Inferring the antigenic epitopes for highly pathogenic avian influenza H5N1 viruses

The evasion of influenza virus from host immune surveillance is mainly mediated through its surface protein hemagglutinin (HA), the main component of influenza vaccine. Thus, identification of influenza virus antigenic epitopes on HA can not only help us understand the molecular mechanisms of viral immune escape but also facilitate vaccine strain selection. Despite previous efforts, there is a lack of systematic definition of the antigenic epitopes for the highly pathogenic avian influenza (HPAI) H5N1 viruses. In this study, we infer the HA antigenic epitopes for H5N1 viruses by integrating the antigenic sites mapped from the HA of human influenza H3N2 viruses, the sites which were reported to be associated with immune escape in H5 viruses and the mutation hotspot sites identified in the evolutionary history of HPAI H5N1 viruses. We show that these inferred antigenic epitopes play significant roles in antigenic variation of HPAI H5N1 viruses. Based on inferred antigenic epitopes, we further develop a computational method to effectively predict antigenic variants for HPAI H5N1 viruses (available at http://biocloud.hnu.edu.cn/predict/html/index.html). Therefore, our work has not only inferred the antigenic epitopes for HPAI H5N1 viruses but also provided an effective computational method to assist vaccine recommendations for protection against the deadly bird flu.     

Antigenic sites of H1N1 influenza virus hemagglutinin revealed by natural isolates and inhibition assays

The antigenic sites of hemagglutinin (HA) are crucial for understanding antigenic drift and vaccine strain selection for influenza viruses. In 1982, 32 epitope residues (called laboratory epitope residues) were proposed for antigenic sites of H1N1 HA based on the monoclonal antibody-selected variants. Interestingly, these laboratory epitope residues only cover 28% (23/83) mutation positions for 9 H1N1 vaccine strain comparisons (from 1977 to 2009). Here, we propose the entropy and likelihood ratio to model amino acid diversity and antigenic variant score for inferring 41 H1N1 HA epitope residues (called natural epitope residues) with statistically significant scores according to 1572 HA sequences and 197 pairs of HA sequences with hemagglutination inhibition (HI) assays of natural isolates. By combining both natural and laboratory epitope residues, we identified 62 (11 overlapped) residues clustered into five antigenic sites (i.e., A-E) which are highly correlated to the antigenic sites of H3N2 HA. Our method recognizes sites A, B and C as critical sites for escaping from neutralizing antibodies in H1N1 virus. Experimental results show that the accuracies of our models are 81.2% and 82.2% using 41 and 62 epitope residues, respectively, for predicting antigenic variants on 197 paring HA sequences. In addition, our model can detect the emergence of epidemic strains and reflect the genetic diversity and antigenic variant between the vaccine and circulating strains. Finally, our model is theoretically consistent with the evolution rates of H3N2 and H1N1 viruses and is often consistent to WHO vaccine strain selections. We believe that our models and the inferred antigenic sites of HA are useful for understanding the antigenic drift and evolution of influenza A H1N1 virus.     

An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses

A newly developed lectin neuraminidase test (LNT) and a panel of mouse monoclonal and post-infection ferret antibodies have been used to analyse antigenic drift in N1 neuraminidases of influenza A viruses isolated between 1933 and 1957 and also between 1977 and 1980. Significant antigenic differences were detected among the 'early' (1933-57) viruses since the NA of viruses isolated one year apart could be distinguished serologically. The NA of the 're-emerged' virus A/USSR/92/77 (H1N1) was antigenically related but not identical to influenza A viruses isolated in 1949 (A/Paris/49 (H1N1), A/Geneva/49 (H1N1) which thus predates the previously observed antigenic similarity of A/USSR/77 with A/FW/50 (H1N1) virus.     

