6 : 2 or 5 : 3 reassortants between PR8 and two pdm09 strains, A/California/7/2009 (Cal7) and A/England/195/2009, differing in the source of segment 2. The 5 : 3 viruses replicated better in MDCK-SIAT1 cells than the 6 : 2 viruses, but the 6 : 2 CVVs gave higher haemagglutinin (HA) antigen yields from eggs. This unexpected phenomenon reflected temperature sensitivity conferred by pdm09 segment 2, as the egg HA yields of the 5 : 3 viruses improved substantially when viruses were grown at 35 °C compared with 37.5 °C, whereas the 6 : 2 virus yields did not. However, the authentic 5 : 3 pdm09 HGRs, X-179A and X-181, were not markedly temperature sensitive despite their PB1 sequences being identical to that of Cal7, suggesting compensatory mutations elsewhere in the genome. Sequence comparisons of the PR8-derived backbone genes identified polymorphisms in PB2, NP, NS1 and NS2. Of these, PB2 N701D affected the temperature dependence of viral transcription and, furthermore, improved and drastically reduced the temperature sensitivity of the HA yield from the 5 : 3 CVV mimic. We conclude that the HA yield of pdm09 CVVs can be affected by an epistatic interaction between PR8 PB2 and pdm09 PB1, but that this can be minimized by ensuring that the backbones used for vaccine manufacture in eggs contain PB2 701D.

Viral glycoproteins are under constant immune surveillance by a host's adaptive immune responses. Antigenic variation including glycan introduction or removal is among the mechanisms viruses have evolved to escape host immunity. Understanding how glycosylation affects immunodominance on complex protein antigens may help decipher underlying B cell biology. To determine how B cell responses can be altered by such modifications, we engineered glycans onto the influenza virus hemagglutinin (HA) and characterized the molecular features of the elicited humoral immunity in mice. We found that glycan addition changed the initially diverse antibody repertoire into an epitope-focused, genetically restricted response. Structural analyses showed that one antibody gene family targeted a previously subdominant, occluded epitope at the head interface. Passive transfer of this antibody conferred Fc-dependent protection to influenza virus-challenged mice. These results have potential implications for next-generation viral vaccines aimed at directing B cell responses to preferred epitope(s)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Influenza A H3N2 has been linked to annual outbreaks within the human population attributable to continuous structural changes. H3N2 HA contains well identified antigenic sites and receptor-binding sites (RBS) that are possibly correlated to viral evolution and infection. However, the structural significance of amino acid residues associated with both viral evolution and infection were not fully demonstrated. Throughout this study, we generated and analyzed H3N2 HA models that represented the clade 3C.2 population (comprised of clades 3C.2, 3C.2a, and 3C.21 from the transitioning 2014-2018 H3N2 strains) and 3C.3a (from the 2016 H3N2 strain). Model quality estimation, structural analyses and superimposition, and network analytics of H3N2 HA1 evolution were performed. We found that the structural properties of residues 158-160 could influence the overall HA backbone. More specifically, amino acid substitutions at residues 159-160 affected the amino acid orientation at residue 158, thereby, causing the overall HA backbone structure to vary. Our results were consistent with 1968-2018 HA1 evolution. Taken together, we propose that our results would highlight the structural significance of residues 158-160 in HA1 for both antigenic drift and RBS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The hemagglutinin (HA) and neuraminidase (NA) of influenza A virus possess antagonistic activities on interaction with sialic acid (SA), which is the receptor for virus attachment. HA binds SA through its receptor-binding sites, while NA is a receptor-destroying enzyme by removing SAs. The function of HA during virus entry has been extensively investigated, however, examination of NA has long been focused to its role in the exit of progeny virus from infected cells, and the role of NA in the entry process is still under-appreciated. This review summarizes the current understanding of the roles of HA and NA in relation to each other during virus entry.

