MTH1基因表达的半定量RT-PCR检测方法的建立

目的建立一个检测培养MDCK细胞内MutT同源蛋白1（MTH1）基因表达的半定量RT-PCR体系。方法细胞培养后提取总RNA,经优化RT-PCR反应检测细胞内MTH1基因的表达量。条带经ScionImmage软件分析并与内参基因GAPDH相比,对体系的准确性、重复性和灵敏度进行分析,然后运用本体系检测流感病毒感染后MDCK细胞内MTH1的表达。结果扩增产物经测序分析证实与GenBank中该细胞的MTH1基因序列一致;灵敏度检测发现最低可从50ng总RNA中检出目的基因的表达;重复性测定发现,MDCK细胞MTH1与GAPDH灰度值比值的均值为（2.02±0.09,n=10）,CV值为4.31%。结论本方法的准确性、重复性和灵敏度均较好,可用于半定量检测培养细胞尤其是MDCK细胞内MTH1 mRNA表达量。     

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。     

甲型H1N1流感的小知识

甲型H1N1流感又称为A（H1N1）型流感,人感染猪流感。其病原体为甲型H1N1流感病毒,该病毒包含有禽流感、猪流感和人流感三种流感病毒的核糖核酸基因片断。中国卫生部2009年4月30日发布2009年第8号公告,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。     

人巨细胞病毒感染THP-1源性巨噬细胞对IL-1β表达的影响

目的 观察HCMV感染THP-1源性巨噬细胞后,促炎细胞因子IL-1β表达的时序性变化.方法 构建HCMV感染THP-1源性巨噬细胞模型,设立HCMV AD169标准毒株感染组、模拟感染对照组、LPS+ATP对照组和poly(dA:dT)对照组.用ELISA法测定THP-1源性巨噬细胞培养上清IL-1β水平在病毒感染细胞后lh、3h、6h、12h、24 h和48 h的时序性变化.分别用real-time PCR和western blot检测感染后6 h IL-1β基因和蛋白的表达水平.结果 HCMV感染THP-1源性巨噬细胞6h后,促炎细胞因子IL-1β基因的相对表达量是模拟感染组的7.77倍.HCMV感染THP-1源性巨噬细胞1h后,细胞上清IL-1β表达量逐渐显著增高,感染后3h和6h继续升高,感染后12h达到高峰,一直持续到48 h.HCMV感染THP-1源性巨噬细胞6h HCMV感染组IL-1β蛋白表达明显高于模拟感染对照组和poly(dA:dT)对照组,而与LPS+ATP对照组比较无统计学差异.结论 HCMV感染THP-1源性巨噬细胞可诱导IL-1β高表达,且呈时序性增高趋势.     

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.     

甲型H1N1流感病毒疫苗株高适应性MDCK细胞的筛选及鉴定

目的筛选甲型H1N1流感病毒疫苗株高适应的MDCK单克隆细胞株,用于培养生产流感病毒疫苗,为细胞代替鸡胚生产制备流感病毒疫苗提供保证。方法通过有限稀释法将MDCK细胞进行单克隆化,扩大培养建立单克隆化细胞库,通过血凝和TCID50筛选甲型H1N1流感病毒疫苗株的高适应性单克隆化细胞株,并鉴定所获得的细胞株细胞表面NeuAcα2,6 Gal的丰度。结果共制备了97株单克隆化MDCK细胞,经过甲型H1N1流感病毒疫苗株的筛选,共筛选到2株高适应甲型H1N1流感病毒疫苗株的MDCK单克隆化细胞株,其TCID50分别为6.68 log10 TCID50/ml和6.77 log10 TCID50/ml,其表面NeuAcα2,6 Gal的丰度明显提高。结论成功培养了MDCK单克隆细胞株,经筛选获得的单克隆细胞株其血凝滴度,TCID50都比普通MDCK细胞有明显提高,为细胞培养生产流感病毒疫苗奠定了基础。     

