Sequence analysis and receptor specificity of the hemagglutinin of a recent influenza H2N2 virus isolated from chicken in North America

Influenza viruses bind host cells following an interaction between the viral hemagglutinin (HA) protein and host cell sialylated glycoproteins and glycolipids. Differences in binding affinities of the HAs for different types of sialic acid linkages (α2-3 vs. α2-6) contribute to determining the host range of an influenza virus. The ability of an avian influenza virus HA to bind the human form of the receptor may be one requirement for an avian virus to propagate in the human population. In this paper, we describe the characterization of the HA from an H2N2 virus isolated from a Pennsylvania chicken farm in 2004. Sequence analysis revealed that this HA is a member of the Eurasian clade, and receptor binding studies show that it maintains its specificity for the avian influenza virus α2-3 linked sialic acid receptor.     

Structural characterization of the hemagglutinin receptor specificity from the 2009 H1N1 influenza pandemic

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

Positive selection operates continuously on hemagglutinin during evolution of H3N2 human influenza A virus

It has been proposed that antigenic evolution of hemagglutinin 1 (HA1) for H3N2 human influenza A virus was punctuated. In the population genetic analysis, however, it was controversial whether positive selection operated on HA1 in a punctuated manner for the branches of the phylogenetic tree where transitions to new antigenic clusters occurred (C branches), or continuously. In the molecular evolutionary analysis, positive selection was detected for the trunk (T) branches but the relationship between antigenic evolution and positive selection was unclear. Here molecular evolutionary analysis was conducted to examine natural selection operating on HA1 of H3N2 human influenza A virus by dividing HA1 into epitopes A-E and other sites, as well as dividing the phylogenetic tree into the C branches overlapping with the T branches (C-T branches), those not overlapping with the T branches (C-NT branches), the T branches not overlapping with the C branches (NC-T branches), and other branches (NC-NT branches). Positive selection was detected for C, T, and NC-T branches, whereas evolution for the NC-NT branches appeared to be mainly neutral. Positive selection appeared to have operated throughout the trunk, which covered the entire time period of the phylogenetic tree, suggesting that positive selection operated continuously on HA1 during evolution of H3N2 human influenza A virus.     

Protection against divergent influenza H1N1 virus by a centralized influenza hemagglutinin

Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 10(7) virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses.     

Identification of sequence mutations affecting hemagglutinin specificity to sialic acid receptor in influenza A virus subtypes

The attachment of the hemagglutinin protein of the H1N1 subtype of the pandemic influenza A virus to the sialic acid receptor Sia(α2-6)Gal has contributed to the ability of the virus to replicate in the human body and transmit among humans. In view of the pandemic caused by the replication and transmission of the H1N1 virus, more studies on the specificity of hemagglutinin towards sialic acid and how it affects the replication and transmission ability of this virus among humans are needed. In this study, we have applied sequence, structural and functional analyses to the hemagglutinin protein of the pandemic H1N1 virus, with the aim of identifying amino acid mutation patterns that affect its specificity to sialic acid. We have also employed a molecular docking method to evaluate the complex formed between hemagglutinin protein and the sialic acid receptor. Based on our results, we suggest two possible mutation patterns, namely (1) positions 190 and 225 from glutamic acid and glycine to aspartic acid (E190D in A/Brevig Mission/1/18 (H1N1), A/New York/1/18(H1N1) and A/South Carolina/1/1918(H1N1) and G225D in A/South Carolina/1/1918(H1N1), A/South Carolina/1/1918(H1N1), and A/Puerto Rico/8/34(H1N1)), and (2) positions 226 and 228 from glutamine and glycine to leucine and serine, respectively (Q226L and G228S in A/Guiyang/1/1957(H2N2), A/Kayano/57(H2N2), A/Aichi/2/1968(H3N2), A/Hong Kong/1/1968(H3N2) and A/Memphis/1/68(H3N2)) that can potentially contribute to the specificity of hemagglutinin to Sia(α2-6)Gal, thereby enabling the replication and transmission of virus within and among humans.     

Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus

Recurrence of highly pathogenic avian influenza (HPAI) virus subtype H7 in poultry continues to be a public health concern. In 2003, an HPAI H7N7 outbreak in The Netherlands infected 89 people in close contact with affected poultry and resulted in one fatal case. In previous studies, the virus isolated from this fatal case, A/Netherlands/219/2003 (NL219) caused a lethal infection in mouse models and had increased replication efficiency and a broader tissue distribution than nonlethal isolates from the same outbreak. A mutation which introduces a potential glycosylation site at Asn123 in the NL219 hemagglutinin was postulated to contribute to the pathogenic properties of this virus. To study this further, we have expressed the NL219 hemagglutinin in a baculovirus expression system and performed a structural analysis of the hemagglutinin in complex with avian and human receptor analogs. Glycan microarray and kinetic analysis were performed to compare the receptor binding profile of the wild-type recombinant NL219 HA to a variant with a threonine-to-alanine mutation at position 125, resulting in loss of the glycosylation site at Asn123. The results suggest that the additional glycosylation sequon increases binding affinity to avian-type α2-3-linked sialosides rather than switching to a human-like receptor specificity and highlight the mechanistic diversity of these pathogens, which calls attention to the need for further studies to fully understand the unique properties of these viruses.     

Sialic acid recognition of the pandemic influenza 2009 H1N1 virus: binding mechanism between human receptor and influenza hemagglutinin

Quantum mechanical fragment molecular orbital calculations have been performed for receptor binding of the hemagglutinin protein of the recently pandemic influenza 2009 H1N1, A/swine/Iowa/1930, and A/Puerto Rico/8/1934 viruses to α2-6 linked sialyloligosaccharides, as analogs of human receptors. The strongest receptor binding affinity was observed for the 2009/H1N1pdm. The inter-fragment interaction energy analysis revealed that the amino acid mutation of 2009/H1N1pdm, Ser145Lys, was a major cause of such strong binding affinity. Strong ionic pair interaction between the sialic acid and Lys145 was observed only in the 2009/H1N1pdm, in addition to the hydrogen bond between the sialic acid and Gln226 observed in all the HAs. Therefore, pandemic 2009/H1N1pdm has been found to recognize the α2-6 receptor much stronger than the 1930-swine and 1934-human.     

Comparative study of the hemagglutinin and neuraminidase genes of influenza A virus H3N2, H9N2, and H5N1 subtypes using bioinformatics techniques

To investigate the genomic patterns of influenza A virus subtypes, such as H3N2, H9N2, and H5N1, we collected 1842 sequences of the hemagglutinin and neuraminidase genes from the NCBI database and parsed them into 7 categories: accession number, host species, sampling year, country, subtype, gene name, and sequence. The sequences that were isolated from the human, avian, and swine populations were extracted and stored in a MySQL database for intensive analysis. The GC content and relative synonymous codon usage (RSCU) values were calculated using JAVA codes. As a result, correspondence analysis of the RSCU values yielded the unique codon usage pattern (CUP) of each subtype and revealed no extreme differences among the human, avian, and swine isolates. H5N1 subtype viruses exhibited little variation in CUPs compared with other subtypes, suggesting that the H5N1 CUP has not yet undergone significant changes within each host species. Moreover, some observations may be relevant to CUP variation that has occurred over time among the H3N2 subtype viruses isolated from humans. All the sequences were divided into 3 groups over time, and each group seemed to have preferred synonymous codon patterns for each amino acid, especially for arginine, glycine, leucine, and valine. The bioinformatics technique we introduce in this study may be useful in predicting the evolutionary patterns of pandemic viruses.     

Acid stability of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.     

