水痘-带状疱疹病毒临床分离株基因型别分析以及与Oka疫苗株的区别

目的 运用分子生物学方法 对从水痘或带状疱疹患者皮肤疱疹液中分离得到的水痘-带状疱疹(VZV)株进行基闪型研究,并区分感染是南野牛株还是由Oka疫苗株引起的.方法 从水痘或带状疱疹患者的皮肤水疱液中分离VZV,然后利用聚合酶链反应和限制性片段长度多态性分析对病毒株的ORF38、54、62和R5可变区基因进行分析.结果 在所分离的19株VZV中,存在PstⅠ+ Bgl Ⅰ+ R5A和Pst Ⅰ+ Bgl Ⅰ+RSB两种基因型,其中Pst Ⅰ+ Bgl Ⅰ+ R5A占52.7%,Pst Ⅰ+ Bgl Ⅰ+R5B占47.3%,没有发现与Oka疫苗株相同的基因型.结论 本研究中所分离的VZV毒株均系野生株,它们的基因型与欧洲、美国、日本的VZV分离株均不相同.利用病毒基因组中ORF38和ORF62区域的单一核苷酸多态性,能够区分疫苗株和野毒株.     

水痘-带状疱疹病毒基因分型研究进展

水痘-带状疱疹病毒(varicella-zoster virus,VZV)属人类疱疹病毒α亚科,又称人类疱疹病毒3型(HHV-3),是水痘和带状疱疹的病原体.VZV的原发感染引起水痘,水痘痊愈后可获终生免疫.但VZV原发感染后,病毒可沿感觉神经上行至一个或多个脊髓后根和三叉神经节内形成潜伏感染.当机体免疫力降低时病毒可被再激活并沿神经轴突到达相应神经支配皮肤区域,引起带状疱疹[1].     

水痘-带状疱疹病毒基因分型研究进展

水痘-带状疱疹病毒(varicella-zoster virus,VZV)属人类疱疹病毒α亚科,又称人类疱疹病毒3型(HHV-3),是水痘和带状疱疹的病原体.VZV的原发感染引起水痘,水痘痊愈后可获终生免疫.但VZV原发感染后,病毒可沿感觉神经上行至一个或多个脊髓后根和三叉神经节内形成潜伏感染.当机体免疫力降低时病毒可被再激活并沿神经轴突到达相应神经支配皮肤区域,引起带状疱疹[1].     

水痘-带状疱疹病毒减毒机制的研究进展

水痘．带状疱疹病毒（varicella．zostervirus,VZV）属人类疱疹病毒α亚科,亦称人类疱疹病毒3型（humanherpesvirus3,HHV-3）,是水痘和带状疱疹的病原体。     

水痘-带状疱疹病毒基因启动子分析

目的从水痘-带状疱疹病毒(VZV)主要基因的启动子中找到最容易被VZV感染激活的启动子及其最短有效序列以构建VZV报告细胞系。方法构建18个VZV开放读码框架(ORF)启动子的重组报告质粒,用基因转染瞬时表达分析法比较各启动子在VZV感染早期启动报告基因产物表达的活性;将最容易被激活的启动子从5′和3′端截短后构建截短启动子的重组报告质粒,比较各截短启动子的活性以找到启动子的最短有效序列;应用点定向突变技术将最短有效序列中细胞转录因子的结合序列进行碱基置换突变后比较突变启动子的启动活性。结果VZV感染早期ORF9、ORF61和ORF66的启动子最容易被激活;ORF9启动子的最短有效序列位于-127～-34(93 bp),其上游刺激因子(USF)结合序列的碱基置换突变后启动子活性降低50%;ORF61启动子的最短有效序列位于-227～-52(165 bp),其Pbx-1结合序列的碱基置换突变后启动子活性增强一倍,但NF-κB结合序列的突变对启动子活性无影响。结论ORF9启动子的最短有效序列最适合用来构建VZV报告细胞系。     

