2009年上海地区甲型流行性感冒流行及甲型H1N1病毒分离株变异分析

目的 了解2009年上海地区人群流行性感冒(流感)的流行特征和流行期间甲型H1N1分离株基因和抗原的变异.方法 采集2009年上海地区哨点医院和学校聚集性流感样患者咽拭子标本,接种犬肾细胞(MDCK细胞)分离流感病毒,直接荧光免疫法鉴定流感病毒型,RT-PCR法鉴定亚型,对部分甲型H1N1流感病毒进行血凝素(HA)、神经氨酸酶(NA)等片段全基因测序,分析甲型H1N1流感病毒HA、NA等基因变异.结果 2009年上海地区冬春季人群流感中,季节性H1N1和H3N2流感同时存在,进入第32周时,甲型H1N1和季节性H3N2流感同时流行,第40周后主要是甲型H1N1流行.甲型H1N1流行株HA进化分析显示,不同区域、不同月份分离株互有穿插,上海地区分离株聚集成簇形成一个分枝,与西班牙、俄罗斯、丹麦等国的流行株接近.HA演绎推导氨基酸位点虽有变异,但都不位于抗原决定区域;NA基因演绎推导氨基酸位点未观察到274位点及与耐奥司他韦药物相关其他位点的变异;PB2蛋白氨基酸序列分析显示,第627位和第701位氨基酸分别是谷氨酸和天冬氨酸,为禽源流感病毒PB2蛋白氨基酸位点.结论 2009年上海地区冬春季人群流感,季节性H1N1和H3N2同时流行,夏秋季开始甲型H1N1、季节性H3N2在人群中同时流行,之后以甲型H1N1为主.甲型H1N1与早期分离株比较有一定变异,但尚未出现流行病学意义的抗原漂移株,仍表现为对人的高亲和力和低致病性特征.     

上海地区2009年人群甲型流行性感冒病毒抗体水平检测分析

Objective To know the levels of antibodies against influenza A virus subtypes H1 and H3 of population in Shanghai during 2009, and the detection of antibodies against avian influenza virus subtypes H5 and H9 in population which contacts with avian. Methods The serological survey of the antibodies against influenza A viruses subtypes H1, H3, H5 and H9 in 356 close contacts with avian (professional population) and 332 general subjects (general population) at various age groups were carried out using hemagglutinin inhibit (HI) test. Results The positive rates of antibodies against influenza virus A/Brisbane/59/2007 (H1N1) in general population and professional population were 82.8% (275/332) and 73.9% (263/356), respectively; those of A/Brisbane/10/2007 (H3N2)were 50.6% (168/332) and 54.8% (195/356), respectively. The positive rate of antibodies against influenza virus A/Brisbane/59/2007 (H1N1 )was significantly higher than that of influenza A viruses subtype H3, which was consistent with etiological survey of influenza virus in Shanghai during 2008.The positive rates of antibodies against influenza A virus subtype H5 in professional population and general population were 4.2% (15/356) and 0.3% (1/332), respectively; those of influenza A virus subtype H9 were 34.6% (123/356) and 2.4% (8/332), respectively. The positive rates of antibodies against influenza virus A/Brisbane/59/2007 (H1N1 ) and A/Brisbane/10/2007 (H3N2) in age groups of 6 months-5 years and ≥60 years were lower than other age groups. Conclusions The immune protective response against seasonal influenza A subtype H1 and H3 of population in Shanghai is high,while those of children and the elders were low. The levels of antibodies against influenza A viruses subtype H5 and H9 in professinal population present obviously ascending trend, which indicates that the etiological and serological survey of influenza virus in this population should be enhanced.     

