2009年泉州市H1N1流感监测及基因特性分析

目的 了解2009年泉州地区H1N1流感监测情况,分析泉州市H1N1流感病毒的HA和NA基因特征,探讨该病毒的遗传变异及分子特性.方法 对泉州市H1N1流感监测期间的病人咽拭子采用real-time RT-PCR方法检测病毒核酸,MDCK细胞培养进行病毒分离、鉴定,并提取其中2株代表性毒株病毒RNA;采用RT-PCR扩增病毒HA和NA基因,纯化产物进行核苷酸序列测定;用DNAStar Megalign软件进行序列分析.结果 1020份咽拭子中有200份为H1N1流感病毒核酸阳性,70份季节性流感病毒核酸阳性,其中53份为H3N2亚型,14份为H1N1亚型,3份为B型,并分离到29株甲型H1N1流感病毒株.HA基因经核苷酸序列测定显示,该毒株与北美流行株高度同源,由HA基因核苷酸序列推导的氨基酸系列与疫苗株A/Brisbane/59/2007相比,有22个位于抗原决定簇的氨基酸位点发生变异,但受体结合特异性仍为人样受体.NA基因耐药性位点分析,显示对达菲药物依然敏感.结论 2009年泉州市H1N1流感流行毒株与北美流行株高度同源,相对于疫苗代表株出现了HA蛋白抗原性的改变.     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

一起甲型H1N1流感疫情病原检测及其HA与NA基因特性研究

目的 确认引起一起流感暴发疫情的病原,阐明该病原的血凝素基因(HA)和神经氨酸酶基因(NA)的特性.方法 疫情中最早出现流感样症状病例的咽拭子样本用real-time RT-PCR方法检测甲型H1N1流感病毒核酸,采用鸡胚分离法进行病毒培养,选取两病毒分离株进行HA和NA核苷酸序列测定,并进行基因特性分析.结果 此次流感疫情是由甲型H1N1流感病毒引起的,其HA和NA基因均与参比毒株的HA和NA基因高度同源,NA基因没有发生H274Y突变.结论 本研究的甲型H1N1流感病毒分离株为疫苗亲本株和中国分离株的类似株,对神经氨酸酶抑制剂类药物(如达菲)敏感.     

广东从化市首起甲型H1N1流感疫情的调查

目的探讨从化市首例甲型H1N1流感病例的发生过程,为制订防控措施提供依据。方法按照2009年卫生部《甲型H1N1流感流行病学调查和暴发疫情处理技术指南（试行）》的要求进行现场流行病学调查。结果病例潜伏期74h,体温37.5℃,无明显呼吸道症状;临床症状较轻;咽拭子样本检测,甲型H1N1流感病毒核酸阳性,确诊为甲型H1N1流感病例;未见续发二代病例。结论本事件为输入性二代病例引发本地感染甲型H1N1流感疫情,感染者在无防护的环境下,接触保留带有甲型H1N1流感病毒的被褥或尘埃后导致感染。     

2010年广州市甲型H1N1流感病毒分离株HA基因变异分析

目的比较2010年广州市分离到的甲型H1N1流感病毒血凝素（HA）基因和2009年中国大陆甲型H1N1流感病毒HA基因的变异情况,为甲型H1N1流感的监测和防控提供理论依据。方法收集2010年广州市有发热和呼吸道症状病人的咽拭子标本,用H1N1流感特异性引物进行PCR检测,扩增分离到的H1N1病毒HA片段,测序后与2009年的H1N1毒株进行比对和分析,并用生物信息学方法对抗原位点和糖基化位点进行分析。结果共收集到426份标本,甲型流感阳性211份,其中H1N1流感4株,与2009年分离的甲型H1N1流感相比,有12个氨基酸碱基位点发生了有意义突变,其中6个位点位于抗原位点上;4株毒株HA基因145位氨基酸都发生了变异;其中2株毒株在第180位氨基酸位点的抗原位点发生了变异。进化分析表明4株毒株与2009年中国大陆分离的8株毒株进化关系较远。结论 2010年广州市甲型H1N1毒株与2009年相比发生了较大变异。HA基因145位和180位氨基酸位点变异对H1N1毒株抗原变异有重要意义。本文分离的A/Guangdong/ZS03/2010（H1N1）和A/Guangdong/ZS01/2010（H1N1）毒株可能已经发生了抗原性漂移。     

