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. Author manuscript; available in PMC: 2016 Mar 11.
Published in final edited form as: Cell Host Microbe. 2015 Mar 11;17(3):377–384. doi: 10.1016/j.chom.2015.02.006

A human-infecting H10N8 influenza virus retains a strong preference for avian-type receptors

Heng Zhang 1,4,#, Robert P de Vries 2,$,#, Netanel Tzarum 1,#, Xueyong Zhu 1, Wenli Yu 1, Ryan McBride 2, James C Paulson 2,*, Ian A Wilson 1,3,*
PMCID: PMC4359746  NIHMSID: NIHMS665526  PMID: 25766296

SUMMARY

Recent avian-origin H10N8 influenza A viruses that have infected humans pose a potential pandemic threat. Alterations in the viral surface glycoprotein, hemagglutinin (HA), typically allow influenza A viruses to cross the species barrier for adaptation to a new host, but whether H10N8 contains adaptations supporting human infection remains incompletely understood. We investigated whether the H10N8 HA can bind human receptors. Sialoside glycan microarray analysis showed that the H10 HA retains a strong preference for avian receptor analogs and negligible binding to human receptor analogs. Crystal structures of H10 HA with avian and human receptor analogs revealed the basis for preferential recognition of avian-like receptors. Furthermore, introduction of mutations into the H10 receptor-binding site (RBS) known to convert other HA subtypes from avian to human receptor specificity failed to switch the preference to human receptors. Collectively, these findings suggest the current H10N8 human isolates are poorly adapted for efficient human-to-human transmission.

INTRODUCTION

In December 2013, a reassortant H10N8 influenza A virus was identified in Jiangxi province, China [A/Jiangxi-Donghu/346/2013 (H10N8)] (Chen et al., 2014). Three further cases of human infection with H10N8 virus (AIV) have been confirmed, leading to two deaths (Report of Health and Family Planning Commission of Jiangxi Province, 2014). In May 2014, this H10N8 AIV was reported to be infectious among feral dogs in live poultry markets in Guangdong Province, China (Su et al., 2014). Thus, it is of major public health interest to understand the extent to which the current circulating H10N8 viruses have evolved any capability to bind human receptors and thus facilitate human-to-human transmission (Garcia-Sastre et al., 2014).

Several H10 viruses cause disease in mammals. H10N7 viruses caused conjunctivitis in humans in Egypt in 2004 and Australia in 2010 and 2012 (Arzey et al., 2012; PAHO EID Weekly Updates, 2004), whereas aerosol infection of mink with an H10N7 virus led to mild pulmonary lesions (Englund et al., 2000). Very recently, H10N7 virus was detected in dead seals and involved in mass mortality in Denmark, Sweden, Germany and the Netherlands (Zohari et al., 2014). Relatively few studies have been carried out on avian H10N8 infections in humans and other species. An avian H10N8 strain (A/environment/Dongting Lake/Hunan/3-9/2007), isolated from water samples of Dongting Lake wetland, replicated efficiently in the mouse lung, and virulence increased rapidly during adaptation, indicating ability to adapt to a mammalian host (Zhang et al., 2011). Phylogenetic analysis shows that human H10N8 originated through the reassortment of H9N2 strains with other viruses circulating in poultry and in environmental samples (such as wild birds and water samples from their habitat in the wetlands) from Jiangxi Province; its hemagglutinin (HA) and neuraminidase (NA) genes originated from ducks and wild birds, respectively (Chen et al., 2014; Shi et al., 2014; Liu et al., 2015). This type of reassortment is similar to influenza A H5N1 and H7N9 viruses isolated from humans; the H10N8 virus also acquired six internal gene segments from an H9N2 virus (Chen et al., 2014).

HA is the viral surface glycoprotein responsible for viral entry into host cells through binding to sialylated receptors on the cell surface followed by pH-triggered membrane fusion in endosomal compartments. A switch in receptor-binding specificity from avian α2-3 to human α2-6 linked receptors is a major obstacle for influenza A viruses to cross the species barrier for adaptation to a new host. The Gly225-Gln226-Ser227-Gly228 (H3 numbering is used throughout) motif in the receptor-binding site (RBS) of the human H10N8 HA suggested avian-like receptor binding preference. Only one basic amino acid (arginine) in the cleavage site between HA1 and HA2 was consistent with its low pathogenicity in poultry (Chen et al., 2014). However, the H10N8 HA contained Ala135Thr and Ser138Ala substitutions that favor mammalian adaptation; M1 Asn30Asp and Thr215Ala and NS1 Pro42Ser substitutions are also associated with increased virulence in mice (Chen et al., 2014).

To understand the underlying mechanism of human infection by an H10N8 virus and its possible transmission capabilities, we performed a comprehensive study of its receptor-binding properties and determined HA crystal structures with avian and human receptor analogs. The H10N8 HA has a strong preference for avian-like receptors and negligible binding to human-like receptors, which indicates poor adaptation of human-infecting H10N8 influenza viruses for human-to-human transmission.

