人偏肺病毒广州分离株全基因组序列测定及分析

目的 探讨广州地区儿童感染的偏肺病毒基因组分子结构特点和基因型类型.方法 参考GenBank上的偏肺病毒00-1株(AF371337)基因组设计分段扩增引物,进行RT-PCR分段扩增偏肺病毒基因组,克隆于T载体上,序列测定,用Clustal W/X、DNASTAR、MEGA4.1等软件分析基因组序列.结果 偏肺病毒hMPVgz01全基因组为13 327 bp,提交到GenBank上的序列号为GQ153651,有8个开放阅读框(open reading frames,ORFs),基因组结构为:3-N-P-M-F-M2-SH-G-L-5;将hMPVgz01株全基因组核苷酸序列与GenBank上的偏肺病毒全基因组序列进行Clustal W比较,发现与偏肺病毒的A组相似性较高,为92%～97%,与A2b组的BJ1887相似性为最高,而与B组相似性为81%,与C组的禽偏肺病毒为71%.将hMPVgz01株的N、F、G基因与偏肺病毒的A1、A2、B1、B2的相对应基因进行相似性比较,同样也是与A2b型的相似性为最高,因此确认广州地区的hMPVgz01株为A2b型.结论 广州地区儿童感染的偏肺病毒hMPVgz01株全基因组序列为13 327 bp,GenBank 序列号为GQ153651,hMPVgz01与偏肺病毒的A2b型相似性最高,确认hMPVgz01株属于偏肺病毒A2b型.     

2009年肠道病毒71型广州分离株的全基因组序列分析

目的分析2009年肠道病毒71型广州分离株GZHY09的全基因组序列,并与GenBank中的序列进行分析比对。结论从GenBank上选不同地区的EV71全基因组序列,设计相互重叠的覆盖病毒整个基因组序列的12对引物,采用RT-PCR扩增出12个基因片段,通过测序获得全基因组序列,并参考国内外各EV71基因型的分离毒株,用MEGA4软件进行进化树分析,用DNAStar软件中的MegAlign进行同源性分析。结果 12个重叠基因片段序列拼接获得EV71全基因组序列,共7404个核苷酸,同源性分析结果表明在VP1区,GZHY09株与中国大陆的Anhui08、Zhejiang08、Shenzhen08的核苷酸序列同源性比较高,其中以Anhui08最高（97.7%）。结论 GZHY09属EV71病毒的C4亚型,与近年我国大陆地区流行的EV71株在进化上属于同一基因型,与Anhui08、Zhejiang08、Shenzhen08具有高度的同源性。     

7型腺病毒疫苗株76.5～87mu核苷酸序列分析

目的获得７型腺病毒疫苗株（Ａｄ７ｖ）７６５－８７ｍｕ核苷酸序列，分析该区段的基因结构和功能。方法从Ａｄ７疫苗株克隆出６８－８７ｍｕ核苷酸片段，应用Ｓａｎｇｅｒ双脱氧法进行核苷酸序列分析。结果Ａｄ７ｖ７６５～８７ｍｕ核苷酸序列长度为３５５７ｂｐ，推测此片段编码Ｅ３区１２１ｋＤ、１９２ｋＤ、２０１ｋＤ、２０５ｋＤ、１０３ｋＤ和１５２ｋＤ蛋白的一部分。所编码的６个蛋白与Ａｄ７原型株（Ａｄ７ｐ）和Ａｄ７ｈ（１９８６年以来南美流行的毒株）核苷酸高度同源（大于９７２％）。１６１ｋＤ蛋白由于缺失一个核苷酸，造成移码突变，编码提前终止。编码７７ｋＤ蛋白的ＯＲＦ由于缺失一段核苷酸而提前终止编码。结论这一结果为阐明Ａｄ７的基因结构与功能并为构建Ａｄ７载体时对Ｅ３区缺失提供依据     