2009甲型H1N1流感大流行期间北京儿童的流感监测

目的 了解2009年甲型H1N1流感大流行期间北京地区儿童中流感流行的情况.方法 采用WHO推荐的实时荧光定量RT-PCR和国家流感中心推荐的分型方法,对2009年甲型H1N1流感大流行期间因流感样症状来首都儿科研究所附属儿童医院就诊患儿的咽拭子标本进行流感病毒核酸检测.结果 2009年6月1日至2010年2月28日期间共检测了4363份咽拭子标本,其中623例为甲型H1N1阳性,阳性率为14.3%,657例为其他甲型流感病毒阳性(15.1%),所有甲型流感病毒的总阳性率为29.3%.623例中有23例为危重症病例(占阳性患者的3.7%),其中5例死亡.618例信息完整的甲型H1N1病例中,患儿年龄为14天～16岁,性别比例为男比女为1.3:1.1～3岁儿童占25.2%,3～6岁学龄前儿童和6～12岁学龄儿童所占比例相近,各约占30%.在监测期间,仅呈现了一个甲型H1N1的流行波.2009年11月达到最高峰,随后减弱,2010年2月快速下降至2.7%.对监测期间每周20～30份临床标本同时进行季节性流感的监测显示,季节性H3N2、甲型H1N1和乙型流感交替流行.呼吸道合胞病毒(RSV)在甲型H1N1流行趋势减缓后逐渐流行成为流行优势株.结论 2009年6月至2010年2月北京地区儿童中出现甲型H1N1的流行,主要累及学龄前和学龄儿童.季节性流感和RSV与甲型H1N1交替流行.     

2009-2010年山西省流感／甲型H1N1流感的病原学监测分析

目的分析掌握山西省2009--2010年流感／甲型H1N1流感的流行特征,为预测和防控流感／甲型H1N1流感流行提供科学依据。方法对哨点医院和集体发热疫情进行监测采样,采用病毒核酸检测法和细胞培养法分离鉴定流感／甲型H1N1流感病毒,并对2009年5月至2010年4月山西省录入“中国流感监测信息系统”的流感样病例监测报告数据及其样本病原学监测数据进行统计分析。结果山西省全年均有流感病毒活动,2009年流行优势毒株为甲型H1N1流感病毒,流行最高峰在11月（阳性率为58．1％,甲型H1N1占88．1％）,主要导致59岁以下人群发病,其中5-24岁年龄组阳性率较高,进入2010年后乙型（Victoria系）流感病毒的活动有所增加,成为流行株。结论流感样病例监测和病原学监测,可以及时反映流感活动状况,对于掌握该省流感／甲型H1N1流感流行规律有着重要意义。     

2011-2017年河北省甲型H1N1流感流行特征分析

目的掌握河北省甲型H1N1流感的流行特征,为流感防控提供科学依据.方法通过中国流感监测信息系统收集河北省2011年4月至2017年3月流感及流感样病例(influenza-likeillness,ILI)监测数据进行统计分析.结果共检测ILI标本77 008份,甲型H1N1流感病毒核酸阳性2 699份,阳性率3.50％.对2 699例甲型H1N1流感病例进一步分析显示:全省11个地市均有该病例检出,男女比例为1.09:1,各年龄组均可发病,检出率最高的是25～59岁组,最低0～4岁组.2011年11月至2017年3月共出现4次流行,具有明显的季节性,且呈单峰性.各年度均有甲型H1N1流感病例检出,甲型H1N1流感分别是2012-2013、2013-2014和2016-2017年度的优势病原.结论河北省甲型N1N1流感流行具有明显的季节性,冬春季流行,近年来感染以5～14岁年龄组为主.     

2012-2015年北京市顺义区流感监测结果分析

目的 分析顺义区2012-2015年流感病毒核酸的监测结果,掌握流感流行规律.方法 用实时定量PCR法对采集的流感样病例标本进行核酸检测.结果 2012年9月-2013年8月共检测流感样病例标本1040份,核酸检测阳性标本95例,阳性率为9.13％,高峰出现在12月-次年1月,主要以H3N2和新甲型H1N1流感病毒为主;2013年9月-2014年8月共检测流感样病例标本889份,核酸阳性标本138例,阳性率为15.52％,流行高峰出现在1-3月,以H3N2和甲型H1N1流感、乙型流感病毒为主.在2014年9月-2015年8月共检测流感样病例标本798份,核酸阳性标本151例,阳性率为18.92％,流行高峰出现在2014年11月-2015年4月份,以H3N2和乙型流感为主.检测阳性率最高的为60岁以上人群,其次是5-14岁组.结论 2012-2015年,顺义区新甲型H1N1亚型、H3N2亚型、乙型流感均有流行,且各年度优势毒株不一.     

