甲3型、乙型流感病毒在MHL混合细胞中增殖的研究

[目的]建立MHL混合细胞瓶分离培养流感病毒的方法，为进行流感监测及临床快速诊断打下基础。[方法]采用MDCK、HEP—2、LLC混合细胞培养瓶和MDCK单一细胞培养瓶，同时分别接种甲3型和乙型流感病毒标准毒株，并设立阴性细胞对照组，观察甲3型和乙型流感病毒毒株在混合细胞瓶和MDCCK细胞瓶中的细胞病变效应(CPE)、血凝滴度和单克隆抗体荧光染色情况。[结果]病毒血凝滴度分别均为1：160，经1：5稀释的甲3型和乙型流感病毒接种MHL混合细胞瓶和MDCK细胞瓶经37℃、48h培养后，感染甲3型病毒细胞出现细胞拉网、团聚病变现象，感染乙型流感病毒细胞则出现明显细胞固缩、脱落现象，MDCK单一细胞培养瓶CPE强于混合细胞培养瓶：37℃、72h培养后，检测血凝滴度，感染甲3型流感病毒的MDCK细胞和混合细胞均为1：40。感染乙型流感病毒的MDCK细胞为1：120，MHL混合细胞为1：20；单克隆荧光抗体检测，感染甲3型和乙型流感病毒的MDCK细胞和混合细胞均呈阳性。[结论]应用MDCK、HEP—2，IL-2混合细胞瓶分离、培养，不仅可对甲型流感爆发进行流行病学监测，同时还可分离鉴定其它呼吸道病毒，利于临床诊断、治疗和预防。     

磷酸奥司他韦对上海及周边地区不同流行性感冒病毒亚型的体外抗病毒效果

目的 了解上海及周边地区近年来流行性感冒(流感)病毒流行株对磷酸奥司他韦的敏感度及是否存在耐药株.方法 从2004-2006年上海市、江苏省无锡市和浙江省德清县流感监测和暴发病例分离到的流感病毒中,随机抽取B型、H1亚型、H3亚型的部分流感病毒,用中性红吸收法在体外检测磷酸奥司他韦的抗病毒作用,以50%抑制浓度(IC50)表示药物的抗病毒效果.方差分析检验药物对各亚型流感株IC50的差异.结果 磷酸奥司他韦对研究使用的50株不同型和亚型流感病毒皆有抑制病毒感染细胞的效果,IC50均＜25 mg/L.药物对B亚型病毒的效果较差,IC50为19.97 mg/L(15.16～22.36 mg/L),高于A/H1N1亚型的8.15 mg/L(0.02～22.36 mg/L,P＜0.05)和A/H3N2亚型的10.92 mg/L(0.08～19.72 mg/L,P＞0.05);对于不同年份分离的流感病毒,药物效果有所变化.结论 磷酸奥司他韦对上海及周边地区的流感病毒具有较好的体外抗病毒效果,需要建立监测网络来监控药物的效果和病毒的耐药情况.     

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.     

上海某院淋球菌株对常用抗生素耐药性观察

淋病是目前世界上发病人数最多的性传播疾病之一,由于菌株的变异和滥用抗生素,自1976年美国发现2例耐青霉素治疗的淋球菌病例以来,淋球菌对青霉素等抗生素耐药菌株不断增加。目前临床上多应用壮观霉素、头孢三嗪和氟喹诺酮类药物对其治疗。但由于这些药物的广泛使用、细菌染色体突变及耐药基因转移等原因,淋球菌逐渐产生了对这些抗生素的耐药菌株。为了解上海地区淋球菌流行株的耐药现状,我们采用琼脂稀释法对1995年和2005年临床分离的淋球菌菌株对青霉素、头孢曲松及环丙沙星的敏感性进行测定,现将结果报道如下。     