The influenza virus hemagglutinin (HA) protein has HA1 and HA2 subunits, which form an initial complex. HA1's bind host cell sialic acids, which triggers endocytosis, HA1/HA2 separation, and HA2-mediated fusion between virus and endosome membranes. We report hydrogen-deuterium exchange mass spectrometry (HDX-MS) on the HA2 subunit without HA1. HA2 contains the fusion peptide (FP), soluble ectodomain (SE), transmembrane domain (TM), and endodomain. FP is a monomer by itself, while SE is a trimer of hairpins that includes an interior bundle of residue 38-105 helices, turns, and residue 154-178 strands packed antiparallel to the bundle. FP and TM extend from the same side of the SE hairpin, and fusion models often depict a FP/TM complex with membrane traversal of both domains that is important for membrane pore expansion. The HDX-MS data of this study do not support this complex and instead support independent FP and TM with respective membrane-interfacial and traversal locations. The data also show a low level of aqueous exposure of the 22-38 segment, consistent with retention of the 23-35 antiparallel β sheet observed in the initial HA1/HA2 complex. We propose the β sheet as a semirigid connector between FP and SE that enables close membrane apposition prior to fusion. The I173E mutant exhibits greater exchange for residues 22-69 and 150-191, consistent with dissociation of SE C-terminal strands from interior N-helices. Similar trends are observed for the G1E mutant as well as less exchange for G1E FP. Fusion is highly impaired with either mutant, which correlates with reduced membrane apposition and, for G1E, FP binding to SE rather than the target membrane.

Cell-penetrating peptides (CPPs) show promise as an attractive delivery vehicle for therapeutic molecules-including nucleic acids, peptides, proteins, and even particulates-into several cell types. It is important to identify new CPPs and select the optimal CPP for each application, because CPPs differ in their internalized efficiency and internalization mechanisms. Here, we identified new CPPs derived from the peptides with the hemagglutinin cleavage site (pHACS) of highly pathogenic influenza viruses. We compared the potential of peptides from the pHACS of four subtypes of influenza A virus (H1, H3, H5, and H7) and an influenza B virus (H1-pHACS, H3-pHACS, H5-pHACS, H7-pHACS, and B-pHACS, respectively) to serve as CPPs. H5-pHACS and H7-pHACS, but not the other peptides, bound to mouse dendritic cells and human epithelial cells and were internalized efficiently into these cells. H5-pHACS and H7-pHACS required glycosaminoglycans, especially heparan sulfate and neuropilins, to bind to the cells. In addition, we designed a mutant H7-pHACS with superior cell-binding capability by changing a single amino acid. Furthermore, when conjugated with antigen, H5-pHACS and H7-pHACS induced antigen-specific antibody responses, demonstrating the usefulness of this antigen-delivery vehicle. Our results will improve our understanding of the mechanisms of CPPs and facilitate the development of novel drug-delivery vehicles designed to improve therapeutic efficacy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Since 2013, the H7 subtype avian influenza virus (AIV-H7) has seriously endangered human life and health, and has had a serious impact on the poultry industry in China. A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects the antibody for AIV-H7 was developed, basing on a monoclonal antibody (mAb) against the neutralizing epitopes on hemagglutinin (HA)gene. Twelve hybridoma cell lines were screened by cell fusion. Hemagglutination inhibition (HI) assay and indirect ELISA were used to identify the competitive effect of the mAbs. High-affinity mAb 1H11 was selected as a competitive antibody. The reaction conditions for the C-ELISA were optimized for AIV-H7 antibody detection. The cross-reactivity of the C-ELISA was determined by AIV-(H1H15), NDV, IBV and IBDV positive serum. A total of 1294 field samples (chicken (462), duck (318), goose (219), quail (203) and pigeon (92) were simultaneously detected by C-ELISA and HI assay. The C-ELISA was found to have a high specificity of 93.23% and a sensitivity of 96.24%. These results reveal a positive coincidence between C-ELISA and HI assay at a coincidence rate of 97.52%. In addition, It confirmed that this method can be used for the diagnosis of AIV-H7 antibodies from chicken, ducks, goose, quail and pigeons., (Copyright © 2019. Published by Elsevier Inc.)