惠州市流感病原学监测结果分析

目的对我市流感样病例标本进行病原学检测,分析其病原学特点,为流感防控提供病原学依据。方法采用MDCK细胞（狗肾细胞）培养法分离流感病毒,用血凝抑制试验对病毒株进行分型鉴定。结果从哨点医院采集的520份标本中共分离出流感病毒4l株,分离率为7．9％,以新甲型H1N1为主。其中新甲型H1N126株（63．4％）,H3N2型5株（12．2％）,B（Yamagata）型1株（2．4％）．B（Victoria）型9株（22．0％）。流感流行高峰出现在春季的1—3月和秋季的8-9月。健康人群血清中流感抗体的阳性率不高,最高为新甲型H1N1抗体阳性率41．9％,最低为B（Yamagata）的抗体阳性率,仅8．1％。对2010年2株新甲型H1N1进行基因测序,结果显示甲型H1N1基因未发生变异,暂时不会造成大的流行。结论惠州市流感病毒的流行时间有明显的季节性,活动相对平缓,新甲型H1N1流感病毒是春季的优势毒株,下半年逐渐转变为H3N2型流感病毒。     

Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1

Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.     

The validation of a real-time RT-PCR assay which detects influenza A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-resistant (H275Y) influenza A H1N1 (2009)

Influenza A H1N1 (2009) was declared by the World Health Organisation (WHO) as the first influenza pandemic of the 21st century. Rapid detection of influenza A and differentiation of influenza A H1N1 (2009) and seasonal influenza A is beneficial. In addition the rapid detection of antiviral resistant strains of influenza A H1N1 (2009) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and type simultaneously for influenza A H1N1 (2009) and oseltamivir resistant (H275Y) influenza A H1N1 (2009). This multiplex assay will allow laboratories to screen respiratory samples for all types of influenza A, influenza A H1N1 (2009) virus and oseltamivir resistant (H275Y) influenza A H1N1 (2009) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. Since most virology laboratories already offer a molecular service for influenza A this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing.     

Viral load and epidemiological profile of patients infected by pandemic influenza a (H1N1) 2009 and seasonal influenza a virus in Southern Brazil

Correlation between virologic profile and clinical features of patients infected by influenza virus provides important information for epidemiological control and clinical management of future disease outbreaks. Samples from patients in Southern Brazil, from June to December 2009, were examined and the viral load was correlated with epidemiological data. All samples were analyzed by qRT-PCR for detection of the 2009-pandemic Influenza A (H1N1). Relative viral loads were assessed based on the 2(-ΔCT) method and epidemiological data were obtained for each patient, following ethical policies. A total of 933 samples were positive for pH1N1 (2009) influenza; 172 were positive for seasonal influenza A; 13 were undetermined; 1992 samples were negative for influenza A. Combined molecular and epidemiological data were available for 38 seasonal and 198 pandemic samples. The median viral load was higher in pandemic than in seasonal influenza samples; in patients infected with pH1N1 (2009), viral load associated positively with chills, myalgia and rhinorrhea, and negatively with dyspnea, but no association was observed with other symptoms, nor with clinical conditions such as pregnancy, smoking, immunodepression and co-morbidities. Regarding patients infected with seasonal influenza, viral loads did not show statistically significant association with any of the symptoms. This is the first study in Brazil that examines epidemiological and molecular data from the 2009 influenza pandemic. The results may serve as a basis for developing strategies to control human-to-human infection and viral dissemination, and for implementing effective measures and public health policies against future novel disease outbreaks.     

Differential diagnosis of pandemic (H1N1) 2009 infection by detection of haemagglutinin with an enzyme-linked immunoassay

A sensitive and convenient immunoassay that can directly differentiate pandemic (H1N1) 2009 (pH1N1) virus from seasonal influenza virus can play an important role in the clinic. In the presented study, a double-sandwich ELISA (pH1N1 ELISA), based on two monoclonal antibodies against haemagglutinin (HA) of the pH1N1 virus, was developed. After laboratory determination of the sensitivity and specificity characteristics, the performance of this assay was evaluated in a cohort of 904 patients with influenza-like illness. All seven strains of pH1N1 virus tested were positive by pH1N1 ELISA, with an average lower detection limit of 10^{3.0 ± 0.4} tissue culture infective dose (TCID)₅₀/mL (or 0.009 ± 0.005 HA titre). Cross-reaction of the assay with seasonal influenza virus and other common respiratory pathogens was rare. In pH1N1-infected patients, the sensitivity of the pH1N1 ELISA was 92.3% (84/91, 95% CI 84.8–96.9%), which is significantly higher than that of the BD Directigen EZ Flu A + B test (70.3%, p <0.01). The specificity of pH1N1 ELISA in seasonal influenza A patients was 100.0% (171/171, 95% CI 97.9–100.0%), similar to that in non-influenza A patients (640/642, 99.7%, 95% CI 98.9–100.0%). The positive predictive value for pH1N1 ELISA was 97.7% and the negative predictive value was 99.1% in this study population with a pH1N1 prevalence of 10.1%. In conclusion, detection of HA of pH1N1 virus by immunoassay appears to be a convenient and reliable method for the differential diagnosis of pH1N1 from other respiratory pathogens, including seasonal influenza virus.