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。     

甲型H1N1流感病人外周血免疫细胞和细胞因子水平变化

目的:观察甲型H1N1流感患者和流感症状患者外周血中B淋巴细胞(B)、自然杀伤细胞(NK)、辅助性T淋巴细胞(CD4+T)、细胞毒性T淋巴细胞(CD8+T)绝对数的变化以及血清中细胞因子水平的变化,探讨甲型H1N1流感病毒对机体免疫反应的影响。方法:收集68名病人(流感症状26名,轻症甲流29名,重症甲流患者13名),健康体检者20名;应用流式细胞术检测B%、CD4+T%、CD8+T%以及NK%;应用酶联吸附免疫法检测其血清中TNF-α、IL-12、IL-18的浓度。结果:与健康对照组相比较,流感症状组、轻症甲流组及重症甲流组外周血中B、CD4+T、CD8+T细胞绝对数显著降低(P<0.05);流感症状组和轻症甲流组的NK细胞高于健康对照组(P<0.05),重症甲流组的NK与健康对照组无显著差异(P>0.05);流感症状组、轻症甲流组及重症甲流组血清中细胞因子TNF-α、IL-12、IL-18的浓度均高于健康对照组(P<0.05);而流感症状组、轻症甲流组、重症甲流组之间差异无显著性(P>0.05)。结论:甲型H1N1流感病毒感染后,外周血中TNF-α、IL-12、IL-18等相关细胞因子上升;以及NK细胞增加,B、CD4+T、CD8+T细胞减少。     

2009H1N1流感病毒神经氨酸酶基因克隆与表达

目的制备2009H1N1流感病毒神经氨酸酶（NA）重组蛋白,为建立H1N1快速检测方法和神经氨酸酶抑制剂筛选模型奠定基础。方法采用MDCK细胞方法分离2009H1N1流感病毒,提取病毒RNA,RT-PCR生成NA基因,构建原核表达载体PET-102/D-TOPO-NA,IPTG诱导表达重组蛋白,SDS-PAGE凝胶电泳、WersternBlotting鉴定重组蛋白。结果成功分离2009H1N1流感病毒,NA蛋白119、152、275、292位氨基酸分别为Glu、Arg、His和Cys。SDS-PAGE凝胶电泳蛋白分子相对分子质量约为64000,与预期一致。WesternBlotting证实该蛋白具有神经氨酸酶抗原活性。结论 IPTG诱导原核表达神经氨酸蛋白的最适浓度为0.1mmol/L。实验得到的蛋白尚需进一步纯化。     

1999-2007年佛山市流感病毒活动情况及甲3亚型病毒HA1基因分析

目的总结1999-2007年佛山市流感病毒流行情况,分析甲3亚型（H3N2）毒株流行与其HA1基因进化的关系。方法用MDCK细胞分离流感病毒,培养后血凝阳性者进行型别鉴定。随机抽取每年2～3株甲3亚型毒株的细胞培养物提取RNA后进行HA1基因的逆转录,对其核苷酸和氨基酸序列及亲缘关系进行分析。结果甲型和乙型流感病毒在佛山市人群中同时流行。甲型流感毒株是人群感染流感的主要型别。HA1区氨基酸序列与历年的流感疫苗推荐株相比,点突变率为0.3%～6.08%。发生替换的重要位点包括了抗原决定簇的17个位点、抗体结合部位的10个位点和1个糖基化位点。结论佛山市的甲1和甲3亚型流感毒株活动呈此起彼伏的优势株转换现象。甲3亚型新旧毒株交替迅速,大致按照毒株分离的年代聚类成进化树的不同小侧枝,表明新的流行株出现后可能迅速突破地域局限。提示地区实验室及时监测和研究流感毒株的发生和发展是流感监测网络建设的基础。     

利用反向遗传学技术构建H5N9重组流感病毒株

目的 利用反向遗传学技术构建来源人感染禽流感病毒H5N1和H7N9 HA和NA基因的H5N9亚型禽流感病毒.方法 全基因合成A/Beijing/01/2003(H5N1)禽流感病毒HA基因片段和A/Zhejiang/DTID-ZJU10/2013(H7N9)禽流感病毒NA基因片段,插入到pHW2000载体,与携带有A/Puerto Rico/8/34(H1N1)的6个内部基因的pHW2000重组质粒一起转染293T和MDCK混合细胞,拯救H5N9重组病毒.结果 核酸测序、HA和NA基因转录和表达检测、细胞病变分析确定利用该反向遗传学系统可以成功拯救H5N9病毒.重组H5N9病毒在MDCK细胞上复制增殖能力低于相同方法拯救H1N1病毒.结论 利用反向遗传学技术成功构建一株H5N9重组病毒.     

MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells

The ability to isolate and propagate influenza virus is an essential tool for the yearly surveillance of circulating virus strains and to ensure accurate clinical diagnosis for appropriate treatment. The suitability of MDCK-SIAT1 cells, engineered to express increased levels of alpha-2,6-linked sialic acid receptors, as an alternative to conventional MDCK cells for isolation of circulating influenza virus was assessed. A greater number of influenza A (H1N1 and H3N2) and B viruses from stored human clinical specimens collected between 2005 and 2007 were isolated following inoculation in MDCK-SIAT1 cells than in MDCK cells. In addition, a higher titer of virus was recovered following culture in MDCK-SIAT1 cells. All A(H1N1) viruses recovered from MDCK-SIAT1 cells were able to agglutinate both turkey and guinea pig red blood cells (RBC), while half of the A(H3N2) viruses recovered after passage in MDCK-SIAT1 cells lost the ability to agglutinate turkey RBC. Importantly, the HA-1 domain of the hemagglutinin gene was genetically stable after passaging in MDCK-SIAT1 cells, a feature not always seen following MDCK cell or embryonated chicken egg passage of human influenza virus. These data indicate that the MDCK-SIAT1 cell line is superior to conventional MDCK cells for isolation of human influenza virus from clinical specimens and may be used routinely for the isolation and propagation of current human influenza viruses for surveillance, diagnostic, and research purposes.     

Influenza virus is not restricted by tetherin whereas influenza VLP production is restricted by tetherin

Tetherin (ST2/CD317) is a cellular protein that restricts the release from cells of some enveloped viruses including HIV-1. To examine if influenza virus is affected by tetherin, MDCK cells constitutively expressing human tetherin and control MDCK cells were infected with influenza virus. No difference was observed in infectious titers, at 24 h or 48 h post-infection. In contrast, tetherin expression inhibited influenza virus-like particle (VLP) release into the media. Expression of the HIV protein Vpu overcame the tetherin block of influenza virus VLPs. A human tetherin mutant that lacks a C-terminal GPI anchor attachment signal (tetherin-ΔGPI) was constructed to test if this mutant could be incorporated into the released virus or VLP particles. Whereas tetherin-ΔGPI was incorporated into influenza VLPs it was not incorporated into influenza virions. Taken together these data suggest that influenza virions may contain a tetherin antagonist.     

Subtype-specific identification of influenza virus in cell cultures with FITC labelled egg yolk antibodies

We report on results obtained with a direct immunofluorescence test for subtype-specific identification of influenza virus in detached cells of MDCK cultures after inoculation of 281 clinical specimens from patients with influenzalike disease. Influenza virus antibodies were produced in eggs from immunized hens and labelled with FITC. In 157 cases CPE was found in MDCK cells. A total of 57 cases of influenza A (H3N2), 86 cases of influenza A (H1N1), and 14 cases of influenza B were identified. In 33 cases of influenza A (H1N1) infection with massive CPE guinea pig but not chicken erythrocytes were agglutinated by the cell culture supernatants. The single step immunofluorescence test described proved easy to perform and results were obtained within 1 h after CPE was observed in contrast to the conventional HIT which is very time-consuming.Dedicated to Professor Rudolf Rott on the occasion of his 60th birthday     

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus

BackgroundDespite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny.Study designWe document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09.ResultsComplete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID₅₀ of 10⁴/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia.ConclusionsIt is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities.     

江门市居民甲型H1N1流感病毒血清抗体的调查分析

目的了解江门市各年龄段人群甲型H1N1流感病毒抗体水平,为完善甲型H1N1流感疫情防控措施提供科学依据。方法采取多阶段分层随机抽样方法抽取研究对象480人,进行血清标本采集和问卷调查,应用红细胞血凝抑制方法检测甲型H1N1流感病毒抗体。结果人群甲型H1N1流感病毒抗体阳性率为19.38%,0～、6～、16～、25～和60岁以上组的甲型H1N1流感病毒抗体阳性率分别为22.09%、27.36%、24.47%、14.58%和8.16%。6～24岁的甲型H1N1流感病毒抗体阳性率较高,为51.83%,无症状抗体阳性率达12.61%。抗体阳性率在不同年龄、性别和职业上差异均有统计学意义（P〈0.05）。结论江门市甲型H1N1流感病毒感染状况各年龄段不同,青少年抗体阳性率较高,老年人抗体阳性率相对较低,男性抗体阳性率高于女性。