An avian influenza H5N1 virus that binds to a human-type receptor

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-alpha2,3-galactose (SAalpha2,3Gal), whereas human influenza viruses preferentially recognize SAalpha2,6Gal. A conversion to SAalpha2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAalpha2,3Gal to both SAalpha2,3Gal and SAalpha2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAalpha2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.     

Recognition of N-glycolylneuraminic acid linked to galactose by the alpha2,3 linkage is associated with intestinal replication of influenza A virus in ducks

The hemagglutinin (HA) of H3 human influenza viruses does not support viral replication in duck intestine despite its avian origin. A Leu-to-Gln mutation at position 226 and a Ser-to-Gly mutation at position 228 in the HA of human A/Udorn/307/72 (H3N2) permit a reassortant virus [human Udorn HA, with all other genes from A/mallard/New York/6750/78 (H2N2)] to replicate in ducks. To understand the molecular basis of this change in host range restriction, we investigated the receptor specificity of duck influenza viruses as well as of human-duck virus reassortants. The results indicate that the recognition of a glycoconjugate moiety possessing N-glycolneuramic acid (NeuGc) linked to galactose by the alpha2,3 linkage (NeuGcalpha2,3Gal) is associated with viral replication in duck intestine. Immunofluorescence assays with NeuGcalpha2,3Gal-specific antiserum detected this moiety primarily on the crypt epithelial cells of duck colon. Such recognition, together with biochemical evidence of NeuGc in crypt cells, correlated exactly with the ability of the virus to replicate in duck colon. These results suggest that recognition of the NeuGcalpha2,3-Gal moiety plays an important role in the enterotropism of avian influenza viruses.     

Amino Acid Residues Contributing to the Substrate Specificity of the Influenza A Virus Neuraminidase

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.     

Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.     

Molecular dynamics simulation of the effects of single (S221P) and double (S221P and K216E) mutations in the hemagglutinin protein of influenza A H5N1 virus: a study on host receptor specificity

Avian influenza viruses of subtype H5N1 circulating in animals continue to pose threats to human health. The binding preference of the viral surface protein hemagglutinin (HA) to sialosaccharides of receptors is an important area for understanding mutations in the receptor binding site that could be the cause for avian-to-human transmission. In the present work, we studied the effect of two receptor binding site mutations, S221P singly and in combination with another mutation K216E in the HA protein of influenza A H5N1 viruses. Docking of sialic acid ligands corresponding to both avian and human receptors and molecular dynamics simulations of the complexes for wild and mutant strains of H5N1 viruses were carried out. The H5N1 strain possessing the S221P mutation indicated decreased binding to α2,3-linked sialic acids (avian receptor, SAα2,3Gal) when compared to the binding of the wild-type strain that did not possess the HA-221 mutation. The binding to α2,6-linked sialic acids (human receptor, SAα2,6Gal) was found to be comparable, indicating that the mutant strain shows limited dual receptor specificity. On the other hand, the S221P mutation in synergism with the K216E mutation in the binding site, resulted in increased binding affinity for SAα2,6Gal when compared to SAα2,3Gal, indicative of enhanced binding to human receptors. The in-depth study of the molecular interactions in the docked complexes could explain how co-occurring mutations in the HA viral protein can aid in providing fitness advantage to the virus, in the context of host receptor specificity in emerging variants of H5N1 influenza viruses.     