流感监测中流感病毒优势株在犬肾细胞系（MDCK）细胞模型中竞争性复制的意义

 【摘要】目的 通过流感病毒优势株在犬肾细胞系（Madin Daby canine kidney cells,MDCK)细胞中竞争性复制,探讨季节性流感流行株感染、复制能力强弱对主导流行株的贡献。方法 在对2011年至2012年上海市闵行区季节性流感监测中,监测到流感样病例775例,确诊流感病例119例。选取2012年1月至2月流感病例中分离的3株B型和3株A/H3N2亚型流感流行株,两两交叉配对成9组,共同感染MDCK,细胞培养上清液抽提核酸,mRT-PCR扩增特异性产物。 结果 有7组共感染组合中A/H3N2 (210 bp)电泳条带逐渐变强、B型条带(390 bp)逐渐变弱至第3代完全消失,2组中A/H3N2与B的条带至第3代均明亮。 结论 流感病毒共感染竞争性复制结果与当时人群流感监测中优势株转换相符。该方法为探讨可能引起主导流行株在人群中变迁的原因提供了研究思路。     

实时细胞动态监测的流感病毒株耐药性研究

目的:了解实时细胞动态监测(RTCA)系统,由细胞病变导致金电极板电阻抗变化的研究;评价磷酸奥司他韦对上海地区不同亚型流感流行株体外抗病毒效果。方法:随机选取2009年-2010年上海地区流感不同亚型流行株,采用实时细胞动态监测(RTCA)系统和传统倒置显微镜观察细胞CPE方法,检测磷酸奥司他韦的抗病毒效果。结果:实时细胞动态监测(RTCA)系统通过病变细胞引起电极板的电阻抗变化的观察,结果连续、实时、客观、可信;磷酸奥司他韦对研究中选取的14株上海地区不同亚型流感流行株有抑制病毒感染细胞的效果。结论:实时细胞动态监测系统比较于传统观察CPE方法,获取的实验信息实时、客观、敏感,但是实验费用昂贵。磷酸奥司他韦(达菲)对上海地区不同亚型流感病毒具有较好的体外抗病毒效果。     

乙型肝炎实验动物模型达乌尔黄鼠初探

1986年本室对我市黄鼠进行了乙肝病毒自然携带调查。发现有类乙肝病毒;并用人的乙肝病毒,对健康幼??黄鼠进行人工感染,连续观察,结果感染两个月后出现病毒血症,并有6只黄鼠连续10～17周检测到HBV-DNA,在国内属首次报告,此结果证实了黄鼠肝炎病毒与人乙墅肝炎病毒有交叉免疫反应。本工作对黄鼠作为一种较为理想的动物模型提供了科学依据。     

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.     

高氧化还原电位酸性水对微生物消毒效果的试验观察

高氧化还原电位酸性水的氧化还原电位>1100mv,pH值<2.7。经检测,该水原液作用5min,可使含量10μg/mlHBsAg转阴;其20％稀释浓度作用2min或10％稀释浓度作用15min,对白色念珠菌的杀灭率分别为100％和99.97％;其10％稀释浓度作用2min,对大肠杆菌杀灭率仍可达100％。     

无锡地区流感病毒病原学监测及H3亚型血凝素基因变异研究

目的 监测无锡地区2005年至2008年季节性流感型与亚型分布并了解2008年部分A/H3N2分离株血凝素(8A)基因变异状况.方法 无锡地区医院门诊流感样患者及集体单位聚集性流感样暴发患者采集鼻咽拭标本,接种MDCK细胞,采用标准抗血清鉴定阳性分离株型与亚型,并对2008年部分H3亚型流感病毒进行HA全基因测序,分析HA基因变异状况.结果 2005年至2008年9月,在无锡地区流感样患者中共分离到435株流感病毒,其中164株为A/H1N1亚型,80株为MH3N2亚型,B型191株.型与亚型有明显季节性分布强弱特征.H3亚型HA序列分析,无锡地区9株分离株与上海地区同期分离株接近,有很多序列相互穿插归属于进化树的同一分支,与WHO 2008-2009年疫苗推荐株相近.结论 近年无锡地区散发和局部暴发流感病毒感染仍主要为A/H1N1、A/H3N2和B型,A/H3N2 HA基因与上海地区同期分离株接近,与WHO 2008-2009年疫苗推荐株相近.