2009年辽宁省虫媒病毒分离鉴定

目的 调查辽宁省部分地区虫媒病毒的种类.方法 2009年8月,在辽宁省鞍山市、朝阳市、葫芦岛市和锦州北镇市采集蚊虫标本.蚊虫经分组研磨后接种C6/36和BHK-21细胞,连续3代观察细胞病变情况;对细胞病变阳性的分离物,进行抗原性检测及分子生物学鉴定.结果 在辽宁省上述4个城市共采集蚊虫3600只,主要为中华按蚊(47...     

广西北流市分离到基因Ⅰ型流行性乙型脑炎病毒

目的从广西流行性乙型脑炎(乙脑)监测点之一的北流市分离乙脑病毒并研究其基因分型。方法2006年在广西北流市采集蚊虫标本,在C6/36和BHK-21细胞上进行病毒分离,对病毒分离物进行多种虫媒病毒抗体的酶联免疫吸附试验,获得的乙脑病毒进行E基因扩增、测序和基因分型。结果在北流市采集骚扰阿蚊2588只和三带喙库蚊230只,分61批进行处理,获得3个病毒分离物,酶联免疫吸附试验与乙脑抗体反应,E基因测序后基因分型显示为基因Ⅰ型乙脑病毒。结论从广西蚊虫标本分离到基因Ⅰ型乙脑病毒。     

云南省保山市蚊虫中检测到基因Ⅰ型流行性乙型脑炎病毒

目的 调查云南省保山市蚊虫及其自然感染虫媒病毒状况，为蚊媒病毒病防控提供参考依据。方法 2015年9月采用诱蚊灯法在保山市隆阳区农村人房和畜圈采集蚊虫标本，反转录聚合酶链反应（RT-PCR）用于检测蚊虫标本中黄病毒属、甲病毒属、布尼亚病毒属和流行性乙型脑炎病毒（乙脑病毒）等相关病毒核酸，阳性者进行C/prM基因序列测定、同源性和系统进化分析。结果 共捕获蚊虫3种8 202只，三带喙库蚊（Culex tritaeniorhynchus）、中华按蚊（Anopheles sinensis）和骚扰阿蚊（Armigeres subalbatus）构成比依次为88.50%（7 259只）、11.43%（937只）和0.07%（6只）。蚊虫标本分30批进行RT-PCR检测，14批三带喙库蚊中检测到乙脑病毒核酸，并获得14株乙脑病毒C/prM基因核苷酸序列。同源性和进化分析表明，14株乙脑病毒与基因Ⅰ型乙脑病毒同在一个进化分支，并与2007年保山市腾冲县和2010年德宏傣族景颇族自治州（芒市和瑞丽市）的基因Ⅰ型乙脑病毒具有较近的进化关系。结论 三带喙库蚊为保山市的优势蚊种和乙脑病毒主要传播媒介，当地存在基因Ⅰ型乙脑病毒流行，此为近10年保山市首次检测到基因Ⅰ型乙脑病毒。     

2005年广西媒介蚊虫中流行性乙型脑炎病毒的分离鉴定

目的对广西流行性乙型脑炎(乙脑)流行区的蚊虫进行乙脑病毒分离鉴定。方法2005年6、7月在北流市和靖西县居民区的畜圈内采集蚊虫标本,用C6/36和BHK21细胞分离乙脑病毒,用ClustalX(1.8)、Mega3.1等软件进行分离株E基因序列比对和系统进化分析。结果两地共采集三带喙库蚊771只、骚扰阿蚊425只,从三带喙库蚊中分离到3株基因1型乙脑病毒,3株病毒之间E基因片段核苷酸和氨基酸的最大同源性为99.3%和99.8%,与越南毒株之间核苷酸和氨基酸的最大同源性为98.4%和99.6%。结论从2005年采集自广西的蚊虫中分离到3株基因1型乙脑病毒,这是首次报道在广西分离到基因1型乙脑病毒。     

我国西北地区蚊虫标本中广平病毒的检测

目的 对我国西北地区蚊虫携带病毒开展调查,并快速发现重要的蚊媒病毒.方法 通过形态学方法对采集蚊虫进行分类鉴定,深度测序和宏基因组分析方法分析蚊虫携带的病毒,结合Sanger测序法补全病毒基因组全序,应用系统进化分析方法分析病毒分子遗传特征.结果 2019年7月采集自陕西省的中华按蚊标本和宁夏回族自治区的三带喙库蚊标本...     

