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2009年24卷3期

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Review

多功能蛋白:单纯疱疹病毒Ⅰ型(HSV-1)内膜蛋白VP22*
A Multiple Functional Protein: the Herpes Simplex Virus Type 1 Tegument Protein VP22*

李美丽, 郭洪, 丁琼, 郑春福*Mei-li LI#

2009, 24(3): 153 doi: 10.1007/s12250-009-3035-2

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VP22是单纯疱疹病毒Ⅰ型(HSV-1)数量最多的内膜蛋白之一,平均每个病毒颗粒有2400个拷贝,在-疱疹病毒亚科中高度保守。VP22具有核定位,结合染色质,结合微管,诱导微管重组和细胞间穿梭的功能。VP22可以与细胞蛋白如模板激活因子1(TAF-I)和非肌性肌球蛋白IIA(NMIIA)相互作用,也可以与许多病毒蛋白相互作用,包括内膜蛋白VP16、pUS9和pUL46以及糖蛋白gE和gD。最近的一些研究表明VP22还具有新的功能,比如在感染晚期促进蛋白合成,感染早期聚集病毒mRNA,并且还可能具有转录调控功能。
Abstract:The herpes simplex virus type 1 (HSV-1) VP22, is one of the most abundant HSV-1 tegument proteins with an average stoichiometry of 2 400 copies per virion and conserved among alphaherpesvirinae. Many functions are attributed to VP22, including nuclear localization, chromatin binding, microtubule binding, induction of microtubule reorganization, intercellular transport, interaction with cellular proteins, such as template activating factor I (TAF-I) and nonmuscle myosin II A (NMIIA), and viral proteins including tegument protein VP16, pUS9 and pUL46, glycoprotein E (gE) and gD. Recently, many novel functions performed by the HSV-1 VP22 protein have been shown, including promotion of protein synthesis at late times in infection, accumulation of a subset of viral mRNAs at early times in infection and possible transcriptional regulation function.

Research Article

2008年中国阜阳爆发的肠道病毒71型VP1的基因分析*
Genetic Analysis of the VP1 Region of Human Enterovirus 71 Strains Isolated in Fuyang, China, During 2008*

马绍辉, 刘建生, 王晶晶, 施海晶, 杨卉娟, 陈俊英, 刘龙丁, 李琦涵*Shao-hui MA

2009, 24(3): 162 doi: 10.1007/s12250-009-3033-4

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肠道病毒 71型是小RNA病毒科肠道病毒属成员,其感染主要引起患者手足口病,还能够引起无菌性脑膜炎、脑干脑炎和脊髓灰质炎样的麻痹等多种与神经系统相关的疾病。对2008年春从阜阳地区爆发的9株EV71型肠道病毒分离毒株进行完整 VP1序列分析,发现在核苷酸水平上,9株阜阳分离株的同源性大于98%,与其它C4基因型分离株之间有93-100%的同源性;在氨基酸水平上,阜阳分离株之间的同源性为99-100%,与其它C4基因型分离株之间有97%-100%的同源性,而且第22位均为组氨酸。这提示EV71阜阳分离株为C4基因型,并且22位组氨酸为阜阳株的特征性标志。
Abstract:Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.

鸭肠炎病毒UL4基因的分子特征
Molecular Characterization of the Duck Enteritis Virus UL4 Gene

潘华奇, 王楠, 刘丽, 刘磊, 胡江春, 陈溥言, 王书锦, 曹瑞兵

2009, 24(3): 171 doi: 10.1007/s12250-009-3002-y

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鸭肠炎病毒是一种疱疹病毒,能引起鸭等水禽急性、致死性的传染性疾病。为了研究鸭肠炎病毒(Duck enteritis virus , DEV) UL4的特征,以中国鸭肠炎病毒疫苗株的基因组为模板,分析疱疹病毒UL3基因的保守区域,设计简并引物,进行PCR扩增,得到2086 bp的目的基因片段。序列分析发现,一个714 bp的开放阅读框编码239个氨基酸与疱疹病毒甲亚科的UL4基因同源。序列比对分析发现,疱疹病毒甲亚科的UL4蛋白有10个非常保守的氨基酸残基。进化树分析表明:17个疱疹病毒分成了四个组群,鸭肠炎病毒单独成一支,且与鸡疱疹病毒2型、鸡疱疹病毒3型和火鸡疱疹病毒进化关系最为密切。UL4蛋白的进化关系对疱疹病毒甲亚科成员的分类是有用的。
Abstract:Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.