诺如病毒衣壳蛋白重组人3型腺病毒的构建

目的 制备表达诺如病毒衣壳蛋白的重组人3型腺病毒.方法 将诺如病毒衣壳蛋白基因(Noro-orf2)克隆到腺病毒穿梭载体pBSE3CMV-egfp上,与线性化人3型腺病毒骨架质粒pBRAdv3共电转化感受态大肠杆菌BJ5183,使其在细菌内发生同源重组,带Noro-orf2基因的表达框置换腺病毒E3区,PCR及酶切筛选得到重组腺病毒质粒,将重组腺病毒质粒转染Hep-2细胞进行包装,获得感染性的重组腺病毒粒子,免疫组化分析重组腺病毒中诺如病毒衣壳蛋白的表达.结果 同源重组后经酶切和PCR鉴定证明插入Noro-orf2基因的重组腺病毒质粒pBRAdv3E3dNor成功构建,并经转染包装得到高滴度的重组腺病毒Adv3E3dNor,免疫组化证明诺如病毒衣壳蛋白得到表达.结论 成功构建表达诺如病毒衣壳蛋白的重组3型腺病毒Adv3E3dNor,为研制人3型腺病毒-诺如病毒双价疫苗奠定了基础.     

7型腺病毒疫苗株87mu-97.4mu片段核苷酸序列分析

目的获得７型腺病毒（Ａｄ７）疫苗株８７ｍｕ－９７．４ｍｕ核苷酸序列，分析该区段的基因结构和功能。方法应用Ｓａｎｇｅｒ双脱氧法进行核苷酸序列分析。结果Ａｄ７疫苗株８７ｍｕ－９７．４ｍｕ全长３６９８个核苷酸，推测编码纤维蛋白（３２５个氨基酸）和Ｅ３区１５．４ｋＤ蛋白，Ｅ４区５个蛋白（ＯＲＦ１４．２，ＯＲＦ１５．７，ＯＲＦ８．１，ＯＲＦ４２．８和ＯＲＦ１０．３）。结论这一结果为阐明Ａｄ７的基因结构与功能及利用该病毒做为基因工程疫苗载体打下一定的基础     

广州检出星状病毒4型的全基因组序列测定及分析

目的 探讨广州地区儿童感染的星状病毒基因组分子结构特点和基因型.方法 参考GenBank上的星状病毒基因组设计分段扩增引物,进行RT-PCR分段扩增星状病毒基因组,克隆于T载体上,序列测定,用Clustal W和DNAStar软件分析基因组序列.结果 星状病毒HASTVgz01全基因组为6721 bp,提交到GenBank上的序列号为DQ344027,其中5＇端非编码区（5＇UTR）长82bp,3＇端非编码区末端长81 bp,病毒基因组编码区全长6558个核苷酸,分别编码ORF1a、ORF1b、ORF2,ORF1a基因长2763 bp（83～2845 nt）,ORF1b基因长1557bp（2785～4332 nt）,其中ORF1a、ORF1b两基因有71个核苷酸的重叠区;ORF2全长2316 nt,位于基因组中4325～6640 nt.ASTVgz01与GenBank中8种基因型星状病毒的ORF2基因氨基酸序列同源性比较发现,HASTVgz01与4型的同源性为93%,其他的同源性在61%～70%之间.结论 广州地区儿童腹泻感染的星状病毒HASTVgz01全基因组为6721bp,GenBank序列号为DQ344027,HASTVgz01与4型星状病毒的ORF2基因氨基酸序列比较同源性为最高,确认HASTVgz01是4型星状病毒.     

