福建省首次从病人血清及蚊体分离出登革病毒的研究

目的　１９９９ 年入夏以来, 我省某地发生不明原因发热、头痛、肌痛、乏力、皮疹等症状的病人, 拟诊为登革热。为了从病原学上证实, 我们进行了病毒分离, 为防治工作提供科学依据。方法　应用早期病人血清７ 份, 白纹伊蚊１ 份经处理后接种Ｃ６／３６ 细胞, 并应用恢复期病人血清、双相血清、免疫血清及单克隆抗体进行免疫荧光法鉴定及分型。结果　在７份早期病人血清中经２ ～３ 代分离出５ 株登革病毒, 并从白纹伊蚊体内分离出１ 株登革病毒。结论　此分离的毒株经鉴定均为登革ＩＩ型病毒。此系福建省首次从病人血清及蚊媒体内分离出病毒的报告。不仅为防制工作提供科学依据, 而且为今后的研究奠定了基础。     

福建省1株登革Ⅱ型病毒的鉴定

目的对2005年福建省分离的1株登革病毒(DV)进行鉴定,并从分子水平追踪其可能的传染源。方法采用酶联免疫吸附试验(ELISA)检测疑似登革热患者血清中DV(IgM、IgG抗体;同时应用C6/36细胞、单克隆抗体间接免疫荧光(McAb-IFA)、逆转录(套式PCR法分别进行病毒的分离和鉴定,并对分离株的部分基因进行核苷酸序列分析。结果患者血清登革病毒特异性IgM抗体阳性、IgG抗体可疑,表明该患者在近期感染过登革病毒。患者血清接种C6/36细胞,观察到登革病毒特有的CPE。受感染的C6/36细胞能与登革病毒Ⅱ型单克隆抗体反应,表明分离的病毒株为登革Ⅱ型病毒。分离株的核酸提取物经RT(PCR扩增,登革病毒通用引物可扩增出511bp的特异性条带,型特异性引物扩增出119bp的特异性条带,进一步证实分离的病毒株为登革Ⅱ型病毒。分离株RT(PCR扩增产物的核苷酸序列与30株不同地域来源的登革Ⅱ型病毒相应序列构建的系统发生树表明,此毒株与东南亚地区的毒株比较接近。此次分离株的序列与1999年登革Ⅱ型病毒福建株的对应序列在亲缘关系上有一定程度距离。结合流行病学调查资料,进一步确定此病例为输入性感染病例。结论福建省首次从输入性登革热患者血清中分离出登革Ⅱ型病毒,该病毒来源于东南亚地区。     

2015-2016年深圳市登革Ⅲ、Ⅳ型分离病毒株E/NS1基因序列特征

目的 了解2015-2016年深圳市分离的登革Ⅲ、Ⅳ型病毒E/NS1基因序列特征及登革Ⅲ、Ⅳ型病毒流行规律,探究其可能的传播来源。方法 收集2015-2016年深圳市登革热患者病例资料及急性期血清,用C6/36细胞培养分离登革病毒,用FQ-PCR对其进行血清分型,分离成功的病毒株用反转录-聚合酶链反应(PCR)方法扩增E基因和NS1基因,进行序列分析,绘制系统进化树,氨基酸比对分析4个不同血清型NS1蛋白。结果 从5份Ⅲ型的登革病毒标本中成功分离4份病毒株,3份Ⅳ型登革病毒标本中成功分离2份登革病毒;BLAST分析保守E基因结果表明,与DENV-3分离株同源性最高(99%)的毒株主要是印度尼西亚2010和2015年分离株、菲律宾2015年分离株。与DENV-4分离株同源性最高(99%)的毒株主要是菲律宾2013年分离株、印度尼西亚2010分离株、意大利2009年分离株。NS1基因序列分析显示所选4株不同血清型的病毒株氨基酸相似性为77.69%,4个不同血清型的登革热NS1抗原存在5-7个氨基酸保守区域。结论 2015-2016年深圳市输入的登革Ⅲ、Ⅳ型病毒可能来自印度尼西亚和菲律宾,应加强出入境人员的监测工作,避免输入引起本地感染的风险。     