猪流行性腹泻病毒DX株结构蛋白基因的克隆分析及N蛋白的
原核表达*
Cloning the Structure Genes and Expression the N Gene of
Porcine Epidemic Diarrhea Virus DX*

李建强, 柳纪省, 兰喜, 程杰, 娄忠子, 李学瑞, 殷相平, 李宝玉, 杨彬, 李志勇,

2009, 24(3): 179 doi: 10.1007/s12250-009-2982-y

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对猪流行性腹泻病毒(porcine epidemic diarrhea virus)PEDV DX株结构蛋白基因进行了克隆与测序。结果表明结构蛋白基因S、sM、M 和 N 核苷酸序列长度分别为 4152、231,681 和 1326 碱基 。结构蛋白基因 S、M 和 N 阅读框起始密码子 ATG 前都有冠状病毒特有的转录调控序列。S、M 和 N 蛋白分别含有 30、3 和 7 个 N-糖基化位点。 系统发生树和同源性比较分析结果显示 DX 株与中国JS-2004-2株、LJB/06 和 CH/HLJH/06亲缘关系最为相近,因为JS-2004-2 株分离自中国南方,LJB/06 和 CH/HLJH/06分离自中国东北,而 DX 株分离自中国西北,暗示PEDV 一些毒株在中国流行的范围特别。用含有Nco I 和BamH I 酶切位点的上下引物扩增N基因ORF,定向克隆到pET30载体中,pET30-pN在大肠杆菌Rossta中表达得到大小为55 KDa 的蛋白,Wsetern blot 表明此蛋白有生物学活性。
Abstract:The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM,M and N genes open reading frame (ORF) of DX were 4 152, 231,681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.

运用实时荧光定量PCR方法对SARS-CoV核酸检测
灵敏度和特异性的评估*
Evaluation of Sensitivities and Specificities of SARS-CoV Detection by Real-time Quantitative Reverse Transcription-PCR Assays*

许黎黎, 胡志红, 王华林, 韩霄, 邓菲*Li-li XU

2009, 24(3): 187 doi: 10.1007/s12250-009-3021-8

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灵敏、快速且可靠的病原检测手段,对于SARS的预防、诊断具有至关重要的意义。在本研究中,我们开发并优化了一种基于一步法实时荧光定量RT-PCR的SARS-CoV核酸检测系统,并针对SARS-CoV的所有基因,分别设计不同的引物和探针,通过使用EvaGreenTM染料法和Taqman-MGB探针法,对SARS-CoV感染的Vero E6细胞上清及沉淀分别定量,来综合评估不同引物及探针检测灵敏度和特异性的差异,同时GAPDH基因被用作内参对照。结果显示基于S基因的引物及探针检测灵敏度最高,但由于S基因在不同物种间的保守程度不如N基因,致使基于N基因的引物及探针在检测过程中也可广泛使用。对比显示细胞沉淀中mRNA水平显著高于上清中,故更适合于用作检测标本。
Abstract:The etiological agent of severe acute respiratory syndrome (SARS) was identified as a new coronavirus, termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study, we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreenTM dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities, 13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection, but because of its sequence variability in the different viral strains, primers and a probe based on the N gene were suitable substitutions. Meanwhile, we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.

草鱼呼肠孤病毒非结构蛋白NS80包涵体形成保守域
在原核细胞中的高效表达
Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus*

范超, 张岚岚, 类承凤, 方勤

2009, 24(3): 194 doi: 10.1007/s12250-009-3028-1

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在病毒侵染宿主细胞的过程中,通常在细胞质内形成一种称为病毒复制基质(viral factories)的包涵体结构。它的作用与病毒侵染后期的病毒组装有密切关系。基因序列同源性比对表明,草鱼呼肠孤病毒(GCRV)非结构蛋白NS80,特别是NS80蛋白C端部分,很可能在GCRV侵染细胞形成病毒发生基质方面起着关键性的作用。为探讨GCRVNS80非结构蛋白在病毒侵染中的包涵体形成功能,本文建立了稳定的NS80(335-742) 高效原核表达体系及相关纯化方法,并制备了兔抗NS80抗体。在被GCRV侵染细胞裂解液中,以NS80(335-742)抗体为标记的western blot 显示,被侵染细胞中存在至少两种不同大小的NS80表达形式的相关蛋白,推测其在包涵体的形成过程中扮演重要角色。
Abstract:Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335-742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335-742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

卡波氏肉瘤相关疱疹病毒ORFK8.1基因原核表达产物
的免疫原性分析*
Expression of Kaposi’s Sarcoma-associated Herpesvirus ORFK8.1 and Its Preliminary Diagnostic Application*