RESULTS

Receptor Binding of H10 HA

We analyzed binding of recombinant H10 HA to avian and human linear glycan receptor analogs, α2-3-sialylated di-N-acetyllactosamine (SLNLN) and α2-6 SLNLN, respectively. The ELISA-like binding assay showed that H10 HA has specific recognition for avian analog α2-3 SLNLN, but no detectable binding to human analog α2-6 SLNLN, even at high concentrations (up to 50 μg/ml, Figure 1A). Similarly, by biolayer interferometry, specific binding was observed to avian analog α2-3 SLNLN (apparent Kd of 0.86 μM and 0.65 μM (for Kd1 and Kd2)) with no detectable binding to human analog α2-6 SLNLN (Figure 1B and Figure S1A). This binding affinity is similar to human H7N9 HA (A/Shanghai/2/2013) with apparent Kd >1 μM to α2-3 SLNLN and no detectable binding to α2-6 SLNLN (Xu et al., 2013). However, human H10N8 HA was recently reported to have similar binding affinities to avian-like receptor 3′-SLN and human-like receptor 6′-SLN (1.81 and 1.39 mM, respectively) (Vachieri et al., 2014).

Figure 1. Receptor Binding Properties of Human H10N8 HA (A/Jiangxi-Donghu/346/2013).

Figure 1

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(A) Glycan binding specificity of H10 HA by an ELISA-like assay. The micro-well glass slides were imprinted with either avian receptor α2-3- or human receptor α2-6-linked SLNLN-polyacrylamide (PAA) and probed with recombinant HAs produced in human embryonic kidney (HEK293S GnTI−) cells.

(B) Glycan binding specificity of H10 HA by biolayer interferometry on an Octet Red system (ForteBio) using recombinant HAs produced in insect cells. HA binding at 0.55 mg/ml was measured against immobilized biotinylated glycans α2-3- and α2-6-SLNLN. The binding curve for α2-3-SLNLN was best fitted using the 2:1 heterogeneous ligand binding model (Concepcion et al., 2009), with apparent Kd values of 0.86 μM and 0.65 μM. See also Figure S1A.

(C) Receptor binding analysis of H10 HA by a sialoside glycan microarray using recombinant HAs produced in human embryonic kidney (HEK293S GnTI−) cells. Recombinant HAs from H5N1 and human seasonal H1N1 viruses were used as controls. The mean signal and standard error were calculated from four independent replicates. Nonsialylated controls (#1 and 2), α2-3 sialosides are in white bars (#3-35), α2-6 linked sialosides in black bars (#36-56), and mixed biantennary glycans in gray bars (#57 and 58). Glycans imprinted on the array are listed in Table S1. See also Figure S1B.

We therefore further characterized H10 HA receptor binding on a custom influenza receptor glycan microarray comprised of diverse α2-3 sialosides (#3 to 35) and α2-6 sialosides (#36 to 56) that correspond to biologically relevant N- and O-linked glycans and linear fragments on mammalian glycoproteins and glycolipids (Table S1). Human H10 HA expressed in mammalian cells showed strong selective recognition of avian α2-3 receptors analogs, with negligible binding to human-like α2-6 linked receptors (Figure 1C), similar to H5N1 HA (A/Viet Nam/1203/2004), and in marked contrast to a human seasonal H1N1 virus (A/Kentucky/UR06-0258/2007) (Figure 1C). The bound α2-3 sialosides include sulfated linear glycans (#3, 4, and 7), linear and short branched O-linked glycans (#17 and 18), and long branched biantennary O-linked and N-linked glycans (#23 to 26). Additional avian α2-3 receptors analogs can be bound by H10 HA expressed in insect cells (Figure S1B), which can be attributed to less hindrance from the smaller high mannose glycans on the HA surface (Stevens et al., 2006b; Xu et al., 2013). Thus, human H10 HA exhibits strong avian-type receptor specificity, with no detectable binding to human-type receptors, similar to HAs from other recent human-infecting, avian-origin H7N9, H5N1 and H6N1 viruses analyzed by us and others (Paulson and de Vries, 2013; Shi et al., 2013; Xu et al., 2013; de Vries et al., 2014; Tzarum et al., 2015).

Structural Characterization of H10N8 HA and its RBS

Sequence alignment of human H10N8 HA with those in avian H10N8 and H10 viruses isolated from mammals, showed the H10 HA RBS residues are highly conserved (Figure 2A), although many substitutions can be observed (Table S2). To explore the structural characteristics of human H10N8 HA, we first determined its crystal structure at 2.60 Å resolution (Figure 2B; Table S3) using human H7N9 HA (A/Shanghai/2/2013) [Protein Data Bank (PDB) entry 4N5J] as the molecular replacement search model. The H10 and H7 HA structures are similar with an overall Cα root mean square deviation (RMSD) of 1.3 Å and only 0.4 Å for the receptor binding subdomain (residues 117-265) (Ha et al., 2002). Superimposition of the H10 HA monomer on other avian and human HAs confirms that H10 is structurally closest to Group 2 H3 and H7 HAs (PDB entries 4FNK and 4N5J) with overall Cα RMSDs of 1.3-1.8Å, compared to 2.6-2.9 Å for Group 1 H1, H2 and H5 HAs (PDB entries 3AL4, 3KU5 and 2FKO). Electron density for two N-linked glycosylation sites was observed at the conserved Asn35, as well as at Asn242, in each HA1 (Figure 2B).