2006年河南省南阳市流行性乙型脑炎病毒的分离与分子特征分析

目的在河南省南阳市采集蚊虫标本,进行西尼罗病毒基因检测,分离流行性乙型脑炎(乙脑)病毒并分析分离株的分子生物学特征。方法 2006年从南阳市采集蚊虫标本,将标本研磨处理后进行巢式PCR检测西尼罗病毒核酸,用细胞培养法对标本进行病毒分离,通过血清学与分子生物学方法鉴定新分离到的乙脑病毒,扩增分离株PrM和E基因区,使用生物学软件进行序列和系统发生分析。结果共采集蚊虫标本6231只,巢式PCR检测西尼罗病毒结果均为阴性。从标本中分离到7株病毒,经鉴定均为乙脑病毒。基因分型显示7株病毒均为基因Ⅰ型,新分离株在E基因之间的核苷酸同源性为98.5%～100%,氨基酸同源性为98.4%～100%。新分离株与P3株和SA14-14-2的E基因核苷酸同源性分别为87.7%～88.2%和87.3%～87.9%。新分离株与P3株相比存在10个共同氨基酸差异位点,但在决定毒力的关键位点不同于SA14-14-2。结论河南省南阳市2006年乙脑病毒流行株为基因Ⅰ型,毒株的毒力基因没有明显改变,所有标本均未检测到西尼罗病毒阳性。     

手足口病病原微生物EV71病毒株的分离与鉴定

目的：探讨手足口病病原体EV71病毒的分离方法。方法：取手足口病患儿的粪便,经磷酸盐缓冲液（PBS）稀释后用0.22μm滤膜过滤,将悬液接种于Vero细胞。通过EV71病毒致细胞病变效应（CPE）分析、电镜分析、EV71病毒特异性核酸及蛋白的聚合酶链反应（PCR）及Western Blotting分析来鉴定病毒分离和致病效果。检测病毒悬液对于宿主细胞Vero增殖抑制的效果。结果：标本悬液接种Vero细胞48h后出现CPE。收集感染细胞的培养基上清液,连续感染2次,均出现同样的CPE。电镜检测发现在感染组Vero细胞的细胞质和细胞核周围均有大量聚集的病毒颗粒。EV71特异性核酸RT-PCR检测证实,感染组Vero细胞中可以扩增出EV71病毒特异性的核酸条带。Western Blotting检测发现,在感染组细胞中有高表达EV71特异性抗原蛋白,而未感染组细胞不表达该病毒抗原。MTT检测结果显示,EV71病毒显著抑制宿主细胞Vero的体外增殖和生长,并且呈时间依赖性。结论：成功分离出人手足口病致微生物EV71病毒株（该EV71病毒株编号为EV71-Tj-100623）。     

