Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have ...examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.
The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed ...from a preference for avian receptors (α2,3-linked sialic acid) to a preference for human receptors (α2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA–receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225→Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.
Influenza A(H7N9) viruses have caused a large number of zoonotic infections since their emergence in 2013. They remain a public health concern due to the repeated high levels of infection with these ...viruses and their perceived pandemic potential. A major factor that determines influenza A virus fitness and therefore transmissibility is the interaction of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) with the cell surface receptor sialic acid. Typically, the HA is responsible for binding to the sialic acid to allow virus internalization and the NA is a sialidase responsible for cleaving sialic acid to aid virus spread and release. N9 NA has previously been shown to have receptor binding properties mediated by a sialic acid binding site, termed the hemadsorption (Hb) site, which is discrete from the enzymatically active sialidase site. This study investigated the N9 NA from a zoonotic H7N9 virus strain in order to determine its possible role in virus receptor binding. We demonstrate that this N9 NA has an active Hb site which binds to sialic acid, which enhances overall virus binding to sialic acid receptor analogues. We also show that the N9 NA can also contribute to receptor binding due to unusual kinetic characteristics of the sialidase site which specifically enhance binding to human-like α2,6-linked sialic acid receptors.
The interaction of influenza A virus glycoproteins with cell surface receptors is a major determinant of infectivity and therefore transmissibility. Understanding these interactions is important for understanding which factors are necessary to determine pandemic potential. Influenza A viruses generally mediate binding to cell surface sialic acid receptors via the hemagglutinin (HA) glycoprotein, with the neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release. Previous studies showed that the NA proteins of the N9 subtype can bind sialic acid via a separate binding site distinct from the sialidase active site. This study demonstrates for purified protein and virus that the NA of the zoonotic H7N9 viruses has a binding capacity via both the secondary binding site and unusual kinetic properties of the sialidase site which promote receptor binding via this site and which enhance binding to human-like receptors. This could have implications for understanding human-to-human transmission of these viruses.
The interaction of influenza A viruses with the cell surface is controlled by the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins have opposing activities: HA ...is responsible for binding the host receptor (sialic acid) to allow infection, and NA is responsible for cleaving the receptor to facilitate virus release. Several studies have demonstrated that compatible levels of HA and NA activity are required for a virus to replicate efficiently. This is consequently of great interest for determining virus transmissibility. The concurrent role of these two proteins in receptor binding has never been directly measured. We demonstrate a novel biophysical approach based on bio-layer interferometry to measure the balance of the activities of these two proteins in real time. This technique measures virus binding to and release from a surface coated with either the human-like receptor analog α2,6-linked sialic acid or the avian-like receptor analog α2,3-linked sialic acid in both the presence and absence of NA inhibitors. Bio-layer interferometry measurements were also carried out to determine the effect of altering HA receptor affinity and NA stalk length on receptor binding.
Background: Influenza A viruses contain the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), responsible for receptor binding and virus release, respectively.
Results: The contribution of HA and NA to virus interactions with receptor-coated surfaces was measured using bio-layer interferometry.
Conclusion: The balance between the activities of the two glycoproteins controls virus-cell interactions, therefore transmissibility.
Significance: This technique can be used to examine factors underlying emergent virus transmissibility.
A plasmonic bioassay for the specific detection of human influenza virus has been developed based on gold nanoparticles functionalised with a designed and synthesised thiolated trivalent ...α2,6-thio-linked sialic acid derivative. The glyconanoparticles consist of the thiolated trivalent α2,6-thio-linked sialic acid derivative and a thiolated polyethylene glycol (PEG) derivative self-assembled onto the gold surface. Varying ratios of the trivalent α2,6-thio-linked sialic acid ligand and the PEG ligand were used; a ratio of 25:75 was found to be optimum for the detection of human influenza virus X31 (H3N2). In the presence of the influenza virus a solution of the glyconanoparticles aggregate following the binding of the trivalent α2,6-thio-linked sialic acid ligand to the haemagglutinin on the surface of the virus. The aggregation of the glycoparticles with the influenza virus induces a colour change of the solution within 30 min. Non-purified influenza virus in allantoic fluid was successfully detected using the functionalised glyconanoparticles. A comparison between the trivalent and a monovalent α2,6-thio-linked sialic acid functionalised nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the trivalent ligand for the detection of the X31 virus. Importantly, the glyconanoparticles were able to discriminate between human (α2,6 binding) and avian (α2,3 binding) RG14 (H5N1) influenza virus highlighting the binding specificity of the trivalent α2,6-thio-linked sialic acid ligand.
Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which ...commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.
Background
Two new subclades of influenza A(H3N2) viruses became prominent during the 2014‐2015 Northern Hemisphere influenza season. The HA glycoproteins of these viruses showed sequence changes ...previously associated with alterations in receptor‐binding properties. To address how these changes influence virus propagation, viruses were isolated and propagated in conventional MDCK cells and MDCK‐SIAT1 cells, cells with enhanced expression of the human receptor for the virus, and analysed at each passage.
Methods
Gene sequence analysis was undertaken as virus was passaged in conventional MDCK cells and MDCK‐SIAT1 cells. Alterations in receptor recognition associated with passage of virus were examined by haemagglutination assays using red blood cells from guinea pigs, turkeys and humans. Microneutralisation assays were performed to determine how passage‐acquired amino acid substitutions and polymorphisms affected virus antigenicity.
Results
Viruses were able to infect MDCK‐SIAT1 cells more efficiently than conventional MDCK cells. Viruses of both the 3C.2a and 3C.3a subclades showed greater sequence change on passage in conventional MDCK cells than in MDCK‐SIAT1 cells, with amino acid substitutions being seen in both HA and NA glycoproteins. However, virus passage in MDCK‐SIAT1 cells at low inoculum dilutions showed reducing infectivity on continued passage.
Conclusions
Current H3N2 viruses should be cultured in the MDCK‐SIAT1 cell line to maintain faithful replication of the virus, and at an appropriate multiplicity of infection to retain infectivity.
Influenza A(H3N2) viruses are associated with outbreaks worldwide and can cause disease with severe complications. The impact can be reduced by vaccination, which induces neutralizing antibodies that ...mainly target the haemagglutinin glycoprotein (HA). In this study we generated neutralizing mouse monoclonal antibodies (mAbs) against A/Victoria/361/2011 and identified their epitopes by generating and sequencing escape viruses. The epitopes are located in antigenic site B, which is near the receptor-binding site and is immunodominant in humans. Amino acid (aa) substitutions at positions 156, 158, 159, 189, 190 and 193 in antigenic site B led to reduced ability of mAbs to block receptor-binding. The majority of A(H3N2) viruses that have been circulating since 2014 are antigenically distinct from previous A(H3N2) viruses. The neutralization-sensitive epitopes in antigenic site B of currently circulating viruses were examined with these mAbs. We found that clade 3C.2a viruses, possessing an additional potential glycosylation site at HA1 position N158, were poorly recognized by some of the mAbs, but other residues, notably at position 159, also affected antibody binding. Through a mass spectrometric (MS) analysis of HA, the glycosylated sites of HA1 were established and we determined that residue 158 of HA1 was glycosylated and so modified a neutralization-sensitive epitope. Understanding and monitoring individual epitopes is likely to improve vaccine strain selection.
Abstract As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission ...between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.
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