Figure 2. Crystal Structure of Human H10N8 HA (A/Jiangxi-Donghu/346/2013).

Figure 2

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(A) Sequence alignment of the RBS of HAs from representative H10N8 strains from Asia, North America and Europe. Human A/Jiangxi-Donghu/346/2013 virus was isolated from a similar geographic area to avian A/environment/Dongting Lake/Hunan/3-9/2007 and Guangdong/12 (A/duck/Guangdong/E1/2012) in China. Different residues are shown in red. Residues between human and avian H10 HAs in RBSs are highly conserved. See also Figure S2A, B.

(B) HA trimer is shown in cartoon representation. For one protomer, HA1 is colored in green and HA2 in yellow. The other two protomers are in gray. N-glycosylation sites and N-linked glycans are highlighted in sticks and numbered at the Asn attachment site.

(C) The RBS of human H10N8 HA with a characteristic avian glutamine (Q226).

The RBS base consists of highly conserved Tyr98, Trp153, His183, and Tyr195, and its sides are formed from three secondary elements 130-loop, 190-helix and 220-loop (Figure 2C). The main differences in the H10 RBS arise from Ala135Thr, Ser137Arg and Leu226Gln substitutions compared to human H7 HA (A/Shanghai/2/2013) (Table S4).

Structure of the Human H10 HA in Complex with an Avian Receptor Analog

To uncover the basis for preferential recognition of avian-like receptors, we determined the H10 HA structure with avian receptor analog 3′-SLN at 2.85 Å (Table S3). Clear, interpretable, electron density was observed for all sugars of 3′-SLN: sialic acid (Sia-1), galactose-2 (Gal-2) and glucoseN-acetylglucosamine-3 (GlcNAc-3) (Figure 3A).

Figure 3. Crystal Structures of Human H10N8 HA in Complex with Avian and Human Receptor Analogs.

Figure 3

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(A) Glycan structures and unbiased omit electron density maps of avian receptor analog 3′-SLN and human receptor analog 6′-SLN in human H10 HA complexes, contoured at a 1σ level. In the glycan structures, purple diamonds represent sialic acid (Sia), yellow circles represent galactose (Gal) and blue squares represent glucose N-acetylglucosamine (GlcNAc). In 3′-SLN complex structure, electron density for all three sugars is well defined in chains A and C, while Sia-1 and Gal-2 are visible in chain E. In 6′-SLN complex structure, electron density for all three sugars is well defined in chain A, while Sia-1 and Gal-2 is interpretable in chain C and no sugar is observed in chain E (RBS may be partially occluded).

(B and C) Interactions between the H10 HA RBS and avian and human receptor analogs. Conserved secondary elements of the RBS (130-loop, 190-helix and 220-loop) are labeled and shown in cartoon representation. Selected residues and receptor analogs are labeled and shown in sticks. The RBS is colored in gray and receptor analogs in orange. Hydrogen bond interactions of Sia-1, Gal-2 and GlcNAc-3 of 3′-SLN (B) and 6′-SLN (C) with the H10 HA RBS are shown. See also Figures S3 and S4.

Seven highly conserved hydrogen bonds are formed between Sia-1 and the 190-helix, 130-loop and 220-loop (Figure 3B). The Gln226 side chain makes three direct contacts with the O1-hydroxyl and O3-carboxyl group of Sia-1 (2.4 Å, 2.8 Å and 3.4 Å). This mode of recognition is conserved in avian HA subtypes (Xu and Wilson, 2012). The Tyr98 hydroxyl hydrogen bonds to Sia-1 8- and 9-hydroxyls and Trp153 makes hydrophobic interactions with Sia-1 C7 and C8 (Figure 3B). A slightly stronger conserved hydrogen bond (2.9 Å) between His183 Nε-2 and Sia-1 9-hydroxyl can be observed compared to most HA-avian receptor structures (3.1-3.6 Å). The major RBS substitution between avian and human H10 HAs is Lys137Arg. In human H10 HA, Arg137 Nε-2 and its main-chain amide hydrogen bond with the 1-hydroxyl of Sia-1 (2.9 Å) and 2-hydroxyl of Gal-2 (3.1 Å), respectively. However, no contact was observed between Lys137 and the avian receptor in the recent avian H10N2 HA structure (PDB entry 4CZ0) (Vachieri et al., 2014).

Similar to most avian HA structures, including avian wild-type H5 (Liu et al., 2009; Lin et al., 2009; Xiong et al., 2013; Xu et al., 2013), 3′-SLN binds in a trans conformation (Figure 3B and Figure S3A). The second (Gal) and the third (GlcNAc) sugars in 3′-SLN exit above the 220-loop. This conformation is stabilized by hydrogen bonding of Gal-2 to Gln226 in most avian HA-receptor analogs (Liu et al., 2009; Lin et al., 2009; Xiong et al., 2013). Similarly, the trans conformation in H10 complex is mediated by hydrogen bonding of Gal-2 (O3 and O4) to Gln226 and also from Gal-2 (O2) to Arg137 (Figure 3B), which has only been reported for an avian analog with a human-transmissible H5 mutant (Zhang et al., 2013). The Q226 interactions with the avian receptor resemble those of avian H1, and are distinct from those in human H1 HAs (Lin et al., 2009; Xu et al., 2012) (Figure S3B). The preference of human H10 for avian receptors therefore correlates with the presence of glutamine 226 as in avian H1N1 HAs.

