中国分离的甲属病毒YN87448毒株非结构区基因的核苷酸序列测定及分析

目的　研究我国首例从发热病人血清中分离的YN87448病毒的分子致病基础。方法　甲属病毒基因序列保守区设计引物,用逆转录聚合酶链反应(RTPCR)方法扩增,再将扩增片段克隆到pGEMT载体。测定YN87448毒株非结构基因序列。结果　YN87448病毒非结构区基因序列为7613个核苷酸(不包括5′端帽子结构),编码非结构蛋白1(nsP1),非结构蛋白2(nsP2),非结构蛋白3(nsP3),非结构蛋白4(nsP4);有一个起始密码子(ATG)位于第60位碱基;含有2个终止密码子(TGA),分别位于基因组nsP3基因和nsP4基因的末端。YN87448病毒非结构区基因与辛德毕斯病毒家族中唯一能致成年小鼠死亡的S.A.AR86株相应基因的核苷酸序列同源性为988%,但YN87448毒株不致成年小鼠死亡。与S.A.AR86株相比,基因结构上有三点明显差异:YN87448毒株在nsP3区位于基因组5256bp5309bp含54个核苷酸的插入;位于基因组5603bp处3个核苷酸(AGT)的缺失;在nsP3和nsP4之间有乳白突变终止密码子(TGA)。此序列已输入GeneBank,序列号为AF103734。结论　YN87448为一株新的辛德毕斯病毒株。     

E基因在辛德毕斯病毒与辛德毕斯样病毒细胞感染特性中的作用

目的 寻找辛德毕斯病毒(YN87448病毒)与辛德毕斯样病毒(XJ-160病毒)对细胞感染特性差异的分子基础.方法 生物信息学比较两种病毒糖蛋白基因一级结构及二级结构,分析二者之间的差异,并利用病毒基因重组等现代分子生物学技术构建重组病毒来研究糖蛋白基因在病毒感染细胞特性中的贡献.结果 生物信息学分析显示,YN87448病毒和XJ-160病毒基因组分别具有11 717和11 626核苷酸序列,具有相同的基因组结构特征;两种病毒E基因氨基酸的疏水性主峰基本相同但存在82个散在分布的氨基酸差异位点.病毒基因重组研究结果显示,将XJ-160病毒E基因替换为YN87448病毒E基因的重组病毒(XJ-160/YE1E2)无论在致细胞病变时间,空斑形成直径,还是在病毒功能蛋白的表达等方面均完全体现YN87448病毒特征,而与XJ-160病毒野毒株的表现完全不同.结论 E基因在辛德毕斯病毒与辛德毕斯样病毒细胞感染特性差异方面起着重要作用,这一结果为从分子生物学角度解释两病毒间生物学差异提供了分子生物学理论依据.     

4株蚊媒病毒多重RT-PCR快速检测方法的建立

目的建立登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒的多重RT-PCR快速检测方法。方法参照病毒核酸序列设计多重RT-PCR引物,并检索GenBank国际基因序列数据库初步验证其特异性,随后对Mg2＋、dNTP及引物浓度,RT-PCR反应条件进行优化,建立稳定、特异的多重RT-PCR快速检测4株病毒方法 ,并以同属于黄病毒科的登革3型病毒、登革4型病毒及甲病毒属基孔肯亚病毒、辛德毕斯病毒为对照,验证其特异性。结果应用多重RT-PCR反应体系,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;采用多重RT-PCR引物对登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒进行扩增,分别获得574、251、879、422 bp片段,与设计相符,而对照病毒组均无非特异性扩增条带。结论实验证明,所建立的多重RT-PCR方法能够快速地检测登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒,为其检测提供了一种方便易行的方法 。     

Banna病毒TaqMan探针法RT-PCR检测方法的建立

目的 建立Banna病毒核酸特异的快速、敏感的TaqMan探针RT-PCR检测方法。方法 首先对从GenBank中检索到的所有Banna病毒的序列进行同源性分析及引物、探针的设计。病毒在BHK21细胞或C6／36细胞上繁殖扩增后提取病毒RNA和逆转录。以cDNA为模板分别进行TaqMan探针法RT-PCR和传统的PCR检测，并对方法的种内广谱性和特异性、检测灵敏性以及用于蚊虫和临床标本的适用性等进行评价。结果 Banna病毒核酸特异的TaqMan探针RT-PCR检测方法具有较好的种内广谱性和特异性，可以检出所有12株Banna病毒，而其他虫媒病毒如辛德毕斯病毒、乙型脑炎病毒、bated病毒和环状病毒检测结果均为阴性。与传统PCR相比，敏感性高100倍以上，可以检出每50止标本中含有0.5CCID50的Banna病毒。单链RNA变性条件（65℃）进行逆转录使检测结果的敏感性比99℃变性法最少低10倍。病毒在室温保存1天和4℃保存1周后，检出敏感性会比原来低10倍左右。该方法在检测模拟感染Banna病毒的人血清标本以及蚊虫标本时，均显示出较好的特异性。112份蚊标本中检出6份标本阳性，4份可疑阳性，病毒分离结果3份阳性。结论 本实验成功建立了Banna病毒特异性核酸的TaqMan探针RT-PCR检测方法，并初步证实可用于血清标本和媒介蚊虫标本的检测，为开展Banna病毒的流行监测及临床早期诊断提供了新的技术手段。     