Use of GFP-expressing influenza viruses for the detection of influenza virus A/H5N1 neutralizing antibodies

The hemagglutination inhibition (HI) assay is used most commonly for the detection of antibodies to influenza viruses. However, for the detection of antibodies to avian influenza viruses of the H5N1 subtype either induced by infection or by vaccination, the HI assay is insensitive. Therefore, the virus neutralization (VN) assay has become the method of choice to detect human serum antibodies directed to these viruses. However, this assay requires a second assay for the detection of residual virus replication, which makes it laborious to perform and less suitable for high throughput testing of large numbers of samples. Here we describe an alternative method for the detection of these antibodies, which is based on the use of reporter viruses that express the green fluorescent protein (GFP) upon infection of target cells. GFP-expressing viruses were generated carrying the HA of a variety of antigenically distinct H5N1 influenza viruses. The method proved easy to perform and could be carried out rapidly. Using a panel of antisera raised against H5N1 influenza viruses, the assay based on GFP expressing viruses was compared with the classical virus neutralization assay and the hemagglutination inhibition assay. In general, the results obtained in these assays correlated well. It was concluded that the assay based on the reporter viruses is an attractive alternative for the classical virus neutralization assay and suitable for large sero-epidemiological studies or for the assessment of vaccine efficacy in clinical trials.     

北京市甲型H1N1流行性感冒疫苗预防接种不良反应分析

目的 评价甲型H1N1流行性感冒(简称流感)疫苗在大规模人群预防接种中的安全性.方法 2009年9-12月期间北京市共接种甲型H1N1流感疫苗2 113 280人,通过监测系统收集疑似预防接种不良反应患者的个案信息,采用描述性方法对一般反应及异常反应特征进行流行病学分析.结果 北京市共报告疑似预防接种不良反应612例,其中不良反应321例,偶合症203例,心因性反应82例,待定6例.与疫苗相关的不良反应中,异常反应、一般反应报告发生率分别为5.54/10万(117/2 113 280)、9.65/10万(204/2 113 280);严重异常反应报告率为0.19/10万(4/2 113 280).城区、近郊及远郊县的不良反应报告率分别为:16.87/10万(36/213 519)、17.81/10万(187/1 049 817)及11.53/10万(98/849 944).不同年龄组异常反应、一般反应报告率介于3.65/10万(6/164 604)～8.99/10万(27/300 176)和0.61/10万(1/164 604)～22.06/10万(85/385 275)之间.117例异常反应中主要临床诊断为过敏性皮疹(107例),204例一般反应主要临床诊断为发热(176例).91.90%(295/321)不良反应发生于接种24 h以内,所有病例转归良好.结论 本市甲型H1N1流感疫苗接种后报告的不良反应以过敏及发热为主,不良反应类型及严重程度与预期一致,未发现新的或罕见的不良反应.     

An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses.

A newly developed lectin neuraminidase test (LNT) and a panel of mouse monoclonal and post-infection ferret antibodies have been used to analyse antigenic drift in N1 neuraminidases of influenza A viruses isolated between 1933 and 1957 and also between 1977 and 1980. Significant antigenic differences were detected among the ''early'' (1933-57) viruses since the NA of viruses isolated one year apart could be distinguished serologically. The NA of the ''re-emerged'' virus A/USSR/92/77 (H1N1) was antigenically related but not identical to influenza A viruses isolated in 1949 (A/Paris/49 (H1N1), A/Geneva/49 (H1N1) which thus predates the previously observed antigenic similarity of A/USSR/77 with A/FW/50 (H1N1) virus.     

2009年上海市中小学生甲型H1N1流感疫苗大规模接种流行病学效果评价

目的 评价2009年上海市中小学生接种甲型H1N1流行性感冒疫苗(简称甲流疫苗)的流行病学效果.方法 研究对象为长宁、杨浦、闸北、普陀、浦东新区和崇明县内接受甲流疫苗接种(接种组)和未接受甲流疫苗接种(对照组)的在校中小学生,分别收集接种组与对照组学生每日的发热和流感样病例数,通过计算疫苗保护率和保护效果指数,以评价甲流疫苗的流行病学效果.结果 甲流疫苗接种前,流感样病例的发病高峰是在2009年11月,流感样病例罹患率达6.19%.接种疫苗后,接种组发热的罹患率为0.98%,流感样病例的罹患率为0.61%;对照组发热的罹患率为2.37%,流感样病例的罹患率为1.75%.与对照组相比,接种组甲流疫苗接种后控制发热的保护率为58.65%,保护效果指数为2.42,控制流感样病例的保护率为65.14%,保护效果指数为2.87.结论 接种甲流疫苗可以降低中小学生发热及流感样病例的发生率,具有一定的保护作用.     

