Molecular Mechanisms of Serum Resistance of Human Influenza H3N2 Virus and Their Involvement in Virus Adaptation in a New Host

H3N2 human influenza viruses that are resistant to horse, pig, or rabbit serum possess unique amino acid mutations in their hemagglutinin (HA) protein. To determine the molecular mechanisms of this resistance, we characterized the receptor-binding properties of these mutants by measuring their affinity for total serum protein inhibitors and for soluble receptor analogs. Pig serum-resistant variants displayed a markedly decreased affinity for total pig serum sialylglycoproteins (which contain predominantly 2-6 linkage between sialic acid and galactose residues) and for the sialyloligosaccharide 6′-sialyl(N-acetyllactosamine). These properties correlated with the substitution 186S→I in HA1. The major inhibitory activity in rabbit serum was found to be a β inhibitor with characteristics of mannose-binding lectins. Rabbit serum-resistant variants exhibited decreased sensitivity to this inhibitor due to the loss of a glycosylation sequon at positions 246 to 248 of the HA. In addition to a somewhat reduced affinity for 6′-sialyl(N-acetyllactosamine)-containing receptors, horse serum-resistant variants lost the ability to bind the viral neuraminidase-resistant 4-O-acetylated sialic acid moieties of equine α₂-macroglobulin because of the mutation 145N→K/D in their HA1. These results indicate that influenza viruses become resistant to serum inhibitors because their affinity for these inhibitors is reduced. To determine whether natural inhibitors play a role in viral evolution during interspecies transmission, we compared the receptor-binding properties of H3N8 avian and equine viruses, including two strains isolated during the 1989 to 1990 equine influenza outbreak, which was caused by an avian virus in China. Avian strains bound 4-O-acetylated sialic acid residues of equine α₂-macroglobulin, whereas equine strains did not. The earliest avian-like isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian viruses; a later isolate, however, displayed binding properties more similar to those of classical equine strains. These data suggest that the neuraminidase-resistant sialylglycoconjugates present in horses exert selective pressure on the receptor-binding properties of avian virus HA after its introduction into this host.Influenza A viruses possess two envelope glycoproteins:hemagglutinin (HA) and neuraminidase (NA). HA binds to cell surface sialylglycoconjugates and mediates virus attachment to target cells (19, 30). NA cleaves the α-glycosidic linkage between sialic acid and an adjacent sugar residue, facilitating elution of virus progeny from infected cells and preventing self-aggregation of the virus (1, 13). Natural sialylglycoconjugates are structurally diverse (37, 40), and the preferential recognition of distinct sialyloligosaccharides by HA and NA correlates with the host species from which the viruses are isolated (reviewed in references 19, 30, and 38; see also references 4, 6, 7, 11, and 28).The receptor-binding activity of influenza viruses can be inhibited by certain molecules present in the sera and fluid secretions of animals (see references 14 and 21 for reviews). These inhibitors are classified as α, β, and γ types based on their thermal stability, virus-neutralizing activity, and sensitivity to inactivation by NA and periodate treatments. The β inhibitors are thermolabile mannose-binding lectins that interact with the oligosaccharide moieties on viral glycoproteins. They neutralize virus by steric hindrance of HA and by activation of the complement-dependent pathway (2, 3). By contrast, the α and γ inhibitors are heat-stable sialylated glycoproteins that mimic the structure of the cellular receptors of influenza viruses and competitively block the receptor-binding sites of HA. Influenza viruses are neutralized by γ inhibitors but not by α inhibitors, which are considered to be sensitive to viral NA. However, the distinction between α and γ inhibitors is strain dependent and rather arbitrary, as described by Gottschalk et al. (14). Although inhibitors in serum or other body fluids are believed to influence the selection of influenza virus receptor variants in natural hosts, no direct experimental support for this hypothesis has been presented.A potent γ inhibitor of H2 and H3 human influenza viruses, equine α₂-macroglobulin (EM), contains a Neu4,5Ac₂2-6Gal moiety that is insensitive to viral NA and thus resists inactivation by this enzyme (16, 24, 31). Cultivation of human H3 influenza viruses in the presence of horse serum results in the selection of variants that have a decreased affinity for the Neu5Ac2-6Gal-specific receptors due to a single amino acid substitution (226L→Q) in their HA (32, 33). One of these mutants (X31/HS strain) does not bind the Neu4,5Ac₂ (4-O-acetylated sialic acid) species (25). Therefore, there are at least two mechanisms by which a virus can become resistant to the horse serum inhibitor: a change in the recognition of the type of Sia-Gal linkage, and a change in the recognition of the 4-O-acetylated sialic acid. The relative contributions of these mechanisms to the resistant phenotype are yet to be defined.We have previously shown that horse, pig, and rabbit sera all contain distinct heat-resistant inhibitors of the H3N2 human influenza virus A/Los Angeles/2/87 (LA/87), because variants resistant to these sera possess unique mutations in their HA receptor-binding regions (34). The major inhibitor in pig serum was later identified as α₂-macroglobulin that contains predominantly 2-6 linkage between sialic acid and galactose (35). Gimsa et al. (12) recently showed that pig serum-resistant human and swine strains exhibit decreased affinity for human erythrocytes that had been modified to contain terminal Neu5Ac2-6Gal residues. However, the nature of the rabbit serum inhibitor and the mechanisms of influenza virus resistance to each serum inhibitor remain unknown.To understand the molecular mechanisms by which influenza viruses become resistant to horse, pig, and rabbit serum inhibitors, we compared the receptor-binding characteristics of LA/87 and its serum-resistant variants and analyzed these data in relation to the known amino acid substitutions in the HA of the mutants. We then analyzed the receptor-binding properties of viruses isolated during an equine influenza outbreak that was caused by an avian virus, in order to evaluate the influence of natural inhibitors on the evolution of virus in a new host.     