Peptide mapping of the monoclonal antibodies against the heavy chain hemagglutinin from influenza virus H3N2

Interaction of the synthetic peptides corresponding to the regions 122-133, 136-147, 154-164 and 314-328 of the virus A/Aichi/2/68 hemagglutinin heavy chain with monoclonal antibodies specific for this hemagglutinin was assayed in a variety of tests, e.g., ELISA, competition RIA, hemagglutinin-inhibition and virus-neutralization assays. The monoclonal antibody 152 reacted with the area 136-147 (epitope A), three monoclonal antibodies 3, 19 and 63 reacted exclusively with the area (154-164) Glu (epitope B). Mapping of two monoclonal antibodies IV A1 and IV G6 specific for the influenza virus A/Dunedin/ 4/73 hemagglutinin heavy chain and cross-reacting with a number of the H3 subtype viruses was carried out. The specificity of the interaction of the conservative peptide H3(314-328) with monoclonal antibodies IV A1 and IV G6 was confirmed by competition RIA and by competition hemagglutinin inhibition and virus neutralization.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Positive selection for gains of N-linked glycosylation sites in hemagglutinin during evolution of H3N2 human influenza A virus

The number of N-linked glycosylation sites in the globular head of hemagglutinin (HA) has increased during evolution of H3N2 human influenza A virus. Here natural selection operating on the gains of N-linked glycosylation sites was examined by using the single-site analysis and the single-substitution analysis. In the single-site analysis, positive selection was not inferred at the amino acid sites where the substitutions generating N-linked glycosylation sites were observed, but was detected at antigenic sites. In contrast, in the single-substitution analysis, positive selection was detected for the amino acid substitutions generating N-linked glycosylation sites. The single-site analysis and the single-substitution analysis appeared to be suitable for detecting recurrent and episodic natural selection, respectively. The gains of N-linked glycosylation sites were likely to be positively selected for the function of shielding antigenic sites from immune responses. At the antigenic sites, positive selection appeared to have operated not only on the radical substitution but also on the conservative substitution in terms of the charge of amino acids, suggesting that the antigenic drift is not a by-product of the evolution of receptor binding avidity in HA of human H3N2 virus.     

Evolution of the hemagglutinin gene of influenza A virus H1N1-subtype (1977-1983

A scheme for evolutionary interrelations of the H1-subunits of influenza hemagglutinin genes is proposed for the natural variants of influenza A virus of the H1N1-subtype. It is based on experimental data obtained by the authors and those reported in the literature. Differences among these viral isolates in their amino acid sequences and in the reaction of hemagglutinin inhibition obtained with a set of monoclonal antibodies are compared. The distinctions in the ability of the viruses to react with several monoclonal antibodies are attributed to differences in the primary structures of their hemagglutinins. Some aspects of hemagglutinin gene evolution are discussed in relation to vaccination.     

流感病毒H1N1血凝素基因和神经氨酸酶基因在酵母菌中的表达

在成功克隆流感病毒H1N1全长HA(Hemagglulinin,HA)、NA(Neuramidinase,NA) 基因并测序的基础上,将部分基因序列克隆到表达载体pMETA上,构建了重组表达质粒pMETA/HA(52~1 557 bp)、pMETA/NA(121~1 263 bp),电转化真核酵母菌pMAD16,甲醇诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。SDS-PAGE显示重组蛋白在酵母菌中可以高效表达,蛋白纯度占总蛋白的95％以上,ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。成功克隆和表达了流感病毒H1N1 HA、NA基因序列,为流感病毒H1N1诊断试剂和疫苗的开发等进一步的研究提供了参考。     

Inhibition of influenza A virus (H1N1) fusion by benzenesulfonamide derivatives targeting viral hemagglutinin

Hemagglutinin (HA) of the influenza virus plays a crucial role in the early stage of the viral life cycle by binding to sialic acid on the surface of host epithelial cells and mediating fusion between virus envelope and endosome membrane for the release of viral genomes into the cytoplasm. To initiate virus fusion, endosome pH is lowered by acidification causing an irreversible conformational change of HA, which in turn results in a fusogenic HA. In this study, we describe characterization of an HA inhibitor of influenza H1N1 viruses, RO5464466. One-cycle time course study in MDCK cells showed that this compound acted at an early step of influenza virus replication. Results from HA-mediated hemolysis of chicken red blood cells and trypsin sensitivity assay of isolated HA clearly showed that RO5464466 targeted HA. In cell-based assays involving multiple rounds of virus infection and replication, RO5464466 inhibited an established influenza infection. The overall production of progeny viruses, as a result of the compound's inhibitory effect on fusion, was dramatically reduced by 8 log units when compared with a negative control. Furthermore, RO5487624, a close analogue of RO5464466, with pharmacokinetic properties suitable for in vivo efficacy studies displayed a protective effect on mice that were lethally challenged with influenza H1N1 virus. These results might benefit further characterization and development of novel anti-influenza agents by targeting viral hemagglutinin.     

Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.     

不同宿主H1N1病毒血凝素蛋白(HA)受体结合位点的变异特征

2009年全球性爆发的H1N1病毒已经导致213个国家和地区受到感染, 有16 226人死亡。病毒与宿主细胞表面受体的结合是病毒感染不可缺少的第一步, 从而导致病毒膜与宿主细胞膜的融合。血凝素(Hemagglutinin, HA)就是介导这种受体结合与膜融合的病毒蛋白, 受体结合位点(Receptor binding sites, RBSs)位于HA蛋白三聚体中每个单体的球形头部, 主要由190位螺旋(190~198aa)、130位环(135~138aa)和220位环(221~228)3个二级结构域组成。文章收集了1918~2009年间1 221株H1N1病毒株的HA1序列(长度为327个氨基酸残基), 通过序列比对、各位点氨基酸残基的熵值以及3D结构模拟等生物信息学研究。结果显示不同宿主的不同病毒RBSs具有不同的熵值, 而且不同宿主的病毒HA1其RBSs具有不同的优势序列。3D结构模拟也显示了H1N1不同HA1之间在190位螺旋构象上的细微差异。该研究揭示了不同HA1上RBSs的一些新的特征, 为进一步探讨病毒感染的机理提供了新的信息     

Evolution of the hemagglutinin gene of human influenza A virus H3 subtype

An evolutional tree of human influenza viruses of the H3N2-subtype is suggested on the basis of combined published primary structures of the hemagglutinin HA1-subunit. Possible differences between natural and sequenced structures are discussed. A tendency to reversions in the course of antigenic draft within the subtype has been revealed to support the hypothesis of limited antigenic evolution within a single subtype.     

一株2011年出现的季节性甲型H1N1流行性感冒病毒血凝素基因特性的研究

本文通过比较2011年分离培养的1株季节性甲型H1N1流行性感冒(简称流感)病毒(A/Shanghai/1167/2011(H1N1))与历年季节性甲型H1N1流感病毒的血凝素(HA)基因,追溯该病毒的基因变异与来源,探讨该毒株的出现对流感防控工作的意义.采用反转录-聚合酶链反应(RT-PCR)方法扩增病毒的HA和神经氨酸酶(NA)片段,并进行测序;应用分子生物学软件对获得的序列进行分析,绘制基因进化树;同时,通过血凝抑制试验检测2011年下半年健康人群中该流感病毒的抗体水平.结果显示,A/Shanghai/1167/2011(H1N1)的HA基因序列与世界卫生组织(WHO)2007～2008年季节性甲型H1N1流感病毒疫苗株A/Brisbane/59/2007(H1N1)最接近,同源性达99.2%,与新型甲型H1N1流感病毒A/California/07/2009疫苗株同源性仅为72.4%.其HA基因裂解位点为PSIQSR↓GLF,尚未出现高致病性的分子特征.HA片段共编码557个氨基酸,有9个潜在的糖基化位点,序列与2009年前WHO疫苗株A/NewCaledonia/20/1999(H1N1)、A/SolomonIslands/3/2006(H1N1)和/Brisbane/59/2007(H1N1)相比,分别有15、12和4处不同,这些差异分布在Sa、Sb、Ca1、Ca2、Cb 5个抗原决定簇的氨基酸差异分别有5、5和2处.该毒株在健康人群血清的抗体阳性率为34.33%,几何平均效价(GMT)为10.38.A/Shanghai/1167/2011(H1N1)是2011年出现在上海地区的一个季节性甲型H1N1流感病毒毒株,其抗原变异与既往季节性甲型H1N1流感病毒相比不大,但在以A(H1N1)pdm09为主要流行株的年份检测到散在发生的既往季节性甲型H1N1流感病毒毒株应当引起重视,其在人群中的抗体水平较低,易引起流行,需要提高对类流感人群中此种毒株的持续监测.