ViroSpot microneutralization assay for antigenic characterization of human influenza viruses

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically.     

2003—2008年河北省H3N2亚型流感病毒HA1基因演变特征

目的了解近几年河北省H3N2亚型流感病毒HA1基因演变概况。方法选取2003—2008年河北省分离的25株H3N2亚型流感病毒,提取病毒RNA,进行逆转录一聚合酶链反应扩增,产物纯化测序,测定的序列用生物信息软件分析。结果每个流行期分离的毒株都较当年的疫苗株或前一年的毒株出现了位于不同抗原决定簇或者受体结合位点的氨基酸替换。2003—2008年不同流行期分离株在3、140、142、144、145、158、159、189、192、193、198、204、225、226和227位点氨基酸发生了变化,多数的改变发生在抗原决定簇A、B及RBS。进化树分析显示,每个流行期分离株均与前一年的毒株或当年疫苗株出现了不同程度的变化,不断出现新的进化分支,同一流行期分离株基本呈现集中分布。结论2003—2008年河北省H3N2亚型流感病毒HA1基因不断发生变异,密切关注其变异对防控流感流行有重要意义。     

妊娠合并重症新型甲型H1N1流感患者抗H1N1抗体IgG亚类的分布

目的探讨妊娠合并重症甲型H1N1流感患者抗H1N1抗体各IgG亚类在血清中的分布特点。方法收集2009年11月14日-12月31日,确诊的女性甲型H1N1流感患者,分为妊娠病例组,非妊娠病例组,同时收集抗甲型H1N1抗体阳性的健康妊娠女性作为妊娠对照组,采用ELISA法检测各组血清特异性IgG及亚类的百分结合率。结果妊娠病例组的IgG2I、gG3I、gG4亚类的百分结合率中位数分别为53.0%、65.0%、61.6%,均低于妊娠对照组,差异有统计学意义(P<0.05);各组的特异性抗甲型H1N1 IgG均以IgG1亚类为主,为>70.0%,妊娠病例组的IgG2亚类占5.0%,低于非妊娠病例组的10.0%和妊娠对照组的8.0%,(P<0.05)。结论妊娠合并甲型H1N1流感患者中,特异性抗甲型H1N1 IgG以IgG1亚类为主,IgG2亚类的绝对及相对下降与甲型H1N1感染相关。     

一起由H3N2流感病毒引起的局部流感暴发的病原学研究

目的 对一起流感局部暴发的病原体进行分离并鉴定，研究其变异情况，为流感的预防和控制提供依据。方法 用MDCK细胞对病人的咽拭标本进行分离培养，并对分离到的流感病毒HAl基因核苷酸序列进行测定及抗原分析。还检测了患者的血清抗体水平。结果在22份病人的咽拭标本中，分离到3株H3N2亚型流感病毒，患者恢复期血清抗体水平较急性期抗体水平升高4倍。将其中1株的HAl基因测序，与国际代表株进行比较，并与国际代表株进行交叉血抑试验，表明分离到的流感病毒发生抗原漂移。结论这种变异具有流行病学意义，造成甲3亚型流感病毒在湛江地区的局部暴发。     