人呼吸道禽流感病毒受体的分布趋势

禽类流感病毒和人类流感病毒具有很强的受体识别特异性,分别与唾液酸α-2,3Gal和α-2,6Gal受体分子结合而感染各自的宿主细胞．这种受体结合特异性是流感病毒在禽类和人类之间跨种属传递的主要障碍．应用凝集素组织化学染色技术,探讨人呼吸道各解剖学部位流感病毒唾液酸受体的分布特征．结果显示,唾液酸α-2,3Gal受体, 即禽类流感受体,主要分布在下呼吸道的呼吸部即呼吸细支气管和肺泡, 而在主气管、支气管和细支气管仅少量分布．相反,人类流感病毒受体,唾液酸α-2,6Gal受体在气管、支气管呈高密度分布,随着支气管分级逐渐降低分布减少,至肺泡分布最少．但比较人呼吸道发育成熟过程中,唾液酸α-2,3Gal和α-2,6Gal受体的表达,未发现明显差别．禽流感H5N1病毒体外感染人呼吸道组织试验结果表明,肺泡上皮较支气管和气管上皮易感染,与唾液酸α-2,3Gal受体分布特点相符合．结果提示,人呼吸道可被禽流感病毒感染,目前H5N1病毒极少发生人传人的特点,可能与个体间上呼吸道唾液酸α-2,3Gal受体表达差异有关．     

Effects of receptor binding specificity of avian influenza virus on the human innate immune response

Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.     

E190V substitution of H6 hemagglutinin is one of key factors for binding to sulfated sialylated glycan receptor and infection to chickens

Avian influenza viruses (AIVs) recognize sialic acid linked α2,3 to galactose (SAα2,3Gal) glycans as receptors. In this study, the interactions between hemagglutinins (HAs) of AIVs and sulfated SAα2,3Gal glycans were analyzed to clarify the molecular basis of interspecies transmission of AIVs from ducks to chickens. It was revealed that E190V and N192D substitutions of the HA increased the recovery of viruses derived from an H6 duck virus isolate, A/duck/Hong Kong/960/1980 (H6N2), in chickens. Recombinant HAs from an H6 chicken virus, A/chicken/Tainan/V156/1999 (H6N1), bound to sulfated SAα2,3Gal glycans, whereas the HAs from an H6 duck virus did not. Binding preference of mutant HAs revealed that an E190V substitution is critical for the recognition of sulfated SAα2,3Gal glycans. These results suggest that the binding of the HA from H6 AIVs to sulfated SAα2,3Gal glycans explains a part of mechanisms of interspecies transmission of AIVs from ducks to chickens.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

Receptor specificities of human respiroviruses

Through their hemagglutinin-neuraminidase glycoprotein, parainfluenza viruses bind to sialic acid-containing glycoconjugates to initiate infection. Although the virus-receptor interaction is a key factor of infection, the exact nature of the receptors that human parainfluenza viruses recognize has not been determined. We evaluated the abilities of human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) to bind to different types of gangliosides. Both hPIV-1 and hPIV-3 preferentially bound to neolacto-series gangliosides containing a terminal N-acetylneuraminic acid (NeuAc) linked to N-acetyllactosamine (Galbeta1-4GlcNAc) by the alpha2-3 linkage (NeuAcalpha2-3Galbeta1-4GlcNAc). Unlike hPIV-1, hPIV-3 bound to gangliosides with a terminal NeuAc linked to Galbeta1-4GlcNAc through an alpha2-6 linkage (NeuAcalpha2-6Galbeta1-4GlcNAc) or to gangliosides with a different sialic acid, N-glycolylneuraminic acid (NeuGc), linked to Galbeta1-4GlcNAc (NeuGcalpha2-3Galbeta1-4GlcNAc). These results indicate that the molecular species of glycoconjugate that hPIV-1 recognizes are more limited than those recognized by hPIV-3. Further analysis using purified gangliosides revealed that the oligosaccharide core structure is also an important element for binding. Gangliosides that contain branched N-acetyllactosaminoglycans in their core structure showed higher avidity than those without them. Agglutination of human, cow, and guinea pig erythrocytes but not equine erythrocytes by hPIV-1 and hPIV-3 correlated well with the presence or the absence of sialic acid-linked branched N-acetyllactosaminoglycans on the cell surface. Finally, NeuAcalpha2-3I, which bound to both viruses, inhibited virus infection of Lewis lung carcinoma-monkey kidney cells in a dose-dependent manner. We conclude that hPIV-1 and hPIV-3 preferentially recognize oligosaccharides containing branched N-acetyllactosaminoglycans with terminal NeuAcalpha2-3Gal as receptors and that hPIV-3 also recognizes NeuAcalpha2-6Gal- or NeuGcalpha2-3Gal-containing receptors. These findings provide important information that can be used to develop inhibitors that prevent human parainfluenza virus infection.     