一株发热伴血小板减少综合征布尼亚病毒的分离鉴定及其生长特性研究

目的 探索疑似发热伴血小板减少综合征病例中病原体的分离鉴定,了解病毒生长特性。方法 利用非洲绿猴肾细胞Vero E6从患者抗凝血标本中分离发热伴血小板减少综合征布尼亚病毒（SFTSV）,并通过血清学、形态学等方法对病毒进行鉴定;将分离的病毒接种不同细胞,利用荧光定量PCR检测不同时间细胞上清中病毒载量的变化。结果 从患者抗凝血标本中分离出SFTSV;免疫荧光显示感染病毒的细胞培养物能与患者血清发生阳性反应;在透射电镜下能观察到布尼亚病毒样颗粒。SFTSV可感染Vero E6、Hep G2、THP-1等多种细胞,但病毒在不同细胞中的复制水平差异明显。其中Vero E6细胞对SFTSV较易感,病毒可增殖至4.89109 copy/ml。而Hep-2和HT29细胞不能被SFTSV感染。结论 从疑似发热伴血小板减少综合征病例血液标本中成功分离出SFTSV。该病毒具有较广泛的细胞嗜性,对肾来源的细胞更易感。这些研究将为后续研究SFTSV的相关基础研究提供重要的线索。     

广州市1985～1990年4株登革热病毒分离和鉴定

1985～1990年应用白纹伊蚊 C6/36细胞,对采自广州市城区109批的白纹伊蚊,致乏库蚊分离虫媒病毒。除分离到4株1型、2型,4型登革热病毒(DFV)外,还分离到4株尚待进一步鉴定的虫媒病毒。     

2006年山西省南部地区虫媒病毒分离鉴定

目的了解山西省南部地区虫媒病毒的种类及分布特征。方法使用诱蚊灯捕蚊,通过组织培养法分离病毒,并对病毒分离物进行血清学和分子生物学鉴定。结果2009年9月在山西省运城市、临汾市和阳泉市的3个标本采集点采集到3属4种共3436只蚊虫标本,从中分离到5株病毒分离物,鉴定结果显示有3株(SX0621、SX0633、SX0635)为淡色库蚊浓核病毒,其余2株分离物有待进一步鉴定。对CppDNV非结构蛋白NS1和NS2的部分核苷酸序列分析显示,3株山西省新分离株的同源性为100%;与中国其他省市的分离株同源性在99.9%～100.0%之间。系统进化分析提示所有中国分离株均位于一个相对独立的进化分支中,并且与AaeDNV的进化关系较近。结论在山西省首次分离到淡色库蚊浓核病毒,与中国其他地区的分离株同源性较高。     

E/NS1 modifications of dengue 2 virus after serial passages in mammalian and/or mosquito cells

OBJECTIVE: Dengue viruses are routinely maintained in nature by transmission cycles involving the passage of virus between humans and AEDES mosquitoes. The number of dengue virus lineages has been increasing over time. The aim of this study was to identify the genetic diversity of dengue 2 virus serially transferred in mammalian and/or mosquito cells. METHODS: The E/NS1 gene of dengue 2 virus variants derived from serial passages in Vero or C6/36 cells, or alternately in both cell systems, was amplified and sequenced in order to observe gene modification after serial passages. RESULTS: Three nucleotides (two in E and one in NS1) or two amino acids (one each in E and NS1) changed in the virus that was continuously cultured in Vero cells for 20 passages, whereas four nucleotides (two each in E and NS1) or three amino acids (one in E and two in NS1) changed in the virus cultured for 30 passages. The genome of dengue 2 virus remained stable even when the virus was serially transferred in C6/36 cells for 30 generations. However, there was one amino acid substitution (E46 I-->V) resulting from a single nucleotide change in the E region of dengue 2 virus alternately transferred in C6/36 and Vero cells for either 20 or 30 passages. In addition, dengue 2 virus obtained from serially cultured Vero cells usually replicated better when it reinfected Vero cells, reflecting its high adaptation fitness to the host cell. CONCLUSIONS: It is concluded that genetic changes of dengue 2 virus are constrained in Vero (mammalian) cells, resulting in a variety of genome-related quasispecies populations. Some populations of the virus are subsequently selected by and genetically (at least in the E/NS1 portion of the viral genome) maintained in C6/36 (mosquito) cells during replicative competition.     