云南省输入性登革热病例登革病毒1型的分离研究

目的对2011年8月采集的从老挝输入的登革热病人急性期血清进行登革病毒分离,以找出病原学证据。方法用C6/36白纹伊蚊细胞进行病毒分离,对有细胞病变的标本进行RT-PCR,检测登革病毒RNA及型别,对PCR产物测序,进行比对分析和进化树分析。结果从8例输入登革热病人的血清中分离到4株能引起细胞病变的病毒,均为登革病毒1型,与泰国毒株的同源性99%。结论云南省首次从输入登革热病人血清中分离到登革病毒1型,为云南省登革热的防控提供了依据。     

广东南海市登革热病原学及血清学检测

目的　为了明确广东省南海市１９９８ 年夏、秋季一批发烧、出疹病人的诊断。方法收集疑似登革热（ＤＦ）患者血清５２份,应用逆转录 聚合酶链反应（ＲＴ ＰＣＲ）、病毒分离和间接免疫荧光技术分别进行了病原学和血清学检测。结果　从２５ 份发病早期患者血标本中分离病毒１０ 份,经用单克隆抗体间接免疫荧光检测和ＲＴ ＰＣＲ检测证实ＤＥＮ２ 型感染。ＩｇＧ抗体检测,阳性率为４６－１５％ （２４／５２） ,最高滴度达１∶１２８０,ＩｇＭ 抗体检测阳性率为６０％ （１５／２５）。部分病人双份抗体检测,抗体滴度４ 倍升高。结论　此次南海市登革热暴发流行为登革２ 型感染所致。     

登革病毒2型广东省分离株的鉴定及NS1基因序列分析

目的 明确广东省南海市1998年夏、秋季一批发热、出疹患者的诊断,并从基因水平分析其流行株的来源。方法 收集疑似登革热（DF）患者 血清52份,应用逆转录－聚合酶链反应（RT－PCR）、病毒分离和间接免疫荧光技术分别进行了病原学和血清学检测;同时采用分子克隆技术将PCR扩增产物克隆到T载体,并进行序列测定。结果 从25份发病早期患者血标本中分离病毒10份,经用单克隆抗体间接免疫荧光检测和RT－PCR检没让实为登革病素2型感染。基因序列分析表明,1998年广东分离的登革2型病毒与ThNH-P28/93、C0166`16681`NGC`JAM`04`GD06/93和S1株核苷酸的同源性分别是：98％、97％、96％、93％、92％、91％。结论 此次南海市登革热暴发流行为 登革病毒2型感染所致。基因序列分析提示：1998年广东省南海市分离的登革病毒2型可能来源于泰国。     

逆转录-套式PCR方法鉴定福建省登革热分离株型别的研究

目的　应用分子生物学手段鉴别福建省登革热分离株的型别。方法　病毒培养上清经浓缩后离心沉淀 ,提取病毒ＲＮＡ ,行ＲＴ -ＰＣＲ ,再用型特异性引物扩增出特异性片段 ,电泳观察判断其型别。结果　从早期病人血液中分离的 3株和从白纹伊蚊中分离的 1株病毒经扩增后其产物均为登革热 2型特异性条带。结论　福建省 4株分离株均为登革热 2型病毒。     

神经系统疾病

20001296聚合酶链反应法诊断中枢神经系统肠道病毒感染/李爱云…//中华内科杂志一199,.邓(8).一几5吕 观察朽例无菌性脑膜炎的住院病人其急性期脑脊液标木31份RT一PCR产物电泳和杂交结果阳性,而恢复期仅l例阳性一急性期的脑脊液同时行病毒分离,14例阳性、共分离到6个不同的血清型,其中埃柯病毒2型6例,柯萨奇病毒BZ型、5型和Ecl,。)6型各2例.Echo3型和11型各1例恢复期的脑脊液病毒培养均阴性。指出R‘r一PCR敏感性明显高于病毒培养(I,<。J.()l)。图l参5(李国芳) 20001297格林一巴利综合征与空肠弯曲菌Penner血清型的关系/李海峰…//中…     