Structure of Human H10 HA in Complex with a Human Receptor Analog

Despite no detectable binding of α2-6 SLNLN to H10 HA in the glycan array (Figure 1A, B), soaking of HA crystals with very high concentrations (5mM; HA: ligand stoichiometry of 1:55) of human receptor analog 6′-SLN enabled visualization of this weakly bound ligand at 3.31 Å resolution (Table S3). Electron density for all three sugars was fairly well defined with weaker density for GlcNAc-3 (Figure 3A). The 6′-SLN binds in a cis conformation (Figure 3C) similar to other human HA-6′SLN structures (Lin et al., 2009; Lin et al., 2012; Shi et al., 2013; Xu et al., 2012; Zhang et al., 2013), but with a different orientation due to rotation around the Gal-2 C6-C5 bond compared to human H1/H3 and ferret-transmissible H5 complexes, but similar to the human H7 complex (Figures S4A-4D). The receptor also has slightly different phi angles (O6Sia-C2Sia-O-C6Gal) from those in H1/H3/H5 complexes (41° in H10 to 60-70° in H1/H3/H5/H7, Figures S4A-D). This conformation differs from the low energy solution conformation of α2-6 sialosides (Sabesan et al., 1991) or the equivalent umbrella-like topology observed in HA-human-like (α2-6) receptor structures (Chandrasekaran et al., 2008). Thus, the H10 HA RBS is not configured for binding of human α2-6 receptors.

The hydrogen bonding interactions of Sia-1 with the RBS are conserved and similar to the 3′-SLN complex (Figure 3B, C), but the Gal-2 interactions differ. Gal-2 3-hydroxyl hydrogen bonds to Gln222 Nε-2 and Oε-1 and Gly225 main-chain carbonyl. Interactions between Gal-2 4-hydroxyl and Gln222 Nε-2 and Ser227 main-chain amide mediated via a well-ordered water molecule (W2) (Figure 3C), also help stabilize the Gal-2 conformation. Overall, our results suggest the current H10 has not yet evolved to preferentially bind human receptors, and Arg137 has no direct contact with the human receptor (Figure 3C), in comparison to another study of human H10 (Figure S4E-G).

Mutation of Key Residues in the RBS of the Human H10 HA

The potential for H10 HA to acquire human type specificity was assed using mutations associated with the switch in human pandemic viruses, namely Q226L/G228S in H2N2/H3N2 and E190D/G225D in H1N1. Neither pair of mutations causes a significant switch in receptor specificity (Figure 4). The Q226L substitution reduces affinity to α2-3 sialosides, but gains only very weak binding to human-type α2-6 receptors (Figure 4A, B). The G228S mutant does not affect binding avidity or overall specificity compared to wild type HA (Figure 4C, D); however, several fucosylated structures (#32-34) are now bound. The double mutant Q226L/G228S shows increased binding to PAA-linked 6′-SLNLN but with no reduction in binding to PAA-linked 3′-SLNLN in the ELISA type assay, but largely abrogates binding to both types of receptors in the glycan array (Figure 4E, F). Such differential binding results obtained by ELISA-like assay and glycan array (presumably due to higher density and, hence, avidity effects with PAA polymers in the ELISA-like assay) have been observed for human H7 HA (A/Shanghai/2/2013) and receptor mutants of human H5N1 (A/Vietnam/1203/2004) (Chen et al., 2012; Xu et al., 2013). The G225D mutation on the H10 HA framework retains specificity for α2-3-sialylated glycans (Figure 4G,H), but with reduced avidity to a different subset of glycan receptors relative to wild-type (Figure 1C). The double E190D/G225D mutant exhibits dramatically reduced avidity in both assays (Figure 4I, J), as observed previously for A/Vietnam/1203/2004 (Stevens, et al., 2006c).

Figure 4. Receptor Binding Specificities of Human H10 HA RBS Mutants by ELISA-like Glycan Binding and Glycan Microarray Assays.

Figure 4

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(A, C, E and G) Micro-well glass slides imprinted with either α2-3– or α2-6–linked SLNLN-PAA were probed with recombinant HA mutants (of RBS residues) produced in human embryonic kidney (HEK293S GnTI−) cells.

(B, D, F and H) The same recombinant proteins were used for assessment of receptor binding specificity on a glycan array, as in Figure 1C. The mean signal and standard error were calculated from six independent replicates. The mutations associated with a switch from avian to human receptor binding in pandemic H1/H2/H3 viruses did not generate human receptor binding for human H10 HA.

DISCUSSION

Analysis of the receptor specificity and x-ray structures of the H10 HA clearly show that the avian-origin human H10N8 influenza virus exhibits a clear preference for avian-type receptors, as for other avian influenza viruses. Using a different assay, receptor binding by avian (mallard) H10N2 viruses was recently reported to exhibit a preference for avian-type receptors, but with almost as strong binding to human receptors as pandemic H1 (1918 Spanish influenza) and H3 (1968 Hong Kong influenza) (Vachieri et al., 2014). This apparent discrepancy in binding specificity is likely a reflection of a difference in the assays in the two studies in this study, such as the use of whole influenza viruses instead of hemagglutinin, and the use of lower avidity short receptor analogs that are not bound in the glycan microarray (α2-3 and α2-6 linked SLN; Fig. 1C, glycans #8, 38).