西藏地区水痘-带状疱疹病毒临床分离株基因型研究

目的 研究西藏地区水痘-带状疱疹病毒临床分离株基因型.方法 将临床采集的水痘或带状疱疹患者皮肤水疱液接种至人胚胎成纤维细胞进行病毒分离;对分离得到的病毒运用间接免疫荧光法鉴定,鉴定结果为阳性的病毒继续体外培养以提取其基因组DNA.PCR法扩增并测序水痘-带状疱疹病毒基因组中开放读码框1、21、50、54的部分片段.测序结果与GenBank中水痘-带状疱疹病毒参考株Dumas进行序列比对分析其中单核苷酸多态性,并利用Primer 5.0软件进行酶切位点分析,从而确定基因型.结果 从临床采集的16份标本中经分离鉴定后得到10株水痘-带状疱疹病毒临床分离株,病毒分离阳性率为62.5％.基因分析结果显示西藏地区10株水痘-带状疱疹病毒临床分离株中基因型A1有3株,基因型A2有4株,基因型J1有3株.结论 西藏地区水痘-带状疱疹病毒基因型分布与西藏地区地理位置密切相关.     

水痘-带状疱疹病毒感染实验检测方法的研究进展

对临床表现不典型的可疑水痘-带状疱疹病毒(VZV)感染,需依靠实验室榆查才能确诊.因此,建立敏感、特异、快速和简便的VZV实验检测方法,对VZV感染者的早期诊断和预后具有重要意义.VZV的实验检测方法包括Tzanck涂片、电镜和免疫电镜、病毒分离、免疫学、分子生物学等方法,各方法均有其优点和不足.     

水痘-带状疱疹病毒免疫逃避机制的研究进展

水痘-带状疱疹病毒属人类a疱疹病毒，在感染机体过程中通过MHE-Ⅰ限制的抗原表达抑制、MHE-Ⅱ限制的抗原表达抑制、树突状细胞和角质形成细胞表面免疫分子改变、干扰凋亡过程以及潜伏等机制逃避免疫监视，使病毒有足够的时间复制、传播和引起皮损，或者长期潜伏于神经节。本文拟就有关内容作一综述。     

水痘-带状疱疹病毒免疫逃避机制的研究进展

水痘-带状疱疹病毒属人类а疱疹病毒,在感染机体过程中通过MHC-Ⅰ限制的抗原表达抑制、MHC-Ⅱ限制的抗原表达抑制、树突状细胞和角质形成细胞表面免疫分子改变、干扰凋亡过程以及潜伏等机制逃避免疫监视,使病毒有足够的时间复制、传播和引起皮损,或者长期潜伏于神经节。本文拟就有关内容作一综述。     

Novel approach to differentiate subclades of varicella-zoster virus genotypes E1 and E2 in Germany

Varicella-zoster virus (VZV) is the causative agent of chicken pox (varicella) in children and reactivation of VZV in elderly or immunocompromised persons can cause shingles (zoster). A subclade differentiation of the most prevalent VZV genotypes E1 and E2 in Germany was not possible with the current genotyping methods in use, but is highly important to understand the VZV molecular evolution in more detail and especially to follow up the routes of infection. Therefore the objective of this study was to develop a simple PCR-based method for differentiation of E1 and E2 subclades. Viral DNA was isolated from vesicle fluid samples of six selected German zoster patients and used to amplify nine complete open reading frames (ORFs) of the VZV genome by different PCR assays. Phylogenetic analysis was performed by a Bayesian approach. Based on the analysis of a total of nine ORFs, a 7482 bp stretch consisting of ORFs 5, 37 and 62 contained informative sites for identification of novel subclades E1a, E2a and E2b for VZV genotypes E1 and E2. Specific single nucleotide polymorphisms (SNPs) were demonstrated for subclades E2a and E2b within the ORFs 5, 37 and 62, whereas a subclade E1a-specific SNP was found in ORF 56. The classification of E1 and E2 subclades may facilitate a more exact and in-depth monitoring of the molecular evolution of VZV in Germany in the future.     