中缅部分边境地区登革热流行情况调查

目的了解中缅边境地区云南省盈江那邦镇和缅甸拉咱市登革热流行状况,为边境地区登革热防控提供科学依据。方法 2017年9-10月,在中缅边境地区中国云南盈江那邦镇和缅甸拉咱市各选3个调查点,采用背负式电动捕蚊器采集成蚊并用形态学方法鉴定蚊虫种类,捕获的蚊虫标本通过接种C6/36细胞分离病毒;采用间接酶联免疫吸附试验(ELISA)检测健康人群血清登革病毒IgG抗体;采用NS1抗原检测试剂检测疑似DF病例血清标本,阳性标本采用RT-PCR方法检测鉴定登革病毒血清型。结果共捕获6属14种703只蚊虫,其中登革热媒介埃及伊蚊为当地优势蚊种(57.75%,406/703),未发现埃及伊蚊携带登革病毒。采集的256份健康人血清中DENV IgG抗体阳性13份(5.08%,13/256),其中盈江那邦DENV IgG阳性率4.17%(4/96),缅甸拉咱5.63%(9/160),差异无统计学意义(χ~(2 )=0.265,P0.05);488份疑似登革热病人血清DENV阳性166份(34.02%,166/488),其中盈江那邦阳性83份(32.0%,83/259),缅甸拉咱阳性83份(36.24%,83/229)。从10份阳性标本中分离出5株DENV病毒,其中4株DENV-1(盈江那邦3株,缅甸拉咱1株),1株DENV-3(盈江那邦)。结论中缅边境地区盈江那邦镇和缅甸拉咱市登革热媒介埃及伊蚊分布广,DENV血清型主要以DENV-1为主,应加强中缅边境地区登革热疑似病例和媒介的监测。     

登革4型病毒深圳分离株E基因序列测定及其系统发生分析

目的对深圳市2005年从登革热患者急性期血液中分离到的1株登革病毒进行型别鉴定,从分子水平分析分离株的生物学特征,追踪其可能的地域来源。方法用C6/36细胞培养增殖病毒株SZ0524,收集病毒液。用逆转录-半套式PCR(RT-semi-nested-PCR)方法和荧光PCR方法对其进行型别鉴定。扩增病毒E基因后进行序列测定,并与不同国家和地区的登革病毒株进行同源性比较和进化树分析。结果深圳市登革病毒分离株用4型特异性引物扩增出392bp的特异性条带,荧光PCR进一步证实了分离的病毒株为登革4型病毒。SZ0524与登革病毒4型国际标准株H241株在E基因上核苷酸同源性为99.7%,而与登革病毒1、2、3型国际标准株HAWAII、NGC、H87同源性分别为57.0%、59.2%和56.2%。基因进化树显示SZ0524株与D4-73NIID株和D4-61NIID株亲缘关系最近,其次为H241,在进化树的同一分支上,属基因Ⅰ亚型。结论从分子水平证明从深圳市登革热患者血清中分离到的毒株确为DEN-4型病毒。结合流行病学调查资料,证实此病例为输入性感染病例,该毒株最有可能来源于东南亚一带。     

First Isolation of Dengue Virus from the 2010 Epidemic in Nepal

Dengue is an emerging disease in Nepal and was first observed as an outbreak in nine lowland districts in 2006. In 2010, however, a large epidemic of dengue occurred with 4,529 suspected and 917 serologically-confirmed cases and five deaths reported in government hospitals in Nepal. The collection of demographic information was performed along with an entomological survey and clinical evaluation of the patients. A total of 280 serum samples were collected from suspected dengue patients. These samples were subjected to routine laboratory investigations and IgM-capture ELISA for dengue serological identification, and 160 acute serum samples were used for virus isolation, RT-PCR, sequencing and phylogenetic analysis. The results showed that affected patients were predominately adults, and that 10% of the cases were classified as dengue haemorrhagic fever/ dengue shock syndrome. The genetic characterization of dengue viruses isolated from patients in four major outbreak areas of Nepal suggests that the DENV-1 strain was responsible for the 2010 epidemic. Entomological studies identified Aedes aegypti in all epidemic areas. All viruses belonged to a monophyletic single clade which is phylogenetically close to Indian viruses. The dengue epidemic started in the lowlands and expanded to the highland areas. To our knowledge, this is the first dengue isolation and genetic characterization reported from Nepal.     