Detection of yellow fever virus nucleic acid in infected mosquitoes by RNA:RNA in situ hybridization

An in situ hybridization technique was developed for the strand-specific detection of yellow fever virus (YFV) RNA. An 35S-labeled, transcribed RNA probe was used to detect positive-sense polarity YFV genomic RNA in infected C6/36 (Aedes albopictus) cells, dissected mosquito tissues, and sections of plastic-embedded, YFV-infected Aedes aegypti mosquitoes. Mosquito tissues fixed in buffered Formalin retained morphological integrity. The low concentrations of probe used yielded high specific signal on infected specimens and low background signal on uninfected specimens.     

Replication and expression of a recombinant Sindbis virus in mosquitoes

A recombinant Sindbis virus, TE/3'2J/ANTI-S, containing LaCrosse virus small segment cDNA in antisense orientation, was inoculated into Aedes triseriatus mosquitoes. Virus replication and LAC-ANTI-S RNA expression were analysed temporally and spatially. TE/3'2J/ANTI-S virus titre peaked at 5.0 log₁₀ TCID₅₀ in heads 6–9 days post infection (p.i.) and decreased to 3.4 log₁₀ TCID₅₀ by 37 days p.i. Salivary glands contained 4.4 log₁₀ TCID₅₀ of virus 6 days p.i.; titres were lower in other organs. LAC-ANTI-S RNA levels paralleled virus titre. SIN E1 antigen was detected in many mosquito organ systems, but in specific cells and tissues of some organs. TE/3'2J/ANTI-S virus exhibited different cellular tropisms in salivary glands of Aedes and Culex mosquitoes.     

Quantitative in situ hybridization using strand specific RNA probes: expression of the bunyavirus Germiston S segment in mosquito cells

Infection of Vero (monkey) cells by Germiston bunyavirus is highly cytopathic with cell lysis and virus production at a high titre, whereas infection of Aedes albopictus C6/36 (mosquito) cells leads, after an acute primary phase, to a persistent non-cytopathic infection with a loss in virus production. In this report we demonstrate that single-stranded RNA probes can be successfully used in an in situ hybridization assay to quantify viral expression during this persistent infection. The steady-state levels of viral S-RNA segment (genomic and messenger sense) during the acute phase were similar to those observed in lytically infected Vero cells, but appeared delayed. Both senses of S-RNA were detected throughout persistent infection but in lower amounts, in less than 10% of the cells and always in the cytoplasm of infected cells. The number of copies per cell of messenger sense S-RNAs remained low during persistent infection whereas a higher fluctuation was observed for genomic S-RNAs. In situ hybridization with specific stranded RNA probes provides both qualitative and quantitative informations, that can lead to a better understanding of virus-cell interactions.     

Detection of dengue viral RNA in mosquito vectors by mixed phase and solution hybridization

A mixed phase hybridization technique was developed to detect dengue virus type 2 (DEN-2) RNA in pools of infected Aedes albopictus mosquitoes using radiolabelled RNA probes. This technique used a guanidine thiocyanate extraction procedure to simplify analyte preparation. The probes contained sequences complementary to portions of the NS-1 or NS-5 genes of the DEN-2 viral genome. One infected mosquito in a pool of 25 could be detected in approximately 48 h. RNAs from DEN serotypes 1-4 were extracted from cultured mosquito (C6/36) cells. The NS-1 RNA probe was highly specific for DEN-2 RNA. The NS-5 RNA probe detected both DEN-2 and DEN-4 RNA. DEN-2 RNA was also detected by molecular hybridization in concentrated solutions of guanidine thiocyanate using the NS-1 probe. Solution hybridization was 10-fold more sensitive when detecting RNA from purified DEN-2 virus than the mixed phase assay and could detect one infected mosquito in a pool of 25 within 6-8 h. Solution hybridizations were performed in 2-3 h vs 16-20 h for mixed phase hybridizations, and solution hybridizations required 5-10 times less mosquito RNA than mixed phase hybridizations to attain comparable sensitivities. However, solution hybridizations did result in a broader probe specificity than mixed phase hybridizations.