Relationship between mitogenic activity of influenza viruses and the receptor-binding specificity of their hemagglutinin molecules

The relationship between the mitogenic activity of influenza type A viruses for murine B lymphocytes and the receptor-binding specificity of their hemagglutinin was examined. Receptor-binding specificity was determined by the ability of the virus to agglutinate erythrocytes that had been sialidase treated and then enzymatically resialylated to contain sialyloligosaccharides with defined sequences. Distinct differences in receptor-binding specificity were observed between strongly and weakly mitogenic viruses of the H3 subtype, with strong mitogenic activity correlating with the ability of the virus to recognize the sequence N-glycolylneuraminic acid alpha 2,6 galactose (NeuGc alpha 2,6Gal). Viruses isolated early in the evolution of the H3 subtype (from 1968 to 1971) are relatively weak mitogens and recognize the sequence N-acetylneuraminic acid alpha 2,6 galactose (NeuAc alpha 2,6Gal) but not NeuGc alpha 2,6Gal. H3 viruses isolated since 1972 are strongly mitogenic, and these viruses recognize both NeuGc alpha 2,6Gal and NeuAc alpha 2,6Gal. The amino acid substitution of Tyr for Thr at residue 155 of HA1 may be critical to this change in receptor-binding specificity and mitogenic activity of the later H3 viruses. Horse serum-resistant variants of H3 viruses, which bind preferentially to the sequence NeuAc alpha 2,3Gal, are poorly mitogenic. Differences were also observed between the receptor-binding specificity of the strongly mitogenic H3 viruses and viruses of the H2 and H6 subtypes, the mitogenic activity of which is limited to strains of mice that express the class II major histocompatibility complex glycoprotein I-E. The results indicate that the receptor-binding specificity of the hemagglutinin plays a critical role in determining the mitogenic activity of influenza viruses.     

唾液酸受体并非流感病毒各亚型在雪貂组织中播散分布的决定因子

目的探讨流感病毒在雪貂组织中的分布与唾液酸受体的关系。方法用病毒分离的方法分析流感病毒H5N1（SZ406H,A/VN/1203/04）,SH1N1,H3N2（Brisbane/09,HK/09）在雪貂各组织中分布,用直接免疫荧光法分析雪貂各组织的唾液酸受体的分布,并通过体外实验证实活病毒与组织上受体的结合。结果 H5N1（SZ）和H5N1（A/VN/1203/04）在雪貂的肝、脾、肺、肠中有分布,H5N1（A/VN/1203/04）在脑组织中也有分布,而SH1N1、H3N2（Brisbane/09,HK/09）只分布于肠组织。而唾液酸受体SAα2,6Gal和SAα2,3Gal的I型受体分布于脾、心、肺、肠、脑组织中,和SAα2,3Gal II型受体分布于肝、脾、心、肺、肠、脑组织。SH1N1病毒与SAα2,6Gal能结合,而H5N1与SAα2,3Gal结合。结论 H5N1能在雪貂的多器官组织组织中分布和繁殖,而H3N2和SH1N1仅能在肠组织中分布繁殖。SAα2,6Gal和SAα2,3Gal受体在雪貂多器官组织中均有表达,说明唾液酸受体是病毒进入的门户,但不是病毒分布的决定因子。     

Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.     

Influenza virus receptors in the human airway

Avian influenza A (H5N1) virus infections have resulted in more than 100 human deaths; yet, human-to-human transmission is rare. We demonstrated that the epithelial cells in the upper respiratory tract of humans mainly possess sialic acid linked to galactose by alpha 2,6 linkages (SA alpha 2,6Gal), a molecule preferentially recognized by human viruses. However, many cells in the respiratory bronchioles and alveoli possess SA alpha 2,3Gal, which is preferentially recognized by avian viruses. These facts are consistent with the observation that H5N1 viruses can be directly transmitted from birds to humans and cause serious lower respiratory tract damage in humans. Furthermore, this anatomical difference in receptor prevalence may explain why the spread of H5N1 viruses among humans is limited. However, since some H5N1 viruses isolated from humans recognize human virus receptors, additional changes must be required for these viruses to acquire the ability for efficient human-to-human transmission.     

Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.     

Host-mediated selection of influenza virus receptor variants. Sialic acid-alpha 2,6Gal-specific clones of A/duck/Ukraine/1/63 revert to sialic acid-alpha 2,3Gal-specific wild type in ovo

Human and animal influenza A isolates of the H3 serotype preferentially bind SA alpha 2,6Gal or SA alpha 2,3Gal linkages (where SA represents sialic acid), respectively, on cell-surface sialyloligosaccharides. Previously, we have demonstrated selection of SA alpha 2,3Gal-specific receptor variants of several human viruses which differed from the parent viruses by a single amino acid at residue 226 of the hemagglutinin which is located in the receptor binding pocket (Rogers, G. N., Paulson, J.C., Daniels, R.S., Skehel, J.J., Wilson, I.A., and Wiley, D.C. (1983) Nature 304, 76-78). In this report, the selection in the reverse direction was accomplished starting with a SA alpha 2,3Gal-specific avian virus, A/duck/Ukraine/1/63 (H3N7), yielding SA alpha 2,6Gal-specific variants that exhibit the receptor binding properties characteristic of the human isolates. Selection was again mediated at residue 226 of the hemagglutinin, in this case changing from Gln in the parent virus to Leu in the variants. Although the SA alpha 2,6Gal-specific avian virus variants were stable to passage in MDCK cells, they exhibited dramatic reversion to the SA alpha 2,3Gal-specific phenotype of the parent virus during a single passage in chicken embryos. This was in contrast to the SA alpha 2,6Gal-specific human virus isolates which were stable to passage in both hosts. The reversion of the avian virus variants in eggs provides compelling evidence for host-mediated selection of influenza virus receptor variants.     

Residue Y161 of influenza virus hemagglutinin is involved in viral recognition of sialylated complexes from different hosts

Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to N-acetylneuraminic acid α3 (NeuAcα3) sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species-specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied, and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved HA surface-located amino acid residues in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell-type-dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharide binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to N-glycolylneuraminic acid (NeuGc). Rescued mutant Y161A viruses demonstrated a 5- to 10-fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism.     

Two glycosylation sites in H5N1 influenza virus hemagglutinin that affect binding preference by computer-based analysis

Increasing numbers of H5N1 influenza viruses (IVs) are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA) on the viral surface to host sialic acid (SA) receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs.     

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.     

The receptor binding specificity of the live attenuated influenza H2 and H6 vaccine viruses contributes to vaccine immunogenicity and protection in ferrets

To prepare for influenza pandemics that may be caused by the H2 and H6 subtype influenza viruses, live attenuated influenza virus (LAIV) H2 and H6 vaccines are being developed and evaluated. The H2 and H6 vaccine candidates with different receptor binding preferences specified by amino acid substitutions at residues 226 and 228 were generated and evaluated for their growth in embryonated chicken eggs and their immunogenicity and protection against wild-type virus challenge in the ferret model. The viruses containing Q226 and G228 in the hemagglutinin (HA) protein bound to the avian-like α2,3-sialic acid (SA) receptor and replicated efficiently in chicken eggs. The viruses with L226 and G228 bound preferentially to the human-like α2,6-SA receptor. The viruses containing L226 and S228 displayed dual binding to both α2,3-SA and α2,6-SA receptors and replicated efficiently in eggs. The strains containing L226/G228 or L226/S228 that preferentially bound to α2,6-SA receptors replicated efficiently in the upper respiratory tract of ferrets, induced high levels of neutralizing antibody, and conferred a high level of protection against wild-type virus challenge infection compared to the strain with the Q226/G228 residues. Our data suggest that pandemic vaccines with receptor binding preference to both avian- and human-like receptors might be desired for efficient viral replication in eggs and for inducing protective immune responses in humans.