西尼罗病毒荧光RT-PCR检测方法的建立

目的用TaqMan技术建立荧光RT-PCR方法,用于检测西尼罗病毒(WNV)。方法从GenBank上调取WNV的基因序列,设计WNYA、WNYB、WNYC三套荧光RT-PCR,经优化后用于WNV灭活苗、乙型脑炎病毒(JEV)、黄热病毒(YFV)等11种病毒核酸的检测,同时用10倍倍比稀释的双链DNA、质粒DNA、cDNA、RNA进行扩增,以检测其灵敏度。结果建立的WNYA、WNYC荧光RT-PCR检出WNV灭活苗阳性,而JEV等10种非WNV病毒均为阴性,能检出30个拷贝和65个拷贝的双链DNA、420个拷贝和460个拷贝的质粒DNA;WNYB的反转录效率明显低于WNYA和WNYC。结论建立的WNYA、WNYC荧光RT-PCR可作为检测WNV的技术储备。     

Real-time RT-PCR检测西尼罗病毒一步法的建立

目的建立针对西尼罗病毒E基因的一步法Real-time RT-PCR检测方法,用于西尼罗病毒感染样本的快速准确检测和定量。方法针对西尼罗病毒的保守基因E设计引物和探针,建立一步法Real-time RT-PCR反应方法并分析敏感性和特异性。结果本研究建立的一步法Real-time RT-PCR方法可以特异性检测出西尼罗病毒,不与日本脑炎病毒及其他病毒产生交叉反应。该方法的最低检出浓度为3.6×102copies/μl,标准曲线的线性范围为3.6×102～3.6×107copies/μl。结论本研究建立的一步法real-time RT-PCR方法敏感性和特异性较高,且不易出现污染引起的假阳性结果,适合用于西尼罗病毒感染样本的检测。     

TaqMan MGB Real-time RT-PCR 检测西尼罗病毒方法的建立

目的建立一种灵敏、特异、高效的西尼罗病毒（West Nilevirus，WNV）感染诊断和流行病学调查的实验室检测方法。方法用C6／36细胞培养WNV并用Vero细胞蚀斑法滴定病毒浓度；根据wNVC蛋白基因组保守序列，设计一套特异性引物和TaqManMGB探针，用细胞培养病毒液进行方法的条件优化；用不同病毒评价方法的特异性，用系列浓度病毒稀释液和染毒蚊子评价方法的灵敏性。结果建立的TaqManMGBReal-timeRT-PCR方法可检出低于0．01PFU的WNVRNA，并可检出只含3只染毒蚊的标本；用该方法检测4种血清型登革病毒标准毒株、日本脑炎病毒、麻疹病毒、基孔肯亚病毒均为阴性。结论新建TaqManMGBReal-timeRT—PCR方法具有极高的特异性和灵敏性，是实验室早期诊断WNV感染和调查媒介携带WNV情况的理想方法。     

尼帕病毒巢式RT-PCR方法的建立和初步应用

目的 建立灵敏、特异的检测尼帕病毒的巢式RT-PCR方法。方法 根据GenBank公布的尼帕病毒N基因序列,设计2对特异性引物(外引物和内引物),建立巢式RT-PCR方法,优化反应体系,测定特异性和灵敏度,并进行临床样品的检测。结果 该方法扩增的片段序列与源序列同源性均为100%。特异性试验结果表明本试验设计的内外侧引物不能从新城疫病毒、牛瘟病毒和日本乙型脑炎病毒中扩增出条带。敏感性试验结果表明,该方法最低能检测出的标准模板RNA浓度为39 fg/μL。100份临床样品检测结果全部为阴性。结论 初步建立了快速、灵敏、特异的检测尼帕病毒 N基因的巢式RT-PCR方法,可用于动物疫病监测和检验检疫等领域。     

西尼罗病毒研究进展

1999年，西尼罗病毒（West Nile virus，WNV）首次在美国纽约地区出现，随后几年迅速在北美大陆蔓延。近年WNV流行表现出新的特点，部分感染病人出现严重的神经系统症状，死亡率高，引起了人们的高度关注。该文就近年来对WNV的生物学性状、致病性、感染的免疫、流行病学、防治措施及疫苗研制的研究进展进行综述。     