基于基因序列聚类的甲型流行性感冒病毒H3抗原变异规律研究

目的 利用聚类分析方法探讨全球甲型流行性感冒病毒(流感)H3亚型抗原的变异规律。方法下载NCBI GenBank和流感病毒数据库中全部的甲型流感病毒RNA4节段H3亚型基因序列,在Clustalx中进行序列对齐后,使用两阶段聚类法进行分析,并随后探讨各类的三间分布。结果 所有序列可被分为10类,其中7类主要为人流感病毒,人流感病毒和鸟类、其他哺乳动物的流感病毒被明确的分为不同类别,但和猪流感病毒则共存于数个类中。各类呈现出明显的时间分布和宿主分布规律,但并未发现地域分布规律。结论 由于受到人类免疫系统的选择压力,H3抗原呈现出5-7年出现一次较大变异的流行特征,且这一趋势随着近十年来流感疫苗的普遍使用而呈现加速趋势。同时,猪流感病毒和人流感病毒出现在同一类别中,两者的遗传距离较近,这为猪作为病毒重配的载体提供了新的佐证。     

浙江省2006年乙型流感病毒的分子变异和抗原特性研究

2006年浙江省乙型流感的流行强度明显较往年增强,为明确2006年浙江省乙型流感病毒流行株的分子进化特征,分析其发生暴发的原因,我们对当年从各地市分离的乙型流感病毒进行了抗原及基因特征分析,并与2005年分离毒株进行比较.     

2008—2009年南宁市人季节性H1N1流感病毒神经氨酸酶潜在的抗原位点和抑制剂耐药位点分析

目的了解2008—2009年南宁市人季节性H1N1流感病毒神经氨酸酶(NA)基因的遗传进化特征,探讨NA基因潜在的抗原位点和活性位点(抑制剂耐药性位点)变异规律。方法提取2008—2009年20株H1N1流感病毒RNA,采用RT-PCR扩增病毒NA基因后进行序列测定,通过CTLPred软件预测NA基因上潜在的抗原位点,并对NA分子进化和其重要功能位点的遗传变异进行分析。结果将20株H1N1流感病毒毒株与北半球疫苗推荐株A/Solomon/3/2006和A/Bris-bane/59/2007构建进化树,NA进化树共分成3个类群,10株2008年H1N1流感病毒毒株聚集成分支Ⅱ,10株2009年毒株与疫苗株A/Brisbane/59/2007聚集成分支Ⅰ,A/Solomon/3/2006则独立形成1个分支(Ⅲ)。2008年毒株抗原位点替换频率较高,相对于A/Solomon/3/2006氨基酸替换分布在不同的3个区域的3个位点,分别是N64H、Y100H、L367I;2009年度的毒株抗原决定簇位点相对保守。在已知NA的酶活性位点中,2009年所有毒株均在275位点发生了H>Y的突变,而这种变异在2008年的毒株中未有出现。结论 A/Solomon/3/2006落后于2008年毒株,A/Brisbane/59/2007对应的疫苗在2009年能起到较好的保护作用;大量H275Y耐药株的出现提示在流感病毒监测中应密切关注其耐药位点的变异。     

上海市健康人群流感抗体调查

[目的 ]　了解上海市健康人群流感抗体水平 ,分析和预测流感的流行趋势。 [方法 ]　应用血凝抑制试验法对 1999年 10月采集的 15 0份健康人群血清进行了流感抗体测定。 [结果 ]　对流感病毒抗原A/悉尼 / 5 / 97(H3N2 )和A/上海 / 1/ 98(H3N2 )抗体阳性率分别为 98.0 %和 92 .l%。对A/京防 / 2 6 2 / 95 (H1Nl)和A/上海 / 7/ 99(H1N1)抗体阳性率分别为 6 9.2 %和 47.0 %。对B/鲁防 / 7/ 97和B/上海 / 12 1/ 99抗体阳性率分别为 30 .5 %和2 7.4%。 [结论 ]　A/悉尼 / 5 / 97(H3N2 )类毒株已不可能在上海再度引起流行。A/上海 / 7/ 99(H1N1)类毒株今后在上海可能会出现局限性流行和散发病例。乙型流感今后在上海可能会出现较多的散发病例     