The influenza A virus genome consists of eight single-stranded negative-sense RNA segments. The noncoding regions located at the 3'- and 5'- ends of each segment are necessary for genome packaging, and the terminal coding regions are required to precisely bundle the eight segments. However, the nucleotide residues important for genome bundling are not defined. Here, we introduced premature termination codons in the hemagglutinin (HA) or matrix protein 2 (M2) gene and constructed virus libraries containing random sequences in the terminal coding regions. Using these virus libraries, we identified nucleotide residues involved in efficient virus propagation. Viral genome packaging was impaired in viruses that contained single mutations at these identified residues. Furthermore, these single mutations altered the local structure of the viral ribonucleoprotein complex. Our results show that specific nucleotide residues in the viral protein coding region are involved in forming the precise structure of the viral ribonucleoprotein complex., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human antibody-based immunity to influenza A virus is limited by antigenic drift resulting from amino acid substitutions in the hemagglutinin (HA) head domain. Glycan addition can cause large antigenic changes but is limited by fitness costs to viral replication. Here, we report that glycans are added to H1 and H3 HAs at discrete 5-to-7-year intervals, until they reach a functional glycan limit, after which glycans are swapped at approximately 2-fold-longer intervals. Consistent with this pattern, 2009 pandemic H1N1 added a glycan at residue N162 over the 2015-2016 season, an addition that required two epistatic HA head mutations for complete glycosylation. These strains rapidly replaced H1N1 strains globally, by 2017 dominating H3N2 and influenza B virus strains for the season. The pattern of glycan modulation that we outline should aid efforts for tracing the epidemic potential of evolving human IAV strains. IMPORTANCE Frequent mutation of its major antibody target, the glycoprotein hemagglutinin, ensures that the influenza virus is perennially both a rapidly emerging virus and a major threat to public health. One type of mutation escapes immunity by adding a glycan onto an area of hemagglutinin that many antibodies recognize. This study revealed that these glycan changes follow a simple temporal pattern. Every 5 to 7 years, hemagglutinin adds a new glycan, up to a limit. After this limit is reached, no net additions of glycans occur. Instead, glycans are swapped or lost at longer intervals. Eventually, a pandemic replaces the terminally glycosylated hemagglutinin with a minimally glycosylated one from the animal reservoir, restarting the cycle. This pattern suggests the following: (i) some hemagglutinins are evolved for this decades-long process, which is both defined by and limited by successive glycan addition; and (ii) hemagglutinin's antibody dominance and its capacity for mutations are highly adapted features that allow influenza to outpace our antibody-based immunity.

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Though oseltamivir and zanamivir are the active anti-influenza drugs, the emergence of different strains of influenza A virus with mutations creates drug-resistance to these drugs. Therefore, it is essential to find a suitable approach to stop the viral infection. The present study focuses on understanding the conformational changes of the HA2 protein at different pH levels (pH 7, pH 6, pH 5) and on blocking the low pH-induced conformational changes of the HA2 protein with a suitable ligand using molecular docking and molecular dynamics (MD) simulation methods. As the pH value decreases to pH 5, the protein undergoes large conformational changes with less stability in the order of pH 7 > pH 6 > pH 5. The fusion peptide (residues 1-20) and the extended loop (residues 58-75) deviate more at pH 5. The ligand stachyflin bound between the N- and C-terminal helix regions retains the stability of the HA2 protein at pH 5 and blocks the low pH-induced conformational transition. The performance of stachyflin is increased when it directly interacts with residues at the intramonomer binding site rather than the intermonomer binding site. The susceptibility of the HA2 protein of different subtypes to stachyflin is in the order of H1 > H7 > H5 > H2 > H3. Stachflin has a higher binding affinity for H1 (at pH 7, pH 6, pH 5) and H7 subtypes than others. Lys47, Lys58, and Glu103 are the key residues that favor the binding and highly stabilize the HA2 protein at low pH. Graphical abstract Low pH-induced conformational change of influenza HA2 protein.

Most neutralizing antibodies against highly pathogenic avian influenza A virus H5N1 recognize the receptor-binding site (RBS) on the globular head domain and the stem of H5N1 hemagglutinin (HA). Through comprehensive analysis of multiple human protective antibodies, we previously identified four vulnerable sites (VS1-VS4) on the globular head domain. Among them, the VS1, occupying the opposite side of the RBS on the same HA, was defined by the epitope of antibody 65C6. In this study, we report the crystal structures of two additional human H5N1 antibodies isolated from H5N1-infected individuals, 3C11 and AVFluIgG01, bound to the head at 2.33- and 2.30-Å resolution, respectively. These two new antibody epitopes have large overlap with and extend beyond the original VS1. Site-directed mutagenesis experiments identified eight pivotal residues (Ser-126b, Lys-165, Arg-166, Ser-167, Tyr-168, Asn-169, Thr-171, and Asn-172) critical for 65C6-, 3C11-, and AVFluIgG01-binding and neutralization activities. These residues formed a unique "Y"-shaped surface on H5N1 globular head and are highly conserved among H5N1 viruses. Our results further support the existence of a vulnerable site distinct from the RBS and the stem region of H5N1 HA, and future design of immunogens should take this particular site into consideration., (© 2019 Wang et al.)