The nature of the HA receptor binding specificity is believed to be a critical factor for efficient virus transmission between within and between species. Adaptation of avian HAs in group 1 (H1, H2 and H5) and in group 2 (H3 and H7) for human receptor specificity occurs by different mechanisms involving several key RBS mutations (Connor et al., 1994; Gamblin et al., 2004; Matrosovich et al., 2000; Nobusawa et al., 2000; Rogers et al., 1983; Stevens et al., 2006a and 2006c). In H1N1 pandemic influenza viruses, Glu190Asp and Gly225Asp substitutions switch specificity to human α2-6 receptors, while avian Gln226 and Gly228 are maintained (Gamblin et al., 2004; Matrosovich et al., 2000; Nobusawa et al., 2000) (Table S4). However, Gln226Leu and Gly228Ser mutations completely change specificity to human receptors in pandemic H2N2 and H3N2 viruses, representing an adaptation that is essential for efficient human-to-human transmission (Connor et al., 1994; Rogers et al., 1983) (Table S4). Q226L and G228S are also known to affect the receptor binding preference of H5 HA A/Indonesia/5/05 (Chutinimitkul et al., 2010). Loss of glycosylation in H5 HA (A/Vietnam/1203/2004) in combination with these two substitutions enhances virus replication in ferrets (Wang et al., 2010), but not transmission between ferrets (Chen et al, 2012). Recently, E627K in PB2, H99Y in PB1, H107Y in HA, T160A (loss of a glycosylation site) in HA, and either HA-Q226L or HA-G228S (H5 numbering) in HA, were found to constitute minimal sets of substitutions for airborne transmission of A/Indonesia/5/05 virus between ferrets (Herfst et al., 2012; Linster et al., 2014). Another recent study showed four mutations, N158D (affecting the same N-glycosylation sequon as T160A), N224K, Q226L and T318I in H5 HA (A/Vietnam/1203/2004) with the remaining seven gene segments from a 2009 pandemic H1N1 virus, enable efficient airborne transmission in ferrets (Imai et al., 2012). In our study, single or double mutations that occurred in human H1/H2/H3 HAs did not support preferential binding of human-type receptors indicating H10 HA adaptation to human specificity may differ from known pandemic viruses.

Distinct from the finding that H10 virus (avian H10N2) possesses significant avidity for human receptors (Vachieri et al., 2014), our studies at the structural and receptor specificity level revealed that the human H10N8 is a typical avian influenza virus with strong specificity for avian receptors. Our results are in agreement with the recent report from the Gao group (Wang et al., 2015), and suggest the present human H10N8 is poorly adapted for efficient human-to-human transmission, as also for human H7N9, H5N1 and H6N1 viruses.

EXPERIMENTAL PROCEDURES

Expression and Purification of HAs in Insect Cells and Mammalian Cells

Recombinant H10 HA was expressed in Hi5 insect cells using a pFastBac vector and purified by His-tag affinity purification, dialyzed, cleaved by trypsin and gel filtration chromatography, and used for crystallization and determination of binding affinities by biolayer interferometry. Uncleaved proteins were used for glycan microarray analyses. Recombinant HAs were also expressed in HEK293S GnTI−/− cells using the pCD5 vector and purified from cell culture supernatants as described previously (de Vries et al., 2010; Xu et al., 2013), and used for ELISA-like glycan binding and glycan microarray analyses. Additional details are found in Supplemental Experimental Procedures.

Crystallization and Structural Determination of the H10 HA

H10 HA crystals were obtained using vapor diffusion sitting drops at 4 °C with 0.2 M NaSCN, 20% (w/v) PEG 3350. Complexes with receptor analogues were obtained by soaking HA crystals in reservoir solution that contained glycan ligands (5 mM). Diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) (Table S3). The H10 apo structure was solved by molecular replacement method and was used as the starting model for determination of the H10 HA-glycan structures.

Glycan Binding of HAs by Biolayer Interferometry

Recombinant HAs were buffer-exchanged into PBS buffer. Association of HAs was measured on an Octet Red (ForteBio) against immobilized biotinylated 3′-SLNLN and 6′-SLNLN (V-Labs, Covington, LA) at 30 °C.

ELISA-like Glycan Binding and Glycan Microarray Analyses of HAs

Recombinant HAs were pre-complexed with horseradish peroxidase (HRP)-linked mouse anti-Strep-tag antibody (IBA) and with Alexa647-linked anti-mouse IgG (Life Biosciences prior to incubation. The mixtures were incubated on the micro-well slides that contained PAA-linked 3′-SLNLN and 6′-SLNLN (provided by the Consortium of Functional Glycomics) or on the microarray, followed by washing, scanning and analyses.

