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.     

从2009甲型H1N1流行性感冒暴发看流行性感冒病毒的演化

流行性感冒(简称流感)病毒已经与人类“相处”了400多年,可在各年龄段的人群中流行和引起发病,每年在全球引起25万～50万人发病[1]。2009年3月在北美地区暴发了新型H1N1流感,截至2009年6月8日,已有73个国家和地区的25288人被感染,其中139人死亡(http://www.who.int/csr/don/2009_06_08/en/index.html)。世界卫生组织(WorldHealthOrganization,WHO)曾称其为猪流感,原因是其多数基因片段来源于猪流感病毒。猪流感这一名称直接导致了埃及4月30日为预防流感蔓延而扑杀了30万头生猪。随后,WHO将其改名为甲型H1N1流感(下称2009甲型H1N1流感)。因此,甲型H1N1流感病毒的起源毫无疑问成为世界各国关注的热点,本文就2009甲型H1N1流感、1997年人际传播的H5N1禽流感以及人类历史上的几次人流感大流行,对甲型流感病毒的基因组结构特点及其特有的进化机制作一简单介绍......     

2009甲型H1N1流感病毒研究进展

2009年3月在美国和墨西哥爆发的新型甲型H1N1流感在很短的时间内便扩散到世界多个国家,形成了流感的大流行,引起世界卫生组织和各国的高度重视。综述新型甲型H1N1流感病毒的基因组来源、目前主要的检测手段,并对预防和治疗的方法进行简单介绍。     

甲型H1N1流感病毒快速核酸检测技术的建立

美国、墨西哥等国家相继发生甲型H1N1流感疫情后,即刻引起全球关注。WHO日前宣布目前为流感大流行第五期,预示又一次流感大流行可能逼近。正确检测和鉴定病毒是必须解决的首要问题。我们开展了甲型H1N1流感病毒快速核酸检测技术的研制工作,目前已经建立了甲型H1N1流感病毒核酸RT-PCR检测技术,并将其及时用于临床样本的检测。     

新发甲型H1N1流感病毒HA分子的变异分析

目的:从分子进化水平上分析流感的起源及发展问题,研究目前爆发的H1N1病毒的HA分子的变异行为.方法:以GenBank公布的甲型流感H1N1病毒血凝素(hemagglutinin,HA)核酸序列和我国及世界范围内近几年来报告的H1N1流感病毒HA的核酸及氨基酸序列为研究对象,利用CLUSTAL 1.83和NetNGlyc 1.0等生物信息学软件对HA核酸和氨基酸序列进行了比对分析;将其糖基化位点、氨基酸序列和抗原决定簇与以往流感病毒进行了比较.同时,还将人源和猪源甲型H1N1流感病毒的HA氨基酸序列进行了序列比对和系统发育分析.结果:最新爆发的甲型H1N1流感病毒的HA除了在60,259,453,512位点高度保守区域与之前爆发的流感病毒一致外,在249位点新出现1个"-NTT-"的糖基化位点.发现所有的甲型病毒的氨基酸序列在8个氨基酸位点均发生改变,而8个氨基酸位点位于6个抗原抗原决定簇上.结论:糖基化位点的增加,氨基酸位点的改变导致抗原决定簇的改变,即抗原性漂移现象,都成为引起其传染性改变的重要原因.     

53例重症甲型H1N1流感临床分析

目的了解重症甲型H1N1流感的临床特点,探索其治疗方法。方法回顾性分析53例重症甲型H1N1流感患者的临床资料,总结其临床规律及特点。结果重症甲型H1N1流感好发于20～40岁,20例（37.74%）伴有各种基础疾病。51例（96.23%）有发热,且48例（90.57%）为首发症状,多伴随有畏寒、咳嗽、咳痰、乏力、胸闷和气急等症状。发病早期血常规检查白细胞及中性粒细胞多正常或下降;胸部影像学检查提示33例（62.26%）患者继发不同程度的支气管炎或肺炎。患者经奥司他韦或帕拉米韦抗病毒治疗及相应的抗感染、针对基础疾病治疗等,除2例患者自动出院外,余均痊愈出院。结论重症甲型H1N1流感起病急,早诊断、早期积极合理治疗,能改善预后。     

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。     

中国内地首例确诊甲型H1N1流感病例的实验室检测

本实验室针对中国四川省一例输入性疑似甲型H1N1流感病例的临床咽拭子标本进行Real-time PCR和RT-PCR检测,并随后对部分基因片段进行测序,结果表明临床咽拭子标本为甲型H1N1流感病毒阳性,因此该疑似甲型H1N1流感病例成为中国内地首例确诊的甲型H1N1流感病例。     