含有增强型绿色荧光蛋白报告基因的辛德毕斯病毒的制备与鉴定

目的 制备含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的辛德毕斯病毒,并对其进行鉴定.方法 采用融合PCR技术将报告基因EGFP融合到辛德毕斯病毒XJ-160感染性克隆pBR-XJ160的结构基因编码框后,并在报告基因前面添加亚基因启动子SP6,通过反向遗传学技术拯救得到EGFP标记的辛德毕斯病毒.结果 成功拯救并获得了EGFP标记的XJ-160病毒,该重组病毒具有良好的荧光表达特性和遗传稳定性.结论 EGFP标记的辛德毕斯病毒可用于观测病毒的侵染轨迹,为进一步研究辛德毕斯病毒嗜性、生物学功能,以及感染机制奠定了基础.     

2012年发现的新冠状病毒分子检测方法的建立与探针优化

目的 建立2012年确定的新冠状病毒的分子检测方法,并筛选优化引物与探针.方法 依据最先发表的208 bp长的新冠状病毒1b片段核酸序列,比对分析后设计合成常规RT-PCR引物1对及荧光定量RT-PCR引物1对与TaqMan探针2条(TZ1,TZ2),同时合成锁核酸修饰的TaqMan探针2条(LNA-TZ1,LNA-TZ2).建立检测新冠状病毒感染的常规RT-PCR方法与4种荧光定量RT-PCR,分析比较其灵敏度与特异性,同时参照欧洲发表的2种荧光定量RT-PCR引物与探针对(upE,ORF1b)建立相应方法,以合成的阳性模板比较6对荧光定量RT-PCR引物与探针的检出灵敏度.结果 所合成的常规RT-PCR与荧光定量RT-PCR引物与探针皆有较好特异性,不能扩增其他人冠状病毒模板及常见呼吸道病毒模板,检出下限达50 ～ 500拷贝/反应.锁核酸修饰TaqMan探针可改善荧光定量PCR检测方法的反应性能,经锁核酸修饰的TaqMan探针与常规TaqMan探针相比,检出率提高10倍左右,其中LNA-TZ1与upE探针对具最佳反应性能.结论 本研究所建立的常规RT-PCR与荧光定量RT-PCR可用于新冠状病毒的分子检测,并推荐使用LNA-TZ1与upE探针对.本研究为新冠状病毒分子检测方法应用与改进奠定了基础.     

实时荧光PCR检测手足口病病原体方法的建立及初步应用

目的 建立一种能同时检测肠道病毒、肠道病毒71型和柯萨奇病毒16型等手足口病病原体核酸的实时荧光PCR方法.方法 针对上述病毒的基因序列,设计、合成引物、探针.对38例手足口病患者在实时荧光PCR仪上进行扩增、检测和结果分析;并与常规RT-PCR方法的检测结果进行比对.结果 38例患者肠道病毒、肠道病毒71型和柯萨奇病毒16型实时荧光PCR方法与常规RT-PCR方法的阳性率分别为:73.7%、60.5%、13.2%、71.1%、55.3%、13.2%.统计分析表明,两种方法差异不具有统计学意义.结论 成功建立了肠道病毒、肠道病毒71型和柯萨奇16型病毒核酸的实时荧光PCR检测方法.     

登革热病毒多重荧光PCR检测及基因分型方法的研究

目的建立一种登革热病毒双靶基因多重荧光PCR检测方法,用于登革热病毒的实验室诊断和基因分型。方法选取登革热病毒Ⅰ-Ⅳ型病毒保守区设计型特异性引物探针和通用型引物探针。评估多重荧光PCR检测方法的特异性、重复性和检测限;并对20份阳性样本进行检测。结果20个登革热阳性核酸标本在通用型检测全部为阳性,特异性型别检测发现登革热病毒Ⅰ型10例、登革热病毒Ⅱ型3例、登革热病毒Ⅲ型3例、登革热病毒Ⅳ型4例;20名正常无症状人群标本提取的核酸和HIV、HCV和HEV通用型和特异性型别检测全部为阴性。梯度检测的变异系数均小于5％。对登革热Ⅰ-Ⅳ型病毒检测最低检测限达10^3 eopies／ml。结论本研究建立的登革热病毒双靶基因多重荧光PCR检测及分型方法具有特异性好、重复性好、快速易操作等优点,可用于登革热病毒的快速检测和基因分型鉴定。     