Supplementary Material

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ACKNOWLEDGEMENTS

This work was funded in part by National Institutes of Health Grants R56 AI099275 (to I.A.W) and AI099274 (to J.C.P). RPdV is a recipient of Rubicon and VENI grants from the Netherlands Organization for Scientific Research (NWO). We thank Robyn Stanfield, X. Dai and M. Elsliger for crystallographic and computational support, Henry Tien of the Robotics Core at the Joint Center for Structural Genomics for automated crystal screening, staff at the Stanford Synchrotron Radiation Light source (SSRL) beamlines 11-1 and 12-2. GM/CA CAT is funded in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and NIGMS (Y1-GM-1104). The SSRL is a Directorate of Stanford Linear Accelerator Center National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. DOE Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by NIH, NIGMS (including P41GM103393) and National Center for Research Resources (NCRR, P41RR001209). Several glycans used for HA binding assays were provided by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/) funded by NIGMS grant GM62116 (J.C.P.) We acknowledge the authors, originating and submitting laboratories for HA sequences obtained from GISAID’s EpiFlu Database and from the influenza database of the National Center for Biotechnology Information (NCBI).

Footnotes

SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and four tables, and detailed methods and references.

AUTHOR CONTRIBUTIONS Project design by H.Z., R.P.d.V., N.T., J.C.P. and I.A.W.; protein expression and production H.Z., R.P.d.V., W.Y. and N.T.; X-ray work and analysis by H.Z., N.T. and X.Z.; glycan array work by R.P.d.V. and R.McB.; and manuscript written by H.Z., R.P.d.V., N.T., J.C.P. and I.A.W. All authors were asked to comment on the manuscript. This is manuscript 29039 from The Scripps Research Institute.

ACCESSION NUMBERS Atomic coordinates and structure factors are deposited in the Protein Data Bank (PDB) under accession codes 4XQ5 for A/Jiangxi-Donghu/346/2013(H10N8) HA and 4XQU and 4XQO for complexes with 3′-SLN and 6′-SLN, respectively.