应用含内参的多重实时荧光RT-PCR方法快速检测登革病毒和基孔肯雅病毒

目的 建立一种登革病毒、基孔肯雅病毒并含人类基因内参检测的多重实时荧光RT-PCR方法,能在同一反应管内同时检测目前发现的所有来源的登革病毒或基孔肯雅病毒。方法 针对登革病毒3′端非编码区和基孔肯雅病毒结构蛋白E2-6K-E1区以及人体各类组织细胞中均能稳定表达的RNAse P基因,设计了3套特异性引物和探针,建立了1套能同时检测登革病毒、基孔肯雅病毒及含有人类基因检测内参的多重实时荧光RT-PCR方法,对其灵敏性和特异性进行了验证,并对临床发热病人标本进行了应用评估。结果 该方法对检测体外转录合成的登革病毒和基孔肯雅病毒RNA的灵敏性可达最低每个反应10～100拷贝,对检测登革1型病毒和基孔肯雅病毒的灵敏性分别可达最低每个反应0.1 TCID₅₀/mL和1 TCID₅₀/mL。用20株登革病毒、4株基孔肯雅病毒和日本脑炎病毒、西尼罗病毒、黄热病毒、盖塔病毒、辛德毕斯病毒各1株进行检测,方法的特异性均为100%。方法应用于189份发热病人血清标本检测,可准确地鉴定出其中登革病毒或基孔肯雅病毒核酸阳性的标本,且所有血清标本均能被内参引物和探针有效地扩增和杂交。结论 本研究建立了一种高灵敏性、高特异性且含人类基因内参检测的登革病毒和基孔肯雅病毒多重实时荧光RT-PCR检测方法,可作为登革热或基孔肯雅热病人早期快速鉴别诊断的有效工具,也可用于蚊媒携带登革病毒或基孔肯雅病毒的高通量快速筛查。     

TaqMan MGB探针实时聚合酶链反应检测登革病毒

目的建立一种敏感、特异、重复性好的登革病毒(DengueVirus,DV)鉴定方法。方法根据DV3’端非编码区基因保守序列,设计一套特异性引物和TaqManMGB探针。利用1998~2004年收集的26份登革热病人临床血清标本,15例乙脑病人血清,DV4个血清型标准毒株及23株1978~1997年DV地方流行株和相关西尼罗病毒、日本脑炎病毒、麻疹病毒、基孔肯亚病毒等毒株,同时用克隆了1型DV基因组3’端非编码区序列片段的质粒DNA作为阳性对照,检测所建立方法的特异性、敏感性。结果所建立方法的最低检测限约为每反应5个基因拷贝。用该方法检测4个血清型DV标准毒株、23株DV地方流行株和26例分离到DV的阳性血清标本,检出率为100%;用该方法检测15例乙脑病人血清、10例麻疹病人血清和西尼罗病毒、日本脑炎病毒、麻疹病毒、基孔肯亚病毒,结果均为阴性。结论新建的TaqManMGB探针检测DV方法是实验室早期诊断登革热较理想的方法。     

Low rates of antigen detection and virus isolation from the peripheral blood leukocytes of dengue fever patients

We evaluated direct fluorescent antibody (FA) testing of peripheral blood leukocytes (PBL) from patients in Puerto Rico with serologically and/or virologically confirmed dengue fever as a possible rapid diagnostic test and compared rates of dengue virus isolation from PBL with the rates from plasma or serum using the mosquito inoculation technique. Dengue antigen was detected in the PBL of only 1 of 19 patients with confirmed dengue. Virus was isolated from 3 of 19 PBL specimens and from 6 of 19 acute-phase serum or plasma samples. Four viruses were obtained from serum or plasma only and 1 isolate came from PBL only. We conclude that FA testing of PBL from dengue fever patients has little promise as a rapid diagnostic technique. Despite small numbers, our data suggest that virus isolation from PBL is less sensitive than that from serum or plasma. Our results differ considerably from those of previous studies of dengue hemorrhagic fever patients conducted in Thailand.     

Virologic and serologic surveillance for dengue fever in Jeddah, Saudi Arabia, 1994-1999.

Dengue fever infection was first documented in Jeddah, Saudi Arabia, by virus isolation of dengue type 2 virus in 1994 at the virology laboratory of Dr. Soliman Fakeeh Hospital. Dengue virus surveillance was established after that time. Blood samples were collected from 985 patients (710 male patients and 275 female patients) with suspected cases of dengue from February 1994 to December 1999. Dengue virus isolates were obtained in 207 patients (21%; 162 male patients and 45 female patients). Dengue type 2 was the predominant serotype (138 of 207 isolates, 66.7%), followed by dengue type 1 with (56 of 207 isolates, 27%) and dengue type 3 (13 of 207 isolates, 6.3%). The largest number of isolates (186 of 207 isolates, 90%) was in 1994, a year during which there was a dengue epidemic. In the next 5 years, 1995-1999, only 21 isolates (10%) were isolated. Immunoglobulin M capture enzyme-linked immunosorbent assay was positive in 160 acute samples; 52 of them were from virus culture-positive cases and 108 (11%) from culture-negative cases. The total number of cases diagnosed by both methods was 315 (32%). The prevalence of dengue immunoglobulin G antibodies, as assessed on the basis of immunofluorescent assay, hemagglutination inhibition titers > or = 1/20, or both, in the acute samples was 314 (32%) of 985, indicating past Flavivirus infection. Two patients died, one man with dengue hemorrhagic fever and one woman with dengue shock syndrome. Both fatal dengue cases were due to infection with type 2 virus. All other cases were simple dengue fever. To our knowledge, this is the first report confirming the circulation of 3 dengue serotypes in Jeddah.     