Genomic cartography of varicella-zoster virus: a complete genome-based analysis of strain variability with implications for attenuation and phenotypic differences

In order to gain a better perspective on the true variability of varicella-zoster virus (VZV) and to catalogue the location and number of differences, 11 new complete genome sequences were compared with those previously in the public domain (18 complete genomes in total). Three of the newly sequenced genomes were derived from a single strain in order to assess variations that can occur during serial passage in cell culture. The analysis revealed that while VZV is relatively stable genetically it does posses a certain degree of variability. The reiteration regions, origins of replication and intergenic homopolymer regions were all found to be variable between strains as well as within a given strain. In addition, the terminal viral sequences were found to vary within and between strains specifically at the 3' end of the genome. Analysis of single nucleotide polymorphisms (SNPs) identified a total of 557 variable sites, 451 of which were found in coding regions and resulted in 187 different in amino acid substitutions. A comparison of the SNPs present in the two gE mutant strains, VZV-MSP and VZV-BC, suggested that the missense mutation in gE was primarily responsible for the accelerated cell spread phenotype. Some of the variations noted with high passage in cell culture are consistent with variations seen in the IE62 gene of the vaccine strains (S628G, R958G and I1260V) that may help in pinpointing variations essential for attenuation. Although VZV has been considered to be one of the most genetically stable human herpesviruses, this initial assessment of genomic VZV cartography provides insight into ORFs with previously unreported variations.     

Differentiation between vaccine and wild-type varicella-zoster virus genotypes by high-resolution melt analysis of single nucleotide polymorphisms

BACKGROUND: The analysis of single nucleotide polymorphisms (SNPs) of varicella-zoster virus (VZV) has enabled differentiation between wild-type genotypes from the Oka vaccine strain (V-Oka). OBJECTIVES: To genotype VZV strains in Australia using high-resolution melt (HRM) analysis of SNPs in five gene targets. STUDY DESIGN: Extracted DNA from 78 samples obtained from patients with chickenpox and zoster were genotyped by HRM analysis of SNPs in five open reading frames (ORFs): 1 (685 G>A), 21 (33725 C>T), 37 (66288 G>A), 60 (101464 C>A) and 62 (106262 T>C) using a double-stranded (ds) DNA saturating dye, LC Green Plus. RESULTS: For each genotype, melt curve temperature (T(m)) shifts differentiated the nucleotide present at that locus (P<0.0001) with melting curve shifts between alleles ranging from 0.56 degrees C (ORF 37) to 3.34 degrees C (ORF 62). The most common genotypes detected were the European Type C (59%) and B (18%) strains. This was followed by the African/Asian Type A (14%) and Japanese J1 (9%), strains, both prevalent in the Northern Territory and Western Australia. CONCLUSIONS: HRM analysis of SNPs showed that the European B and C genotypes were most prevalent in Australia, with genotypes A and J strains also present. HRM analysis using a dsDNA dye provides a useful tool in classifying varicella-zoster viruses.     

Intracellular transport and stability of varicella-zoster virus glycoprotein K

VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established that gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.     

Distribution of varicella-zoster virus (VZV) wild-type genotypes in northern and southern Europe: Evidence for high conservation of circulating genotypes

Phylogenetic analysis of 19 complete VZV genomic sequences resolves wild-type strains into 5 genotypes (E1, E2, J, M1, and M2). Complete sequences for M3 and M4 strains are unavailable, but targeted analyses of representative strains suggest they are stable, circulating VZV genotypes. Sequence analysis of VZV isolates identified both shared and specific markers for every genotype and validated a unified VZV genotyping strategy. Despite high genotype diversity no evidence for intra-genotypic recombination was observed. Five of seven VZV genotypes were reliably discriminated using only four single nucleotide polymorphisms (SNP) present in ORF22, and the E1 and E2 genotypes were resolved using SNP located in ORF21, ORF22 or ORF50. Sequence analysis of 342 clinical varicella and zoster specimens from 18 European countries identified the following distribution of VZV genotypes: E1, 221 (65%); E2, 87 (25%); M1, 20 (6%); M2, 3 (1%); M4, 11 (3%). No M3 or J strains were observed.     