上海地区2009年人群甲型流行性感冒病毒抗体水平检测分析

目的 了解上海地区人群季节性H1、H3亚型流感病毒抗体水平及职业人群中H5、H9亚型禽流感抗体的检出情况.方法 应用血凝抑制试验对上海地区2009年与禽类密切接触的职业人群356人以及一般人群332人各年龄组进行H1、H3、H5、H9亚型流感病毒抗体的血清学监测.结果 A/Brisbane/59/2007(H1N1)抗体阳性分布,一般人群有275人,占82.8%,职业人群有263人,占73.9%;A/Brisbane/10/2007(H3N2)抗体阳性,一般人群有168人,占50.6%,职业人群有195人,占54.8%;人群中A/Brisbane/59/2007(H1N1)抗体阳性率明显高于H3,与2008年上海地区流感病原学监测情况相吻合.职业人群与一般人群H5亚型抗体阳性率分别为4.2%(15/356)和0.3%(1/332);H9亚型抗体阳性率分别为34.6%(123/356)和2.4%(8/332).不同年龄组中,6个月～5岁年龄组和≥60岁的老年组A/Brisbane/59/2007(H1N1)、A/Brisbane/10/2007(H3N2)抗体阳性率较低.结论 上海地区人群对H1、H3季节性流感有较强的免疫保护,儿童和老年人群H1、H3季节性流感抗体水平较低.职业人群禽流感病毒H5、H9抗体有明显升高趋势,要加强对职业人群流感样患者病原学和血清流行病学监测.     

犬肾传代细胞分离流行性感冒病毒致细胞病变的敏感性

目的 了解犬肾传代细胞系(MDCK)细胞对甲型流行性感冒(流感)患者鼻咽拭标本培养1代、2代、3代致细胞病变的敏感性.方法 甲型流感患者鼻咽拭标本在MDCK细胞盲传3代培养,倒置显微镜观察每代细胞致细胞病变效应产生情况.结果 经胶体金快速检测为甲型流感的279份流感样患者鼻咽拭标本,MDCK培养第1代产生致细胞病变为184份,占65.9%,第2代产生致细胞病变为255份,占91.4%,第3代产生致细胞病变为269份,占96.4%.279份标本的细胞培养液经多重RT-PCR检测鉴定为甲型流感病毒的有271份.结论 MDCK分离培养甲型流感患者鼻咽拭标本经盲传3代后,基本可达到95%以上的阳性分离率.     

上海地区SARS患者冠状病毒样颗粒培养与分离

目的 对上海地区SARS患进行病原体的培养10分离。方法 复旦大学公共卫生学院与上海市疾病预防控制中心合作,对上海地区第1例、第2例SARS确诊病例咽拭标本接种多种细胞,进行病原分离、观察。结果 在第1例SARS确诊病例咽拭标本感染的Vero E6细胞观察到CPE;经超速离心、电镜负染观察见到了直径在60～100nm的冠状病毒样颗粒;感染的VeroE6细胞和病人血清间接荧光抗体显示阳性。此株冠状病毒样颗粒暂时命名为SARS—CoV—Fudan I株。结论 在上海市的第1例确诊SARS病例的咽试培养中,见到新型冠状病毒样的颗粒。     

浙江北部农村孕妇麻疹抗体水平的调查

目的为了解浙江北部小于8月龄幼儿麻疹病例增多的原因,对孕龄妇女麻疹抗体水平进行调查。方法采集外周静脉血分离血清,采用ELISA法和中和实验方法分别检测麻疹IgG抗体和中和抗体的平均水平及阳性率。结果218名孕妇麻疹抗体IgG水平的阳性率为80.7%,与麻疹分离野毒株的中和抗体几何平均滴度为30.36±4.70,其中中和抗体<1∶2的阴性样本占16.51%,<1∶8者所占比例为19.27%,≥1∶16者所占比例为73.39%。结论孕妇人群中麻疹低滴度抗体会影响母传抗体的维持时间,这很可能和近年来麻疹爆发表现为成人和低月龄儿病例,尤其是未满8月龄婴幼儿病例增多有极大的关系。     

甲型H1N1流行性感冒病毒血凝素蛋白基因进化研究

Objective To determine the evolutionary rate and divergence time of influenza A virus HA gene isolated recently worldwide pandemic and explore the origin and its transmission. Methods A total of 344 HI sequences available in the GenBank (including 248 isolated from human, 84 from swine, 11 from avian, and 1 from ferret) and 7 isolated in Shanghai were collected. The nucleotide substitution rate and time to most recent common ancestor (tMRCA) was calculated using molecular clock theory and Bayesian Skyline Plot (BSP) based on Markov chain Monte Carlo. Then genetic phylogeny was constructed referring to posterior distribution. Results It was found that H1 sequences in the US from human, swine and avian were clustered significantly with swine H1 ones from Asia phylogenetieally (Cluster US). The second cluster (Cluster Eurasian Human) nearly consisted of human H1 sequences isolated in other regions. The third cluster (Cluster Eurasian Animal) consisted of swine and avian H1 sequences from China and Italy respectively. As for all the H1 sequences, the evolutionary rate was of 2.57×10-3substitutions/site per year averagely (95% Highest Posterior Density: 1.96×10-3-3.03×10-3/site per year). The estimated dates for tMRCA of human H1 in Europe and swine H1 in the mainland of China were the earliest, with the corresponding rates of 6.46×10-3/site per year and 0.97×10-3/site per year respectively. The tMRCAs of human and swine H1 sequences from the US were similar, with the rates of 5.86×10-3/site per year and 5.02×10-3/site per year. Conclusion The present flu outbreak was possibly induced by long-term circulation of influenza A virus (H1 N1) in human population and swine herds in America. There was no evidence proving that influenza virus in China involved in the present outbreak.     