间接免疫荧光试验在西尼罗病毒抗体检测中的初步应用

目的了解人群血清西尼罗病毒(WNV)抗体存在的本底,探讨WNV与乙型脑炎病毒(JEV)的免疫交叉反应。方法以新兵入伍健康体检时收集的男性血清347份为检测标本,应用建立的间接免疫荧光试验(IFA)对WNV与JEV的IgG抗体进行检测。结果血清WNV和JEV的抗体阳性率分别为3.2%(11/347)和5.7%(14/244);在同时检测了两种病毒抗体的244份标本中,有3份两者均为阳性,分别占WNV和JEV抗体阳性数的60%(3/5)和21%(3/14)。结论人群对WNV缺乏免疫力,JEV抗体对WNV的交叉免疫作用有一定的局限性。     

马达里亚加病毒TaqMan RT-PCR检测方法的建立

目的 建立马达里亚加病毒(Madariaga virus, MADV)早期且快速的TaqMan反转录聚合酶链式反应(RT-PCR)检测方法。方法 从GenBank中下载MADV基因序列,根据多序列比对结果选择MADV的NSP1基因中的高保守序列片段设计引物与探针,利用体外转录的RNA 标准品建立了MADV的绝对定量分析模型,并进行灵敏度和特异性评价以及重复性验证。结果 该检测方法的灵敏度为1.0×10²拷贝/反应,与其它虫媒病毒无交叉反应,重复性检测变异系数均小于1.5%。通过本研究建立的快速检测方法对源自宁夏回族自治区、浙江省和云南省的共计81份不明原因脑炎病例血清标本及161批库蚊标本进行了测定,检测结果均为阴性。结论 成功建立了MADV TaqMan RT-PCR的快速检测方法,可应用于实验室的早期检测。     

Flanders病毒TaqMan RT-PCR检测方法的建立

目的 应用TaqMan PCR技术建立针对Flanders病毒(Flanders virus, FLAV)的实时荧光定量PCR检测方法。方法 下载GenBank中FLAV基因序列资料并进行多序列比对分析,选择其L基因中的高保守序列片段进行FLAV特异引物与探针的设计,使用来自于不同科属的15株病毒验证方法特异性,通过重复/平行实验验证方法稳定性,并利用体外转录的L基因RNA标准品建立基因拷贝数绝对定量分析模型。结果 引物与探针工作特异性良好,同一样品重复检测Ct值的变异系数均小于1.7%,定量分析模型灵敏度达到100 copies/PCR。结论 建立完成针对FLAV的TaqMan PCR检测方法。实验结果显示,本方法具有高特异性、高敏感性、高稳定性、简便、易操作的特点,为今后对FLAV的检测、监测及相关研究提供技术手段。     

西尼罗病毒抗体酶联免疫吸附试验检测方法的评价

目的系统地评价血清西尼罗病毒(West Nile virus,WNV)抗体的酶联免疫吸附试验(ELISA)检测方法,为在人群和宿主动物中进行WNV感染血清流行病学调查提供技术支持。方法利用构建的包膜蛋白重组质粒(pQE-30)表达纯化西尼罗病毒包膜蛋白,以此为抗原进行ELISA检测。在对抗原包被量、酶标抗体浓度、血清稀释度进行优化的基础上,对本方法的灵敏度和特异度进行评价。结果确定最适抗原包被量为0.034μg,酶标抗体工作浓度和血清稀释度分别为1∶4 800和1∶80;批内变异和批间变异分别为6.7%和22.6%;对小鼠WNV抗体阳性血清53份、JEV抗体阳性血清48份和阴性对照血清94份进行检测,灵敏度为86.8%,特异度分别为93.8%和92.6%。结论本研究建立的ELISA方法灵敏、检测抗体特异,结果可重复,是一种有价值的血清学调查方法。     

西尼罗病毒研究进展

1999年,西尼罗病毒(West Nile virus,WNV)首次在美国纽约地区出现,随后几年迅速在北美大陆蔓延。近年WNV流行表现出新的特点,部分感染病人出现严重的神经系统症状,死亡率高,引起了人们的高度关注。该文就近年来对WNV的生物学性状、致病性、感染的免疫、流行病学、防治措施及疫苗研制的研究进展进行综述。     