我国分离的两株病毒为重组甲病毒

目的 明确我国分离的XJ－90260和XJ－91006病毒的分类地位、种系发生和遗传型。方法 特异引物逆转录－聚合酶链反应（RT－PCR）扩增两株病毒的NSP4、E1基因区和3′端非编码区,测序,进行核苷酸序列同源性比较和3′端非编码区核苷酸序列分析,并结合同属其他病毒这些基因区的核苷酸序列进行种系进化分析。结果 两株病毒的核苷酸序列同源性为100％,与西方马脑炎病毒的核种酸序列同源性最高,具有西方马脑炎病毒3′端非编码区结构特征,其NSP4基因区与东方马脑炎病毒同源,E1基因区与辛德毕斯病毒同源,两株病毒均位于西方马脑炎病毒B组,与西方马脑炎病毒俄罗斯分离株进化关系最近。结论 我国分离的XJ－90260和91006病毒属于西方马脑炎病毒同一遗传型,均为重组甲病毒。     

实时荧光PCR检测手足口病病原体方法的建立及初步应用

目的 建立一种能同时检测肠道病毒、肠道病毒71型和柯萨奇病毒16型等手足口病病原体核酸的实时荧光PCR方法.方法 针对上述病毒的基因序列,设计、合成引物、探针.对38例手足口病患者在实时荧光PCR仪上进行扩增、检测和结果分析;并与常规RT-PCR方法的检测结果进行比对.结果 38例患者肠道病毒、肠道病毒71型和柯萨奇病毒16型实时荧光PCR方法与常规RT-PCR方法的阳性率分别为:73.7%、60.5%、13.2%、71.1%、55.3%、13.2%.统计分析表明,两种方法差异不具有统计学意义.结论 成功建立了肠道病毒、肠道病毒71型和柯萨奇16型病毒核酸的实时荧光PCR检测方法.     

Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection

Sindbis virus (SINV) is an arthropod-borne alphavirus found widely in Eurasia, Africa and Oceania. Clinical SINV infection, characterized by rash and arthritis, is reported primarily in Northern Europe. The laboratory diagnosis of SINV infection is based currently on serology. A one-step TaqMan^® real-time RT-PCR assay was developed for the detection of SINV and evaluated its clinical performance with acute-phase serum samples. The specificity and sensitivity of the real-time PCR assay were assessed using cell cultured Finnish SINV strains. The applicability of the assay for diagnostic use was evaluated using 58 serum samples from patients infected with SINV. The real-time RT-PCR assay was specific and sensitive for the detection of SINV in cell culture supernatants with a 95% detection limit of 9 genome copies/reaction determined by probit analysis. However, in the assay only 7/58 (12%) of serum samples were positive of which two were also positive by conventional nested PCR assay and none by virus isolation. This novel assay is specific and sensitive for detection of SINV and can be used for example for screening SINV in wildlife. However, molecular diagnostic techniques using serum samples seem to be of limited value for the diagnosis of human SINV infection due to the short and low viraemia of infection with SINV.     

两种PCR方法检测狂犬病毒的敏感性和特异性比较

目的比较Nested-PCR和SYBR GreenⅠReal-Time PCR两种方法检测狂犬病毒的敏感性、特异性和时效性。方法对10倍连续稀释的狂犬病毒（疫苗株）核酸样品,采用Nested-PCR和SYBR GreenⅠReal-Time PCR方法进行平行检测,比较两种方法的敏感性;同时以登革热2型病毒、诺如病毒、星状病毒、EV71和乙型脑炎病毒核酸对两种方法的特异性进行评价。结果 SYBR GreenⅠReal-Time PCR方法可检测出2.3×106copies/μl核酸分子,与Nested-PCR方法相比,敏感度提高10倍。两种检测方法的特异性均好,与登革热2型病毒、诺如病毒、星状病毒、EV71和乙型脑炎病毒核酸均无交叉反应。SYBR GreenⅠReal-Time PCR方法检测花费3h,检测时间比Nested-PCR方法节省2h。结论与Nested-PCR相比,SYBR GreenⅠReal-Time PCR是相对高效的检测狂犬病毒的方法。     

Inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers

The genus Alphavirus contains members that threaten human health, both as natural pathogens and as potential biological weapons. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) enter cells readily and can inhibit viral replication through sequence-specific steric blockade of viral RNA. Sindbis virus (SINV) has low pathogenicity in humans and is regularly utilized as a model alphavirus. PPMO targeting the 5'-terminal and AUG translation start site regions of the SINV genome blocked the production of infectious SINV in tissue culture. PPMO designed against corresponding regions in Venezuelan equine encephalitis virus (VEEV) were likewise found to be effective in vitro against several strains of VEEV. Mice treated with PPMO before and after VEEV infection were completely protected from lethal outcome while mice receiving only post-infection PPMO treatment were partially protected. Levels of virus in tissue samples correlated with animal survival. Uninfected mice suffered no apparent ill-effects from PPMO treatment. Thus, PPMO appear promising as candidates for therapeutic development against alphaviruses.     