江苏首例甲型H1N1(2009)流感病毒分离及其血凝素分子遗传特征分析

2009年6月12日,江苏确诊首例甲型H1N1(2009)病例。通过细胞和鸡胚分离系统,我们分离到一株具有较高血凝活性的病毒,命名为A/Jiangsu/1/2009。为了跟踪病毒的变异情况,我们开展了病毒的全基因组测序工作,在此基础上对其血凝素基因(Haemagglutinin,HA)的遗传特性进行了详细研究。分离株HA蛋白不具有多碱基HA裂解位点,具有低致病性流感病毒特点。与参考株A/California/04/2009相比,分离株A/Jiangsu/1/2009HA蛋白的有4个氨基酸发生了突变,但都不在已知的抗原位点上。分离株有5个潜在糖基化位点,这与近年来古典猪H1N1和北美三源重配猪H1病毒完全一致,保留了古典猪H1的特点。与禽流感H1病毒相比,分离株HA蛋白受体结合位点上的E190D和G225D发生突变,这可能成为新甲型H1N1(2009)在人际间传播的一个重要分子基础。此外,其它受体结合位点上相关氨基酸同时具有人和猪流感病毒的特点。本研究首次对早期流行的甲型H1N1(2009)流感病毒的HA蛋白的分子遗传特征进行了详细研究,对进一步监测病原变异具有重要指导意义。     

荧光定量RT-PCR检测甲型H1N1病毒核酸的临床意义

目的观察荧光定量RT-PCR技术在检测甲型H1N1病毒核酸的临床意义。方法对135例经确诊为甲型H1N1的感染患者的咽式子采用荧光定量PCR技术检测H1N1病毒核酸,同时对40例健康体检者的咽式子做为对照组一同进行甲型H1N1病毒核酸检测。结果 135例确诊为甲型H1N1的患者经荧光定量PCR检测阳性129例,符合率为96.99%。40例健康体检者结果全阴性。结论荧光定量PCR检测甲型H1N1病毒核酸具有快速、特异性高等特点,在采用此方法诊断甲型H1N1时,阳性即可确诊,阴性者要结合临床。     

上海地区H9N2亚型禽流感病毒的遗传演化分析

为阐明上海地区 H9N2亚型禽流感病毒分离株的遗传变异、分子特征和重组模式,选取2002和2006～2014年分离自活禽市场、家禽养殖场和生猪屠宰场的14株 H9N2亚型禽流感病毒进行分析。这14株病毒分别来源于鸡、鸭、鸽、野鸡咽喉和泄殖腔样品及猪肺脏样品,用 H9亚型荧光反转录‐聚合酶链反应（RT‐PCR）试剂盒检测后,阳性样品经无特定病原体（SPF）级鸡胚尿囊腔接种并分离病毒,用血凝抑制（HI）实验进一步确定其血凝素（HA）亚型。RT‐PCR分别扩增这14株病毒全基因并进行序列测定,分析8个基因片段的遗传发生关系,发现这些分离株主要由 F／98亚系、Y280亚系、G1亚系及未知亚系重组而成。根据8个基因片段的组合情况,这14株病毒可分成5个基因型。2002、2006～2008年分离的5株H9N2亚型禽流感病毒代表了4个不同基因型,2009～2014年分离的9株H9N2亚型禽流感病毒属第5种基因型,推测可能与疫苗免疫选择压力有关。因此,在以后工作中加强H9N2亚型禽流感分子流行病学监测是非常必要的。     

Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.     

H5N1亚型禽流感病毒进化的研究进展

由H5N1流感病毒引起的高致病性禽流感,在禽类之间广泛传播。当人类接触这些禽类时,可能会被感染并产生严重的呼吸道症状,且死亡率高达60%。血凝素(hemagglutinin,HA)是H5N1病毒中和抗体的主要抗原,为了便于对病毒的HA突变进行研究,根据HA遗传基因的差异远近,所有的H5病毒株都被划分在20个分支内。对于H5N1病毒进化的研究在禽流感疫苗的研制、禽流感大流行的预防等方面均具有重要意义。现对禽流感、H5N1病毒特征、血凝素的结构功能、H5N1病毒的分支以及病毒进化的研究进行概述。     

Susceptibility of pandemic influenza virus A 2009 H1N1 and highly pathogenic avian influenza virus A H5N1 to antiinfluenza agents in cell culture

The data on cytotoxicity and antiviral activity of commercial antivirals, such as Remantadine, Oseltamivir, Arbidol and Ribavirin in the MDCK cell culture infected with highly pathogenic (H5N1) and pandemic 2009 (H1N1) influenza A viruses are presented. The study of the antiviral activity of antivirals in the MDCK cells culture demonstrated that Arbidol, Rimantadine and Ribavirin efficiently inhibited reproduction of the highly pathogenic H5N1 influenza viruses isolated from sick birds. Arbidol and Oseltamivir carboxylate selectively inhibited reproduction of the pandemic 2009 H1N1 influenza A viruses with changed specificity to the cell receptors, causing severe influenza in men, while remantadine had no effect on their reproduction.     