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REFERENCES

  1. Arzey GG, Kirkland PD, Arzey KE, Frost M, Maywood P, Conaty S, Hurt AC, Deng YM, Iannello P, Barr I, et al. Influenza virus A (H10N7) in chickens and poultry abattoir workers, Australia. Emerg. Infect. Dis. 2012;18:814–816. doi: 10.3201/eid1805.111852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chandrasekaran A, Srinivasan A, Raman R, Viswanathan K, Raguram S, Tumpey TM, Sasisekharan V, Sasisekharan R. Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat. Biotechnol. 2008;26:107–113. doi: 10.1038/nbt1375. [DOI] [PubMed] [Google Scholar]
  3. Chen H, Yuan H, Gao R, Zhang J, Wang D, Xiong Y, Fan G, Yang F, Li X, Zhou J, et al. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study. Lancet. 2014;383:714–721. doi: 10.1016/S0140-6736(14)60111-2. [DOI] [PubMed] [Google Scholar]
  4. Chen LM, et al. In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology. 2012;422:105–113. doi: 10.1016/j.virol.2011.10.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chutinimitkul S, van Riel D, Munster VJ, van den Brand JM, Rimmelzwaan GF, Kuiken T, Osterhaus AD, Fouchier RA, de Wit E. In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity. J. Virol. 2010;84:6825–6833. doi: 10.1128/JVI.02737-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Connor RJ, Kawaoka Y, Webster RG, Paulson JC. Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology. 1994;205:17–23. doi: 10.1006/viro.1994.1615. [DOI] [PubMed] [Google Scholar]
  7. Couceiro JN, Paulson JC, Baum LG. Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity. Virus Res. 1993;29:155–165. doi: 10.1016/0168-1702(93)90056-s. [DOI] [PubMed] [Google Scholar]
  8. de Vries RP, de Vries E, Moore KS, Rigter A, Rottier PJ, de Haan CA. Only two residues are responsible for the dramatic difference in receptor binding between swine and new pandemic H1 hemagglutinin. J. Biol. Chem. 2011;286:5868–5875. doi: 10.1074/jbc.M110.193557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. de Vries RP, Zhu X, McBride R, Rigter A, Hanson A, Zhong G, Hatta M, Xu R, Yu W, Kawaoka Y, et al. Hemagglutinin receptor specificity and structural analyses of respiratory droplet-transmissible H5N1 viruses. J. Virol. 2014;88:768–773. doi: 10.1128/JVI.02690-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Englund L. Studies on influenza viruses H10N4 and H10N7 of avian origin in mink. Vet. Microbiol. 2000;74:101–107. doi: 10.1016/s0378-1135(00)00170-x. [DOI] [PubMed] [Google Scholar]
  11. Gamblin SJ, Haire LF, Russell RJ, Stevens DJ, Xiao B, Ha Y, Vasisht N, Steinhauer DA, Daniels RS, Elliot A, et al. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science. 2004;303:1838–1842. doi: 10.1126/science.1093155. [DOI] [PubMed] [Google Scholar]
  12. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 2013;368:1888–1897. doi: 10.1056/NEJMoa1304459. [DOI] [PubMed] [Google Scholar]
  13. Garcia-Sastre A, Schmolke M. Avian influenza A H10N8--a virus on the verge? Lancet. 2014;383:676–677. doi: 10.1016/S0140-6736(14)60163-X. [DOI] [PubMed] [Google Scholar]
  14. Ha Y, Stevens DJ, Skehel JJ, Wiley DC. H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. Embo J. 2002;21:865–875. doi: 10.1093/emboj/21.5.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, et al. Airborne transmission of influenza A/H5N1 virus between ferrets. Science. 2012;336:1534–1541. doi: 10.1126/science.1213362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, et al. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature. 2012;486:420–428. doi: 10.1038/nature10831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lin T, Wang G, Li A, Zhang Q, Wu C, Zhang R, Cai Q, Song W, Yuen KY. The hemagglutinin structure of an avian H1N1 influenza A virus. Virology. 2009;392:73–81. doi: 10.1016/j.virol.2009.06.028. [DOI] [PubMed] [Google Scholar]
  18. Lin YP, Xiong X, Wharton SA, Martin SR, Coombs PJ, Vachieri SG, Christodoulou E, Walker PA, Liu J, Skehel JJ, et al. Evolution of the receptor binding properties of the influenza A (H3N2) hemagglutinin. Proc. Natl. Acad. Sci. USA. 2012;109:21474–21479. doi: 10.1073/pnas.1218841110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Linster M, van Boheemen S, de Graaf M, Schrauwen EJ, Lexmond P, Mänz B, Bestebroer TM, Baumann J, van Riel D, Rimmelzwaan GF, et al. Identification, characterization, and natural selection of mutations driving airborne transmission of A/H5N1 virus. Cell. 2014;157:329–339. doi: 10.1016/j.cell.2014.02.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu M, Li X, Yuan H, Zhou J, Wu J, Bo H, Xia W, Xiong Y, Yang L, Gao R, et al. Genetic diversity of avian influenza A (H10N8) virus in live poultry markets and its association with human infections in China. Sci. Rep. 2015;5:7632. doi: 10.1038/srep07632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liu J, Stevens DJ, Haire LF, Walker PA, Coombs PJ, Russell RJ, Gamblin SJ, Skehel JJ. Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957. Proc. Natl. Acad. Sci. USA. 2009;106:17175–17180. doi: 10.1073/pnas.0906849106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maines TR, Chen LM, Van Hoeven N, Tumpey TM, Blixt O, Belser JA, Gustin KM, Pearce MB, Pappas C, Stevens J, et al. Effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza H5N1 viruses. Virology. 2011;413:139–147. doi: 10.1016/j.virol.2011.02.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, Castrucci MR, Donatelli I, Kawaoka Y. Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J. Virol. 2000;74:8502–8512. doi: 10.1128/jvi.74.18.8502-8512.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nobusawa E, Ishihara H, Morishita T, Sato K, Nakajima K. Change in receptor-binding specificity of recent human influenza A viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides. Virology. 2000;278:587–596. doi: 10.1006/viro.2000.0679. [DOI] [PubMed] [Google Scholar]
  25. Pan American Health Organization (PAHO) Avian influenza virus A (H10N7) circulating among humans in Egypt. EID Weekly Updates 2. 2004:2. Available online at: http://www.paho.org/english/ad/dpc/cd/eid-eer-07-may-2004.htm.
  26. Paulson JC, de Vries RP. H5N1 receptor specificity as a factor in pandemic risk. Virus Res. 2013;178:99–113. doi: 10.1016/j.virusres.2013.02.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Report of Health and Family Planning Commission of Jiangxi Province The third human case of H10N8 bird flu was confirmed. http://www.jxwst.gov.cn/gzdt/201402/t20140213_308109.htm.