白黎芦醇体外抑制水痘-带状疱疹病毒作用机制的研究

目的 应用构建的水痘-带状疱疹病毒(VZV)报告细胞系MV9G进一步研究白黎芦醇体外抑制VZV的作用机制.方法 将无细胞VZV直接感染MV9G细胞(CFVs直接感染)或将带细胞VZV与MV9G细胞共培养(CAVs共培养)以激发MV9G细胞表达报告基因萤火虫荧光素酶.在CFVs直接感染前或CAVs共培养不同时间点加入白黎芦醇,通过比较药物对CFVs或CAVs激发荧光素酶的抑制强度分析白黎芦醇直接灭活病毒、抑制病毒黏附和穿透、抑制病毒在细胞内复制及其时间点和可逆性:通过比较药物作用前后VZV即刻早期蛋白62(IE62)mRNA拷贝数和IE62表达强度变化分析白黎芦醇对IE62转录和表达的抑制作用.结果 白黎芦醇＞30.0 μg/ml时MV9G细胞三磷酸腺苷(ATPs)含量随药物浓度升高而逐渐降低,ATPs降低50％时白黎芦醇浓度(CD.)约60.3μg/ml.CFVs与白黎芦醇(25.0μg/ml)预混37℃水浴孵育2h后直接感染MV9G细胞,CFVs激发荧光素酶下降50％.MV9G细胞在含白黎芦醇培养基中37℃孵育2h后直接感染CFVs,CFVs激发荧光素酶随药物浓度升高而逐渐降低,但4℃孵育对无显著变化.在CAVs共培养中加入白黎芦醇后CAVs激发荧光素酶显著降低,药物抑制荧光素酶50％时浓度(IC.)约8.7 μg/ml.分别在CAVs共培养3、6、9、12、24、30和36 h时加入白黎芦醇,3～24h加药各组CAVs激发荧光素酶均显著高于对照组,但1h、3D h和36 h加药组与对照组间差异无统计学意义.CAVs共培养时撤除白黎芦醇后CAVs激发荧光素酶显著高于撤药前,尤以24h和72 h撤药组明显.VZV IE62 mRNA拷贝数和IE62抗体阳性细胞数随药物作用时间延长而逐渐降低.结论 白黎芦醇细胞毒性较强,MV9G细胞可耐受最高浓度为30.0μg/ml.白黎芦醇部分灭活CFVs、抑制CFVs穿透MV9G细胞但对CFVs黏附MV9G细胞无影响,以浓度依赖方式可逆性抑制CAVs细胞内复制.白黎芦醇可能通过抑制1E62基因的转录和表达而抑制VZV感染的早期阶段.     

四川省分离的基因1型乙型脑炎病毒分子特征分析

目的 从四川省巴中市采集的蚊虫标本中分离乙型脑炎(简称乙脑)病毒(JEV),确定其基因型别,并分析相关的基因1型乙脑病毒PrM和E基因区段氨基酸序列特征.方法 对2004年采集蚊虫标本进行病毒分离,对新分离的乙脑病毒进行生物学、血清学及分子生物学鉴定.逆转录聚合酶链反应(RT-PCR)扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X软件做碱基配对分析,MEGA4软件完成病毒进化分析,GENEDOC(3.2)软件完成氨基酸位点分析,根据蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白三维结构模拟预测分析.结果 共采集4668只蚊虫标本,主要是骚扰阿蚊和库蚊,分离到6株病毒,经鉴定均属于基因1型的乙脑病毒.将四川省分离的6个毒株结合我国新分离的基因1型乙脑病毒与减毒活疫苗株SA14-14-2株的PrM区段和E区段氨基酸比较,发现PrM区段在PrM2、64和65位存在基因1型乙脑病毒独有的氨基酸位点差异,E区段存在14处共同的氨基酸位点差异,其中在E129、222、327和366位点为中国目前分离到的基因1型乙脑病毒所特有的位点特征.结论 从四川省巴中市首次分离到基因1型的乙脑病毒,并发现基因1型乙脑病毒与减毒活疫苗株之间PrM、E基因区段存在氨基酸差异,但现行疫苗株理论上可以保护新分离的基因1型乙脑病毒.