新甲型H1N1流感与中国甲型H1流感HA基因进化比较研究

目的比较新甲型H1N1流感与中国甲型H1亚型流感病毒HA基因的进化。方法用非度量多维尺度法分析下载自GenBank的中国人和猪甲型H1亚型流感HA序列及WHO北半球流感疫苗推荐株HA序列和新甲型H1N1流感HA序列,构建HA基因进化图谱。结果新甲型H1N1流感病毒HA基因与猪甲型H1亚型流感病毒的亲缘关系较近,而和中国人甲型H1亚型流感病毒及WHO近年推荐的疫苗株亲缘关系很远,并且新甲型H1N1流感病毒HA序列在进化上与中国猪甲型H1亚型流感病毒仍然存在一定距离。结论从基因层面上提示我国人群既往免疫和疫苗对新甲型H1N1流感可能不能提供有效保护,病毒有可能会在人群中流行。同时分析结果提示了尚无证据表明这次始于北美的新甲型H1N1流感来自中国。     

上海市卢湾区流感监测结果分析

目的通过对2009年流感监测结果进行分析,探讨科学有效的流感防控措施。方法设置流感监测点医院,开展疫情监测、病原学监测和血清学监测,对监测结果进行统计分析。结果上海市卢湾区7~9月为流感流行季节,各型别流感都存在,7、8月以A3型占优势,9~12月份以甲型H1N1流感为主,2010年初,B型流感逐步增多。发病波及到所有人群,各年龄组总体阳性率差异无统计学意义,甲型H1N1流感较多的侵犯60岁以下的各年龄组,以干部职员、学生为主,季节性流感则多侵犯60岁及以上的老年人群,以离退人员为主。性别构成差异无统计学意义。结论应继续加强流感的监测工作和免疫接种工作。     

河南省8株狂犬病毒磷蛋白基质蛋白基因分析

目的分析河南省8株狂犬病毒磷蛋白P及基质蛋白M的基因序列,探讨狂犬病毒流行株毒力与致病性变异的可能原因。方法以免疫荧光法检测2006年采自河南省的121份犬脑,取阳性犬脑组织悬液接种鼠脑分离病毒,以RT-nest-edPCR法扩增病毒P与M基因,克隆测序后进行生物信息学分析。结果分离到8株狂犬病毒,序列分析表明8株病毒均为基因1型狂犬病毒,8株病毒彼此之间P基因与M基因核苷酸序列同源性均为98.9%～99.8%,推导的氨基酸序列同源性分别为98.0%～99.3%和97.0%～99.5%。8株病毒与我国宁夏分离株CNX8511、CNX8601的同源性最高,P基因与M基因核苷酸同源性分别为89.1%～89.7%和91.1%～92.0%,氨基酸同源性分别为93.3%～94.3%和96.6%～98.0%。在核苷酸及氨基酸水平上,8株病毒与CTN疫苗株的P与M同源性均明显高于与其它疫苗株的相应同源性。系统发育分析表明,8株病毒与我国宁夏街毒株、CTN疫苗株、泰国及菲律宾等东南亚株进化关系最近,而与CTN以外的其它疫苗株、标准攻击毒CVS株,以及我国80年代初分离的DRV、MRV株进化关系较远。氨基酸对位分析表明,较之参比的其它基因1型毒株,河南省8株狂犬病毒的P与M出现多处变异。结论8株狂犬病毒仍属基因1型狂犬病毒,但其P基因及M基因在核苷酸及推导的氨基酸水平上均出现了明显变异。