  28. Rogers GN, Paulson JC, Daniels RS, Skehel JJ, Wilson IA, Wiley DC. Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature. 1983;304:76–78. doi: 10.1038/304076a0. [DOI] [PubMed] [Google Scholar]
  29. Russell RJ, Stevens DJ, Haire LF, Gamblin SJ, Skehel JJ. Avian and human receptor binding by hemagglutinins of influenza A viruses. Glycoconj. J. 2006;23:85–92. doi: 10.1007/s10719-006-5440-1. [DOI] [PubMed] [Google Scholar]
  30. Sabesan S, Bock K, Paulson JC. Conformational analysis of sialyloligosaccharides. Carbohydr. Res. 1991;218:27–54. doi: 10.1016/0008-6215(91)84084-r. [DOI] [PubMed] [Google Scholar]
  31. Sauter NK, Bednarski MD, Wurzburg BA, Hanson JE, Whitesides GM, Skehel JJ, Wiley DC. Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study. Biochemistry. 1989;28:8388–8396. doi: 10.1021/bi00447a018. [DOI] [PubMed] [Google Scholar]
  32. Shelton H, Ayora-Talavera G, Ren J, Loureiro S, Pickles RJ, Barclay WS, Jones IM. Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential. J. Virol. 2011;85:1875–1880. doi: 10.1128/JVI.01822-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shi W, Li W, Li X, Haywood J, Ma J, Gao GF, Liu D. Phylogenetics of varied subtypes of avian influenza viruses in China: potential threat to humans. Protein Cell. 2014;5:253–257. doi: 10.1007/s13238-014-0036-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shi Y, Zhang W, Wang F, Qi J, Wu Y, Song H, Gao F, Bi Y, Zhang Y, Fan Z, et al. Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses. Science. 2013;342:243–247. doi: 10.1126/science.1242917. [DOI] [PubMed] [Google Scholar]
  35. Stevens J, Blixt O, Glaser L, Taubenberger JK, Palese P, Paulson JC, Wilson IA. Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol. 2006a;355:1143–1155. doi: 10.1016/j.jmb.2005.11.002. [DOI] [PubMed] [Google Scholar]
  36. Stevens J, Blixt O, Paulson JC, Wilson IA. Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nature Rev. Microbiol. 2006b;4:857–864. doi: 10.1038/nrmicro1530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stevens J, Blixt O, Tumpey TM, Taubenberger JK, Paulson JC, Wilson IA. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science. 2006c;312:404–410. doi: 10.1126/science.1124513. [DOI] [PubMed] [Google Scholar]
  38. Su S, Qi W, Zhou P, Xiao C, Yan Z, Cui J, Jia K, Zhang G, Gray GC, Liao M, et al. First evidence of H10N8 avian influenza virus infections among feral dogs in live poultry markets in Guangdong Province. China. Clin. Infect. Dis. 2014;59:748–750. doi: 10.1093/cid/ciu345. [DOI] [PubMed] [Google Scholar]
  39. Tumpey TM, Maines TR, Van Hoeven N, Glaser L, Solórzano A, Pappas C, Cox NJ, Swayne DE, Palese P, Katz JM, García-Sastre A. A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science. 2007;315:655–659. doi: 10.1126/science.1136212. [DOI] [PubMed] [Google Scholar]
  40. Tzarum N, de Vries RP, Zhu X, Yu W, McBride R, Paulson JC, Wilson IA. Structure and receptor binding of the hemagglutinin from a human H6N1 influenza virus. Cell Host Microbe. 2015 doi: 10.1016/j.chom.2015.02.005. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vachieri SG, Xiong X, Collins PJ, Walker PA, Martin SR, Haire LF, Zhang Y, McCauley JW, Gamblin SJ, Skehel JJ. Receptor binding by H10 influenza viruses. Nature. 2014;511:475–477. doi: 10.1038/nature13443. [DOI] [PubMed] [Google Scholar]
  42. Wang W, Lu B, Zhou H, Suguitan AL, Jr., Cheng X, Subbarao K, Kemble G, Jin H. Glycosylation at 158N of the hemagglutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets. J. Virol. 2010;84:6570–6577. doi: 10.1128/JVI.00221-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang M, Zhang W, Qi J, Wang F, Zhou J, Bi Y, Wu Y, Sun H, Liu J, Huang C, et al. Structural basis for preferential avian receptor binding by the human-infecting H10N8 avian influenza virus. Nature Commun. 2015;6:5600. doi: 10.1038/ncomms6600. [DOI] [PubMed] [Google Scholar]
  44. Xiong X, Coombs PJ, Martin SR, Liu J, Xiao H, McCauley JW, Locher K, Walker PA, Collins PJ, Kawaoka Y, et al. Receptor binding by a ferret-transmissible H5 avian influenza virus. Nature. 2013;497:392–396. doi: 10.1038/nature12144. [DOI] [PubMed] [Google Scholar]
  45. Xu R, de Vries RP, Zhu X, Nycholat CM, McBride R, Yu W, Paulson JC, Wilson IA. Preferential recognition of avian-like receptors in human influenza A H7N9 viruses. Science. 2013;342:1230–1235. doi: 10.1126/science.1243761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Xu R, McBride R, Nycholat CM, Paulson JC, Wilson IA. Structural characterization of the hemagglutinin receptor specificity from the 2009 H1N1 influenza pandemic. J. Virol. 2012;86:982–990. doi: 10.1128/JVI.06322-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Xu R, McBride R, Paulson JC, Basler CF, Wilson IA. Structure, receptor binding, and antigenicity of influenza virus hemagglutinins from the 1957 H2N2 pandemic. J. Virol. 2010;84:1715–1721. doi: 10.1128/JVI.02162-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Xu R, Wilson IA. In: Structural Glycomics. Yuriev E, Ramsland PA, editors. CRC Press; Boca Raton, FL: 2012. pp. 235–257. (Boca Raton, FL, CRC Press) 2012. [Google Scholar]
  49. Yamada S, Suzuki Y, Suzuki T, Le MQ, Nidom CA, Sakai-Tagawa Y, Muramoto Y, Ito M, Kiso M, Horimoto T, et al. Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature. 2006;444:378–382. doi: 10.1038/nature05264. [DOI] [PubMed] [Google Scholar]
  50. Zhang H, Xu B, Chen Q, Chen J, Chen Z. Characterization of an H10N8 influenza virus isolated from Dongting lake wetland. Virol. J. 2011;8:42. doi: 10.1186/1743-422X-8-42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhang W, Shi Y, Lu X, Shu Y, Qi J, Gao GF. An airborne transmissible avian influenza H5 hemagglutinin seen at the atomic level. Science. 2013;340:1463–1467. doi: 10.1126/science.1236787. [DOI] [PubMed] [Google Scholar]
  52. Zohari S, Neimanis A, Härkönen T, Moraeus C, Valarcher JF. Avian influenza A (H10N7) virus involvement in mass mortality of harbour seals (Phoca vitulina) in Sweden, March through October 2014. Euro. Surveill. 2014;19:20967. doi: 10.2807/1560-7917.es2014.19.46.20967. [DOI] [PubMed] [Google Scholar]

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