付碧石, 李宝林, 欧阳新星, 徐帆洪, 王林定,

2009, 24(3): 202 doi: 10.1007/s12250-009-3029-0

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为了纯化卡波氏肉瘤相关疱疹病毒编码的糖蛋白ORFK8.1并分析其免疫原性,我们在原核表达体系中表达了卡波氏肉瘤相关疱疹病毒编码的氨基端orfK8.1基因产品。含重组质粒pQE-80L-orfK8.1的大肠杆菌经由异丙基硫代-β-D-半乳糖苷诱导表达。融合蛋白通过亲和层析纯化。表达的蛋白和纯化的产品经由SDS-聚丙烯酰胺凝胶电泳鉴定。我们建立了一种ELISA方法来鉴定纯化的ORFK8.1的免疫原性。SDS-聚丙烯酰胺凝胶电泳显示纯化的ORFK8.1蛋白约为26 kDa, 与预期一致。ELISA分析证明ORFK8.1具有免疫原性. 我们利用 ORFK8.1 ELISA筛查湖北地区560份普通人群血清,发现KSHV阳性率为6.80%.
Abstract:The ORFK8.1 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate–polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant ORFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 μg/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein’s specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.

HCMV感染对神经胶质细胞NGF表达的影响
HCMV Infection Depress NGF Expression in Human Glioma Cells

王海涛, 王斌, 刘志军, 白志强, 李玲, 钱冬萌, 闫志勇, 宋旭霞

2009, 24(3): 209 doi: 10.1007/s12250-009-3018-3

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人巨细胞病毒(HCMV)是人类最常见的先天性感染病毒,可引起流产、早产、死胎、胎儿宫内发育迟缓及小头畸形、抽搐、脑瘫、智力低下、视力损害及听力损害等后遗症。本研究采用RT-PCR和Western Blotting技术,检测正常培养和感染HCMV的神经胶质瘤(U251)细胞NGF的表达,探讨人巨细胞病毒对神经胶质瘤细胞内源性神经生长因子(NGF)表达的影响。结果发现U251细胞能够表达NGF,感染HCMV后初期NGF表达无明显变化,后期明显下调,由此我们认为HCMV能够下调U251细胞NGF的表达。
Abstract:Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

利用VP1蛋白制备抗Asia I口蹄疫病毒的单克隆抗体*
Application of VP1 Protein to Develop Monoclonal Antibody against Foot-and-mouth Disease Virus Asia1 Type*

林彤, 独军政, 邵军军, 丛国正, 宋帅, 高闪电, 常惠芸*Tong LIN

2009, 24(3): 215 doi: 10.1007/s12250-009-2986-z

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宁夏回族自治区猪繁殖与呼吸综合症病毒
nsp2基因的遗传变异分析
Genetic Variation Analyses of nsp2 Gene of PRRSV in Ningxia Hui Autonomous Region of China

田宏, 吴锦艳, 尹双辉, 尚佑军, 满自萍, 赵娜, 靳野, 刘湘涛*Hong TIAN

2009, 24(3): 221 doi: 10.1007/s12250-009-3017-4

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为更好的了解中国宁夏地区猪繁殖与呼吸综合症病毒的遗传多样性及分子进化。本研究将该地区2007年爆发的PRRSV的nsp2基因进行了克隆和序列分析,并与2006-2007中国南方PRRSV的流行毒株及经典疫苗株ch-1a的nsp2基因进行了比对。结果表明:与经典毒ch-1a比较,中国宁夏2007年的流行毒株在nsp2基因上有87个核苷酸的缺失,并且该缺失区域与两株2006-2007山东的流行毒株的缺失在同一区域,同一缺失数量。在同源性比对中发现,宁夏的流行毒株与ch-1a的同源性为60.3%-79.9%; 与山东的流行毒株的同源性为80.3%-98.8%; 所分离的毒株之间的同源性为74.9%-100%.该毒株的成功分离为高致病性猪蓝耳病流行病学调查分析提供数据,也为预防控制该病积累了资料。nsp2特性分析为揭示高致病性猪蓝耳病PRRSV在宁夏的流行特点提供参考。
Abstract:To gain a better understanding of the genetic diversity and evolution of PRRSV in the Ningxia Hui Nationality Autonomous Region (Ningxia) of China, the nsp2 genes from a series of PRRSV strains collected from the region in 2007 were partially sequenced. These sequences were then analyzed along with the classical strain (ch-1a) and two other epidemic strains SD (3) and SD2006. Comparison of the nucleotide sequence with ch-1a indicated that nsp2 genes of seventeen Ningxia isolates (NX strain) have deletions of 87 nucleotides. Sequence analysis indicated that homology between the Ningxia strain and ch-1a was 60.3%-79.9% in the nucleotide sequence, and homology between the NX strains and SD strains was 80.3%-98.8% in the nucleotide sequence. The nsp2 genes of the seventeen isolates had 74.9%-100% nucleotide sequence identities with each other. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.