Studies on the adaptation of influenza viruses to MDCK cells

The amino acid sequences and biological properties of the haemagglutinin of three variants of the influenza virus X-31 (H3N2) selected for their capacity to grow in MDCK cells are reported. In two variants, amino acid substitutions at HA1 residues 8 and 144 correlated with the loss of a site for glycosylation and specific changes in antigenicity, respectively. In all three variants substitution of an arginine residue for histidine at HA1 position 17 was correlated with increased pH optima of haemolysis. The importance of this substitution for cleavage of the haemagglutinin precursor required to produce infectious virus is discussed in relation to the three-dimensional structure of X-31 haemagglutinin.     

Mechanism of antigenic drift in influenza virus. Amino acid sequence changes in an antigenically active region of Hong Kong (H3N2) influenza virus hemagglutinin

During antigenic drift in influenza viruses, changes in antigenicity are associated with changes in amino acid sequence of the large hemagglutinin polypeptide, HA1. In ten variants of Hong Kong (H3N2) influenza virus selected with monoclonal antibodies, the proline residue at position 143 in HA1 changed to serine, threonine, leucine or histidine. In other variants, asparagine 133 changed to lysine, glycine 144 to aspartic acid and serine 145 to lysine. All these changes are possible by single base changes in the RNA except the last, which requires a double base change. Residues 142 to 146 also changed in field strains of Hong Kong influenza isolated between 1968 and 1977 (Laver et al., 1980). The single amino acid sequence changes in HA1 of the monoclonal variants were detected by comparing the compositions of the soluble tryptic peptides from the variants with the known sequences of these peptides from wild-type virus. Two insoluble tryptic peptides, comprising residues 110 to 140 and 230 to 255 in the HA1 molecule, were not examined and we do not know if additional changes occurred in these regions.In order to determine whether sequential changes at the same position occurred during antigenic drift, antibody prepared against the new antigenic site on the variants in which proline 143 changed to histidine or threonine was used to select second generation variants of these variants. In the first case, the glycine residue (144) next to the histidine changed to aspartic acid, and in the second, the threonine residue at position 143 reverted to proline and the virus regained the antigenicity of wild-type.Although monoclonal antibodies revealed dramatic antigenic differences between the variants and wild-type virus, only those variants with changes at position 144 of glycine to aspartic acid or at position 145 of serine to lysine could be distinguished from wild-type virus using heterogeneous rabbit or ferret antisera. The other variants, including those which showed sequence changes in widely separated positions of HA1, could not be distinguished from wild-type with heterogeneous antisera.These findings suggest that sequence changes in the region comprising residues 142 to 146 of HA1 affect an important antigenic site on the hemagglutinin molecule, but how these changes affect the antigenic properties, or whether this region actually forms part of the antigenic site is not known.     

A novel pathogenic mechanism of highly pathogenic avian influenza H5N1 viruses involves hemagglutinin mediated resistance to serum innate inhibitors

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes.     