江苏省新甲型H1N1流感疫苗免疫后抗体水平变化趋势研究

目的了解新甲型H1N1流感疫苗免疫前后人群抗体水平动态变化情况。方法随机选取部分疫苗接种人群为受试者,微量半加敏血凝抑制实验检测免疫前、免疫后1个月与免疫后3个月的血清抗体水平,计算抗体阳性率与抗体几何平均滴度(GMT),并进行统计学分析。结果免疫前、免疫后1个月与3个月抗体阳性率分别为13.64%(9.58%~18.61%)、83.63%(78.15%~88.20%)、77.06%(71.09%~82.32%),GMT分别为9.36(8.30~10.56)、122.53(100.29~149.70)、96.07(77.85~118.54)。免疫前与免疫后1个月、3个月后相比,抗体阳性率与GMT差异均有统计学意义。免疫后1个月与免疫后3个月两者差异均无统计学意义。结论本研究所用甲型H1N1流感病毒裂解疫苗免疫后抗体水平明显升高,且维持时间较长。     

甲型H1N1流感的临床特点与治疗

2009年3月,墨西哥暴发的甲型H1N1流感疫情,迅速在全球范围内蔓延,严重威胁着人们的健康,影响面积之广,其流行强度已经直追甚至超过禽流感和SARS.到2009年12月底,全球累计死亡病例超过1万余人,且死亡人数还在增加.WHO在短时间内提升流感大流行预警级别为6级,中国卫生部2009年4月30日发布公告,明确将甲型H1N1流感纳入传染病防治法规定管理的乙类传染病,并采取甲类传染病的预防、控制措施.     

2009年山东省四起甲型H1N1流行性感冒样病例暴发疫情病原学分析

目的 通过对2009年山东省济宁市4起流行性感冒(简称流感)样病例暴发疫情进行病原学分离鉴定,以及对血凝素基因(HA)和神经氨酸酶基因(NA)特性分析,研究其基因变异情况.方法 采集4起流感样病例暴发疫情中发热患者的鼻咽拭子标本34份,采用逆转录实时PCR(realtime RT-PCR)方法进行核酸检测,对阳性标本开展病毒分离,并对分离的甲型H1N1流感病毒的HA、NA基因序列进行测序.利用DNAStar软件对序列进行同源性分析,利用Mega 4.0软件进行基因进化分析和氨基酸进化分析.与WHO推荐的疫苗株及国内代表株进行对比.结果 在34份鼻咽拭子标本中,17份甲型H1N1流感病毒阳性,11份标本分离培养出了甲型H1N1流感病毒.将其中的7株进行HA基因和NA基因测序,HA、NA基因的同源性分别为98.4%～99.6%、99.2%～100.0%.与WHO推荐的疫苗株及国内代表株相比,有11个HA基因的氨基酸发生替换,分别为38、40、56、90、100、145、172、173、220、303及338位,其中38、40和303位位于抗原决定簇C区,172和173位位于抗原决定簇D区,56位位于抗原决定簇E区,同时40位为糖基化位点;有7个NA基因的氨基酸发生了替换,为80、106、241、248、351、369和386位,386位为糖基化位点;未发生神经氨酸酶蛋白275位H-Y的替换.结论 山东省甲型H1N1流感暴发流行株HA基因和NA基因均具有高度同源性,HA蛋白和NA蛋白均存在不同程度的氨基酸替换,部分流行株抗原决定簇和糖基化位点发生改变.     

天津地区2005年甲1亚型流感病毒HA1区基因特性分析

目的 了解天津地区夏季一起小学校流感样暴发疫情中分离出2株甲1亚型流感病毒株HA1基因变异情况。方法MDCK细胞和鸡胚双腔法分离流感病毒，收获病毒液提取病毒的RNA，进行RT—PCR，扩增产物纯化后测序，用DNASTAR软件进行序列分析。结果新分离株HA1基因长度为325个氨基酸，与国际疫苗株A／NewCaledonia／20／99相比氨基酸同源性高于98％，氨基酸替换位点有5个，其中165位位于抗原决定簇Cal区。结论天津地区新分离甲1亚型流感病毒抗原性发生进一步漂移，但变异程度不大。