Le concept de maladies virales émergentes : quel risque de zoonose pour La Réunion ?

In Reunion Island, the risk of emerging infectious diseases lies mainly in several viral zoonoses: West Nile fever, Sindbis virus, Nipah virus, Wesselsbron virus, Rift Valley fever and Japanese encephalitis. There morbidity and consequences are more or less important but they all have a non-negligible epidemic potential, so they have to be monitored. Indeed, the struggle against these emerging infectious diseases requires an early detection of the cases, thus a surveillance system capable of detecting them as early as possible, thanks to a real international network of information, warning and prevention.     

Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus

Nucleic acid amplification techniques are used frequently for rapid diagnosis of viral diseases. In this study, a real-time polymerase chain reaction protocol that uses primers specific for the viral VP4 gene and the commercial SYBR Green reagent were evaluated for the quantitative measurement of human rotavirus (HRV) RNA in human stool specimens. SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. The assay resulted in a sensitive and reproducible detection of targets ranging from low (<10(2)rotavirus cDNA copies/reaction) to high numbers (>10(6)rotavirus cDNA copies/reaction). No cross-reaction was found with crude cell culture stocks of coxsackievirus, echovirus, poliovirus, hepatitis A virus and adenovirus. Analysis with the HRV cDNA standard demonstrated high reproducibility with a coefficient of variation (CV) of 0.2-0.9%. Daily performance among three different laboratories showed a CV no greater than 8%, indicating an intermediate level of variation. These results demonstrate the feasibility of this method for quantitative analysis of human rotavirus in clinical samples.     

Ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional RT-PCR techniques.

BACKGROUND: Rabies is an enzootic and fatal disease and is still a major problem in developing countries. Ante mortem diagnosis in human cases is necessary for medical management of the patient and to ensure appropriate post-exposure treatment of contacts. Both conventional RT-PCR and Real time PCR (TaqMan) have been described for the detection of rabies virus RNA from saliva and tissue respectively, however to date, there have been no studies comparing conventional and real time PCR assays for detection of rabies virus nucleic acid using saliva samples for ante mortem diagnosis. OBJECTIVES: In this study, we evaluated the utility of conventional RT-PCR and SYBR Green I Real time PCR in the ante mortem diagnosis of rabies using saliva samples. STUDY DESIGN: Saliva samples collected from twenty-four patients presenting with typical clinical manifestations of rabies were tested in the two assays. RESULTS: Amongst the 24 samples tested, 21 samples (87.5%) were positive by either of the two molecular methods. Of these 21, rabies virus RNA was detected in 6/21 in the conventional RT-PCR assay while SYBR Green I Real time PCR could detect RNA in 18/21 samples. CONCLUSION: Real time PCR assay was more sensitive than conventional RT-PCR assay (sensitivity 75% versus 37%, p=0.0189). This study highlights the utility of molecular diagnostic tests in establishing ante mortem diagnosis of rabies using saliva samples within a few hours.     

Detection and quantitation of Solenopsis invicta virus in fire ants by real-time PCR

A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of S. invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) for cDNA synthesis. Dithiothreitol used in the cDNA synthesis step was found to significantly decrease the detection sensitivity for SINV RdRp and was therefore omitted. SINV RdRp cDNA was then quantified by QPCR using SYBR Green dye and a standard curve generated from SINV RdRp plasmid clones. A standard curve was successfully constructed from clones of the SINV RdRp region. A strong linear relationship [r² = 0.998; y = (−3.63 ± 0.37)x + (39.19 ± 1.33)] between C_T and starting SINV RdRp copy number was observed within a dynamic range of 5–5 × 10⁶ copies. SINV RdRp copy number was determined with the optimized QPCR method in individual S. invicta ants taken from an infected field colony. Worker ants exhibited the highest RdRp copy number (2.1 × 10⁹ copies/worker ant) and pupae exhibited the lowest (4.2 × 10² copies/pupa). Mean RdRp copy number was lowest in early larvae and pupae. Overall, SINV RdRp copy number increased through larval development, sharply declined during pupation, then sharply increased in adults.     

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR

We developed a real‐time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real‐time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 µl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections. J. Med. Virol. 81:146–152, 2009. © 2008 Wiley‐Liss, Inc.