鸽源H5N1亚型禽流感病毒株的HA和NA基因特征性分析

本研究自行设计合成两对特异性引物,通过RT-PCR扩增出1株鸽源H5N1亚型禽流感病毒血凝素（HA）和神经氨酸酶（NA）两个基因的cDNA片段,将它们成功克隆于pMD18-T载体上,然后进行序列测定。结果表明,HA基因全长1707bp,编码568个氨基酸, HA基因有7个糖基化位点,在裂解位点附近有连续6个碱性氨基酸（R-R-R-K-K-R）的插入,具有高致病性毒株的分子特征。受体结合位点的氨基酸分别为YWIHELY,左侧壁氨基酸为SGVSSA,右侧壁为NGQSGR;NA基因全长1350bp,编码446个氨基酸,NA基因有3个糖基化位点。     

Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses

If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.     

Improving Statistical Certainty of Glycosylation Similarity between Influenza A Virus Variants Using Data-Independent Acquisition Mass Spectrometry

Amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large branched polysaccharide molecules on proteins, can modulate the function of HA and shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to quantitatively measure glycosylation changes in variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages, and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 (H3N2): WT and mutant, each expressed in embryonated chicken eggs and Madin–Darby canine kidney cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence variations and changing viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster genomic surveillance already being done, for a more complete understanding of IAV evolution.     

Mutational changes in the hemagglutinin of equine H3 influenza viruses result in the introduction of a glycosylation site which enhances the infectivity of the viruses

The complete amino acid sequences of the hemagglutinin (HA) glycoprotein of three equine-2 influenza viruses from tropical Africa are presented in comparison with that of a well characterized European equine-2 virus (Suffolk/89) and a consensus sequence from the database. The sequences of the tropical African viruses were deduced from the complete nucleotide sequences of their HA genes reported earlier. Mutational changes in the nucleotide sequences resulted in amino acid changes in the HA which led to the introduction of a new asparagine-linked (N-linked) glycosylation site in two viruses. This new glycosylation site enhanced the infectivity of these viruses as investigated by plaque assay, virus titration in embryonated chicken eggs and tunicamycin treatment. The role of N-linked glycosylation of influenza virus HA glycoprotein in virus infectivity, antigenicity and immunogenicity is discussed in the light of the results of our previous and present investigations.     

一株H5N2亚型鹦鹉源禽流感病毒分子进化分析

2005年在广东进行流行病学调查时分离到一株鹦鹉源禽流感病毒,经鉴定为H5N2亚型禽流感病毒(A/Parrot/Guangdong/268/2005)。该毒株的HA裂解位点附近的氨基酸序列为RETRGLF,只含有一个碱性氨基酸,符合低致病性禽流感病毒的HA裂解位点附近氨基酸序列的分子特征;与H5N2亚型禽流感代表毒株相比,该毒株HA和NA基因的糖基化位点、HA基因的受体结合位点编码区、NA基因的耐药性位点均未发生变异。将该毒株全基因组序列与GenBank已公布的19株H5N2亚型禽流感病毒株的相应序列进行比较分析并绘制系统进化树后发现:其与低致病性禽流感毒株A/Pheasant/NJ/1355/1998(H5N2)-like的亲缘关系最近,位于以A/Chicken/Pennsylvania/1/1983(H5N2)为代表的美洲进化分支。     

1981～2005年中国H1N1甲型流感病毒血凝素基因的HA1演变特征

为了解1981~2005年我国H1N1甲型流感病毒血凝素基因的HA1演变特征,选取H1N1甲型流感病毒370株,提取病毒RNA,经逆转录和聚合酶链反应扩增HA1并测序,测定的序列用生物信息软件分析,与GenBank中相关序列比较,并对推导的编码氨基酸序列进行基因特性分析。结果表明:HA1氨基酸的变异表现为抗原决定簇4个区均有变异,Sb区和Ca区变化较大;HA1受体结合位点(RBS)的前壁130环的第134位赖氨酸从1991年起在部分毒株HA1序列上开始缺失,以后缺失株逐步增多,自2000年起测定的所有毒株上该氨基酸全部缺失,同时这些缺失株的第137位氨基酸也全部由苏氨酸替换为丝氨酸;糖基化位点从增多到减少,最后稳定在7个;1981~2004年我国H1N1甲型流感病毒血凝素HA1编码的氨基酸在种系发育树上同年代基本呈现集中分布,与时间和地域无关,2005年毒株分成两个分支在时间上有明显差异。     