西尼罗病毒感染免疫应答反应研究进展

西尼罗病毒病是由西尼罗病毒引起的一种人兽共患传染病,给人类和动物健康带来重大危害。虽现已有疫苗处在研究阶段,但仍没有人用疫苗获批上市。通过感染动物模型,有关西尼罗病毒免疫反应的研究已经开展。本文对固有免疫和获得性免疫在抵抗西尼罗病毒感染中的作用进行综述,为进一步研究西尼罗病毒激发免疫应答反应的机制和新型疫苗研制提供依据。     

加强对西尼罗病毒的认识

西尼罗病毒感染可引起西尼罗热和西尼罗脑炎,是一种新发传染病,为近年来全球公共卫生的新威胁。本文对西尼罗病毒的生物学特性、致病机制、临床症状和疫苗等方面的研究进展进行综述,为我国防治西尼罗病毒感染提供参考。     

West Nile virus: immunity and pathogenesis

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans. WNV is endemic in parts of Africa, Europe, the Middle East, and Asia, and since 1999 has spread to North America, Mexico, South America, and the Caribbean. WNV infects the central nervous system (CNS) and can cause severe disease in a small minority of infected humans, mostly immunocompromised or the elderly. This review discusses some of the mechanisms by which the immune system can limit dissemination of WNV infection and elaborates on the mechanisms involved in pathogenesis. Reasons for susceptibility to WNV-associated neuroinvasive disease in less than 1% of cases remain unexplained, but one favored hypothesis is that the involvement of the CNS is associated with a weak immune response allowing robust WNV replication in the periphery and spread of the virus to the CNS.     

Seroprevalence of West Nile virus infection in solid organ transplant recipients

A.G. Freifeld, J. Meza, B. Schweitzer, L. Shafer, A.C. Kalil. A.R. Sambol. Seroprevalence of West Nile virus infection in solid organ transplant recipients.
Transpl Infect Dis 2010: 12: 120–126. All rights reserved Background. Of people infected with mosquito‐borne West Nile virus (WNV), <1% develop neuroinvasive disease (NID). Population studies suggest that people older than 65 years may be at higher risk for neurologic symptoms. It has been suggested that solid organ transplant (SOT) recipients are also at higher risk for WNV NID, but definitive serologic and epidemiologic data are lacking. Methods. A serologic screening survey, using a US Food & Drug Administration‐approved enzyme‐linked immunosorbant assay to detect WNV immunoglobulin‐G (IgG) antibody responses in cohorts of SOT recipients and non‐immunocompromised controls, was undertaken at a large Midwestern university organ transplant center in the aftermath of the summer 2003 WNV regional outbreak. Hemagglutination‐inhibition testing was used to confirm WNV IgG‐positive results and differentiate them from positive results caused by Saint Louis encephalitis virus, another flavivirus that is endemic in the Midwestern US. Findings. The rate of WNV IgG‐seropositive responses did not differ between SOT recipients and non‐immunocompromised controls, and were 12% and 10%, respectively. Retrospective chart review showed no documented WNV NID in the seropositive SOT recipients, suggesting an incidence of WNV NID may be as low as 0.7% in this population. Interpretation. Asymptomatic WNV infection is common among immunocompromised SOT patients, occurring as often as it does in non‐immunocompromised controls. Our data indicated that severe WNV NID is less frequent in SOT patients, contrary to what has been suggested in other studies.     

A brief report of West Nile Virus neuroinvasive disease in the summer of 2012 in Hamilton,Ontario

West Nile neuroinvasive disease is a severe infectious disease that is associated with a high mortality rate, especially in immunocompromised hosts. Physicians who are aware of its clinical presentations may be able to order diagnostic tests more appropriately and avoid inappropriate treatment. In the present series, the cases of seven patients admitted to Hamilton Health Sciences (Hamilton, Ontario) in the summer of 2012 with a diagnosis of West Nile neuroinvasive disease were retrospectively reviewed based on available medical records. According to the clinical and laboratory criteria published by the Centers for Disease Control and Prevention, five cases were diagnosed as encephalitis, one case as meningitis and one case as meningomyelitis. Patients were managed supportively. Forty-three percent (three of seven) presented with rash, 71% (five of seven) did not report headache despite exhibiting neurological symptoms, 43% (three of seven) did not have fever on presentation and 37.5% of cerebrospinal fluid samples exhibited a neutrophil predominance. The mortality rate in the present series was 14.3% (one of seven), and 57.1% (four of seven) of the patients had residual symptoms on discharge and at follow-up.