Phylogenetic analysis of pandemic influenza A/H1N1 virus

The principle of the present study was to determine the evolution of pandemic novel influenza A/H1N1 2009 virus (NIV) by phylogenetic, comparative and statistical analyses. The phylogenetic trees of eight genomic segments illustrate that, so far, the sequences of the NIVs (outbreak group A) are relatively homogeneous and derived by the event of multiple genetic reassortment of Eurasian and North American swine, avian and human viruses (group B). It implies that some of the influenza viruses in group B had higher potential to evolve and getting the ability to transmit from human-to-human after animal-to-human cross-species transmission. The second analysis shows that NIV had attempted a little evolutionary change among humans and before introduction into human it had long evolutionary history. Statistical analysis shows that viruses from both outbreak and nearest group have homologous genes in their genomes which might be reflecting the phylogenetic relationship of strains, and also the presence of unique mutations between groups A-B may associate with increased virulence of NIVs. Both phylogenetic and cluster analyses confirm that the gene exchange takes place between viruses originated from different species and it could be generated NIV with unpredictable pandemic potential. Hence, we conclude that an extensive study should be made to recognize, which reassortment groups are closely related to NIVs, and to determine the sites in the genes of NIV under greatest or least selection pressure, which will ultimately be important in the effective design of vaccine and drugs for ‘swine flu’.     

Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

ABSTRACT: BACKGROUND: Avian influenza virus H9N2 is a panzootic pathogen that affects poultry causing mild to moderate respiratory distress but has been associated with high morbidity and considerable mortality. Interspecies transmission of H9N2 from avian species to mammalian hosts does occur. The virus possesses human virus-like receptor specificity and it can infect humans producing flu-like illness. METHODS: Recently, mild influenza like symptoms were detected in H5N1 vaccinated flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus evolution was investigated by sequencing the viral genes to screen the possible virus recombination. The viral amino acid sequences from the isolated H9N2 strains were compared to other related sequences from the flu data base that were used to assess the robustness of the mutation trend. Changes in the species-associated amino acid residues or those that enabled virulence to mammals were allocated. RESULTS: Phylogenetic analyses of haemagglutinin and neuraminidase genes showed that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that prevails in Israel. The six internal segments of the isolated virus were found to be derived from the same sub-lineage with no new evidence of reassortment. The results demonstrated conserved genetic and biological constitution of H9N2 viruses in the Middle East. The recently isolated H9N2 virus from chicken in Egypt possessed amino acids that could enable the virus to replicate in mammals and caused severe disease in domestic chickens. CONCLUSION: The study highlights the importance of continuous monitoring of the mutations evolved in avian influenza viruses and its impact on virulence to avian species in addition to its importance in the emergence of new strains with the capacity to be a pandemic candidate.     

Susceptibility of antiviral drugs against 2009 influenza A (H1N1) virus

The recent outbreak of the novel strain of influenza A (H1N1) virus has raised a global concern of the future risk of a pandemic. To understand at the molecular level how this new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely to already be resistant to, homology modeling and MD simulations have been applied on the H1N1 neuraminidase complexed with oseltamivir, and the M2-channel with adamantanes bound. The H1N1 virus was predicted to be susceptible to oseltamivir, with all important interactions with the binding residues being well conserved. In contrast, adamantanes are not predicted to be able to inhibit the M2 function and have completely lost their binding with the M2 residues. This is mainly due to the fact that the M2 transmembrane of the new H1N1 strain contains the S31N mutation which is known to confer resistance to adamantanes.     

一株H1N1亚型猪流感病毒的分离鉴定及全基因序列分析

2006年从辽宁省某猪场采集具有流感症状猪鼻拭子共30份,经9~11日龄SPF鸡胚分离,对分离株进行了血凝试验、血凝抑制试验、RT-PCR亚型鉴定,全基因序列比对和接种试验动物试验。结果表明:该毒株具有凝集0.5%鸡红细胞的活性,且与抗猪流感H1标准血清发生血凝抑制反应,RT-PCR分别扩增得到全基因8个片段,利用DNAStar生物学软件进行序列分析,HA基因与GenBank登录的H1~H16中的H1基因序列同源性最高,NA基因与N1~N9中的N1基因序列同源性最高,故该LN株分离毒为猪流感H1N1亚型病毒。全基因序列中,除了M基因不是猪源的,其它基因片段均与国内H1N1亚型猪流感参照株高度同源,推测LN株可能是由类人和类禽谱系的流感病毒与古典猪谱系的流感病毒重排形成的;用该病毒接种试验动物可成功复制出猪流感典型症状。该病毒的分离鉴定及全基因组序列分析为我国进一步调查猪流感的流行规律提供了基础数据。