山东地区16株H9N2亚型禽流感病毒HA基因的序列分析

为了解H9N2亚型禽流感病毒(AIV)山东分离株的遗传变异情况,采用RT-PCR技术对16株从山东不同地区分离的H9N2亚型禽流感病毒的HA基因进行扩增、克隆和测序,并对所获得的HA全序列进行同源性和遗传进化分析。结果显示,16个分离株的裂解位点均为RSSR↓GLF,符合低致病性禽流感病毒的分子特征;有7～9个潜在糖基化位点;受体结合位点除198位有变异,其他位点均较保守;234位氨基酸均为L,具有与哺乳动物唾液酸α,2-6受体结合的特征;16个分离株HA基因核苷酸及氨基酸序列同源性分别为96.3%～99.9%和97.1%～99.6%;16个分离株同属于欧亚分支中的A/Duck/Hong Kong/Y280/97亚群。     

Identification of amino acids of influenza virus HA responsible for resistance to a fusion inhibitor, Stachyflin

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1-14, 1999). Stachyflin-resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA-expressing cells and octadecylrhodamine (R18)-labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin-resistant phenotype of HA protein. The molecular mechanism of anti-HA conformational change activity of Stachyflin is discussed.     

Hemagglutinin protein of Asian strains of human influenza virus A H1N1 binds to sialic acid--a major component of human airway receptors

Hemagglutinin (HA) protein plays an important role in binding the influenza virus to infected cells and therefore mediates infection. Deposited HA sequences of 86 Asian strains of influenza A (H1N1) viruses during the first outbreak were obtained from the NCBI database and compared. Interaction of the HA protein of influenza A (H1N1) virus with the human sialic acid receptor was also studied using bioinformatics. Overall, not more than three single-point amino acid variants/changes were observed in the HA protein region of influenza A (H1N1) virus from Asian countries when a selected group sequence comparison was made. The bioinformatics study showed that the HA protein of influenza A (H1N1) binds to the sialic acid receptor in human airway receptors, possibly key to air-borne infection in humans.     

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.     

Structural features influencing hemagglutinin cleavability in a human influenza A virus.

The cleavability of the hemagglutinin (HA) molecule is related to the virulence of avian influenza A viruses, but its influence on human influenza virus strains is unknown. Two structural features are involved in the cleavage of avian influenza A virus HAs: a series of basic amino acids at the cleavage site and an oligosaccharide side chain in the near vicinity. The importance of these properties in the cleavability of a human influenza A virus (A/Aichi/2/68) HA was investigated by using mutants that contained or lacked an oligosaccharide side chain and had either four or six basic amino acids. All mutants except the one that contains a single mutation at the glycosylation site were cleaved, although not completely, demonstrating that a series of basic amino acids confers susceptibility to cellular cleavage enzymes among human influenza virus HAs. The mutants containing six basic amino acids at the cleavage site showed limited polykaryon formation upon exposure to low pH, indicating that cleavage was adequate to impart fusion activity to the HA. Deletion of the potential glycosylation site had no effect on the cleavability of these mutants; hence, the oligosaccharide side chain appears to have no role in human influenza virus HA cleavage. The inability to induce high cleavability in a human influenza A virus HA by insertion of a series of basic amino acids at the cleavage site indicates that other, as yet unidentified structural features are needed to enhance the susceptibility of these HAs to cellular proteases.     

Reactivity of human convalescent sera with influenza virus hemagglutinin protein mutants at antigenic site A

How the antibodies of individual convalescent human sera bind to each amino acid residue at the antigenic sites of hemagglutinin (HA) of influenza viruses, and how the antigenic drift strains of influenza viruses are selected by human sera, is not well understood. In our previous study, it was found by a binding assay with a chimeric HA between A/Kamata/14/91 (Ka/91) and A/Aichi/2/68 that convalescent human sera, following Ka/91 like (H3N2) virus infection, bind to antigenic site A of Ka/91 HA. Here using chimeric HAs possessing single amino acid substitutions at site A, it was determined how those human sera recognize each amino acid residue at antigenic site A. It was found that the capacity of human sera to recognize amino acid substitutions at site A differs from one person to another and that some amino acid substitutions result in all convalescent human sera losing their binding capacity. Among these amino acid substitutions, certain ones might be selected by chance, thus creating successive antigenic drift. Phylogenetic analysis of the drift strains of Ka/91 showed amino acid substitutions at positions 133, 135 and 145 were on the main stream of the phylogenetic tree. Indeed, all of the investigated convalescent sera failed to recognize one of them.     

Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

2009−2015年中国甲型H1N1流感病毒血凝素基因进化分析

【目的】为了解中国地区2009?2015年甲型H1N1流感病毒流行态势,分析血凝素(Hemagglutinin,HA)基因的变异情况及其遗传进化特征。【方法】汇集国家流感中心2009?2015年流感周报的流感流行数据,分析甲型H1N1流感的流行病学特征;从全球共享禽流感数据倡议组织数据库及美国国家生物技术中心数据库下载甲型H1N1流感病毒HA基因序列,采用生物学软件进行系统进化和遗传特性的分析。【结果】2009?2015年全国共发生4次甲型H1N1流感的流行高峰。2009?2015年毒株与参考毒株A/California/07/2009(H1N1)的HA基因同源性逐年降低。遗传进化分析显示同一年份的毒株在系统进化树上基本呈现集中分布,2011年的毒株独立形成2个分支。分子特征表现为HA基因的4个抗原决定簇氨基酸位点均有变异,其中Ca区的203位、Sa区的163位和Sb区的185位氨基酸位点逐渐替换为新的氨基酸。除2010年与2012年,其他年份的毒株通过不同模型均得到正向压力选择HA氨基酸位点240。【结论】甲型H1N1流感在中国地区成为主要流行的亚型之一。HA基因与其编码的氨基酸逐年变异,未来进一步的流感监测能力还需加强。     

Molecular genetic characterization of H5N1 influenza virus strains isolated from poultry in the Kurgan region in 2005

In the second half of 2005, a large-scale outbreak of influenza in poultry and wild birds was caused by a highly pathogenic H5N1 influenza virus in Russia. The level of pathogenicity is a polygenic trait, and most individual genes contribute to the influenza A virus pathogenicity in birds, animals, and humans. The full-length nucleotide sequences were determined for H5N1 strains isolated in the Kurgan region (Western Siberia). The structure of viral proteins was analyzed using the deduced amino acid sequences. The receptor-binding site of hemagglutinin (HA) in strains A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. The structure of the basic amino acid cluster located within the HA cleavage site was identical in all isolates: QGERRRKKR. According to the neuraminidase structure, all H5N1 isolates from the Kurgan region were assigned to the Z genotype. Amino acid residues typical for the avian influenza virus were revealed in 30 out of 32 positions of M1, M2, NP, PA, and PB2, determining the host range specificity. One of the strains contained Lys at position 627 of PB2. Isolates from the Kurgan region were shown to have a remantadine-sensitive genotype. Both strains contained Glu at position 92 of NS1, indicating that the virus is interferon-resistant. Phylogenetic analysis related the Kurgan isolates to subclade 2 of clade 2 of highly pathogenic H5N1 influenza viruses.     

我国部分地区H9亚型禽流感病毒血凝素基因序列比较与遗传发生关系分析

为从分子水平掌握我国H9亚型AIV的遗传变异情况和流行规律,本研究汇集近年来从我国12个省、市、自治区的发病鸡群中分离到的23株H9亚型禽流感病毒,通过RT-PCR方法和核苷酸序列测定获得了23个毒株的HA基因cDNA核苷酸序列。核苷酸和推导的氨基酸序列同源性比较结果表明,这些毒株HA基因的核苷酸序列同源性为94．1％～100％,氨基酸序列同源性为95．4％～100%;将这23个毒株和来自亚洲及世界其它地区的另外31株的HA基因cDNA序列同源性进行比较发现,分离自香港的HK170499株与日本的2个毒株关系较近;氨基酸序列分析发现,CKGS199、CKTJ196、CKTJ296、CKSH300和CKBJ197五个毒株各发生了一个潜在的糖基化位点的丢失。54株H9亚型AIVHA基因55bp～1152bp的氨基酸序列分析发现,裂解位点尽管有10种基序,但本研究中的23株和近年来从我国大陆和香港地区的分离的毒株则均为RSSR↓GLF;构成受体结合位点的191位氨基酸有一个规律,即所有中国大陆毒株与部分香港毒株都为N,其它毒株均为H,141aa～143aa处的糖基化位点有与191aa类似的规律,即：凡是191aa为N的毒株,该处均为NVS（CKBJ194除外）,凡是191aa为H的毒株,则该处均为NVT;遗传发生关系分析,中国大陆毒株处于欧亚谱系的第一支。本研究结果表明近年来我国鸡群中H9N2亚型禽流感病毒的感染流行可能有一个共同的来源,这为制定防治该亚型禽流感流行的有效对策提供了重要的科学依据。