Dengue virus infection during post-epidemic period in Delhi, India

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are major public health problems in India. During the period following an epidemic, a study was carried out using virological and serological tests for confirmation of suspected cases of dengue virus infection in fever cases presenting to the All India Institute of Medical Sciences. Serum samples of suspected DF/DHF cases were processed from January to December 1997. In 37 samples from patients with fever of less than 5-day duration, received on ice, virus isolation was attempted in C6/36 clone of Aedes albopictus cell line, followed by indirect fluorescent antibody staining with monoclonal antibodies to dengue viruses 1 to 4. One hundred and forty-three serum samples from patients with more than 5 days fever were tested for dengue specific IgM antibody by either MAC-ELISA or a rapid immunochromatographic assay. Dengue virus type 1 was demonstrated by culture in 8 (21.6%) of 37 serum samples and IgM antibody could be detected in 42 (29.4%) of the 143 serum samples by the serological methods. The peak of dengue virus infection was seen from September to November 1997.     

Isolation and partial characterization of dengue virus type 2 and 4 strains from dengue fever and dengue haemorrhagic fever patients from Mindanao, Republic of the Philippines

OBJECTIVE: Isolation of dengue virus from dengue fever and dengue haemorrhagic fever cases from Mindanao, Republic of the Philippines. METHODS: 12 patients with clinically suspected dengue fever (DF) or dengue haemorrhagic fever (DHF) presenting in four regional hospitals between August and September 1995 on Minadano were enrolled in the study. Dengue virus was isolated by inoculation of Vero/E6 or C6/36 cells with patient serum. IgM antibodies were measured using a commercial test system. Up to 454 bp of the capsid region and 240 bp of the E/NS1 gene junction of different viral isolates were sequenced and phylogenetically analyzed. RESULTS: Virus could be isolated from seven patients, five isolates were typed as dengue virus type 2 and two as dengue virus type 4 by immunostaining with monoclonal antibodies or by RT/PCR. Phylogenetic analysis confirmed a close relationship of the dengue virus type 2 isolates with viruses isolated in the Philippines in 1983 and 1988. CONCLUSION: As observed in studies from other parts of South East Asia, dengue virus type 2 was readily isolated from dengue haemorrhagic fever cases. Dengue virus type 2 and 4 circulate in Mindanao, Philippines, with dengue type 2 being responsible for most of our severe DF or DHF cases.     

广州地区2012年1、2型登革病毒基因型特征研究

目的 测定广州市2012年1、2型登革病毒(DENV 1、DENV 2)的E基因序列,探讨其来源及基因型。方法 收集广州市2012年478例登革热患者急性期血清478份,用C6/36细胞培养分离登革病毒,RT-PCR法扩增全长E基因,测定序列并绘制系统发育树,结合流行病学资料进行分子流行病学分析。结果 478例患者标本中分离到DENV 1 6株,DENV 22株,测序获得E基因序列,E基因长度均为1 485 bp,编码495个氨基酸,DENV 1碱基同源性为97.4%~99.9％,推导的氨基酸同源性为99.2%~100.0％,其同源性与2011、2006和2004年份的国内DENV 1流行株接近。DENV 2碱基同源性为91.3%,推导的氨基酸同源性为97.8%,其同源性与我国DENV 2流行株相对较远。进化分析发现,DENV 1均属于同一亚型,即亚洲型,DENV 2属于两个亚型,即马来西亚/印度次大陆和东南亚型。结论 推测广州市2012年DENV 1和DENV 2均为输入性,DENV 1可能由柬埔寨和广东佛山输入,DENV 2可能由孟加拉国和缅甸输入。