诺如病毒广州株全基因组序列测定及分析

目的 探讨广州地区儿童感染的诺如病毒基因组分子结构特点和基因组类型。方法 参考GenBank上的诺如病毒MD145-12基因组设计分段扩增引物，进行RT-PCR分段扩增诺如病毒基因组，克隆于T载体上，测定序列，用Clustal W／X、DNAStar、RAT（recombination analysis tool）等软件分析基因组序列。结果 诺如病毒NVgz01全基因组为7558bp，提交到C,enBank上的序列号为DQ369797，3’端非编码区末端长45bp，病毒基因组有3个开放阅读框（ORF），ORF1长5100bp（5～5104nt），ORF2基因长1623bp，位于基因组中5085～6707nt之间，ORF3基因长807bp（6707～7513nt），其中ORF1、ORF2两基因有19个核苷酸的重叠区；NVgz01全基因组核苷酸序列与GenBank上诺如GI组Norwalk68、Southampto、BS5、Chiba、WUG1的同源相似性在43％～44％之间，与GⅡ组MD145、Farmington　Hills、B4S5、Hawaii、Lordsdale、SaitamaU1、Mc37同源相似性在76％～90％之间。NVgz01的ORF1与Mc37核苷酸序列同源性为94％，但ORF2与Mc37的同源性只有65％；NVgz01的ORF2与Fannillgton Hills（AY502023）同源性为最高（94％），ORF1的同源性为88％。结论广州地区儿童腹泻感染的诺如病毒NVgz01株全基因组序列为7558bp，GenBank序列号为DQ369797，NVgz01与诺如病毒的GⅡ组同源性较高，确认NVgz01株属于诺如病毒GⅡ组；根据ORF1、ORF2的同源性差异和RAT程序分析初步认为NVgz01可能是重组病毒。     

2014年湖南省C亚属人腺病毒1株临床分离株的全基因组特征分析

目的:了解2014年湖南省C亚属人腺病毒(HAdV-C)分离株的基因特征。方法:2014年从湖南省长沙市严重急性呼吸道感染儿童病例咽拭子标本中分离到一株HAdV-C病毒株(Hunan2014-s024),分段扩增目的基因片段,拼接后获得全基因组序列,同时下载GenBank数据库中国内外流行的HAdV-C代表株全基因组序...     

人乳头瘤病毒16型E5序列进化分析

目的 通过HPV 16 E5序列进化分析,初步研究中日友好医院妇产科宫颈病变诊治中心无宫颈病变的汉族妇女HPV 16型内变异株类型.方法 用PCR法扩增HPV 16 E5基因片段,并进行比对分析测序.比对后的结果结合临床资料进行分析.结果 本研究第一次利用只有236 bp碱基的E5序列进行HPV 16变异株的进化分析,发现单独使用E5序列进行HPV 16变异株进化分析的准确率很高,并且,本研究第一次发现以4075T即可将亚洲株(As)变异株和其他变异株区分开来.结论 从E5基因序列出发进行进化分析,准确率高.单独以4075T即可将As变异株和其他变异株区分开来.     

人副流感病毒广州分离株ZYMgz01全基因组序列测定及分析

目的探讨广州地区副流感病毒的基因组结构和基因型。方法参照GenBank副流感病毒（U51116）全基因组序列设计11对引物,覆盖副流感病毒基因组的全长。以副流感病毒cDNA为模板分段扩增病毒基因组的全长,各片段克隆到T载体上,并进行序列测定和分析。结果人副流感病毒ZYMgz01株全基因组核酸序列为15462bp,提交到GenBank的序列号为EU326526,与3型副流感病毒保守基因同源性为94.0%,具有3型副流感病毒的结构特征。基因组序列同源性比较结果显示,ZHYMgz01株病毒与兰州的LZ22（FJ455842）病毒株的同源性最高,为99.1%;与美国突变病毒株（U51116）同源性为95.1%;与美国病毒株（Z11575）同源性为95.2%;与日本（ABO12132）病毒株的同源性为94.8%,而与加拿大（EU424062）病毒株的同源性为94.8%。系统进化树显示ZHYMgz01病毒株与兰州的LZ22病毒株同属一个进化分支。结论广州地区儿童呼吸道感染的副流感病毒ZHYMgz01全基因组为15462bp,与3型副流感病毒的同源性最高,确定ZHYMgz01是3型副流感病毒。3型副流感病毒系统进化可能与地域差异有一定的关系。     

Phylogenetic analysis two strains of human of the genomes of adenovirus type 3

Human adenovirus type 3 （HAdV-3） is widely prevalent all over the world, especially in Asia. The objective of this study is to carry out complete genomic DNA sequencing and the phylogenetic analysis for two strains （Guangzhou01 and Guangzhou02） of HAdV-3 wild virus isolated from South China. Nasopharyngeal secretion aspirate specimens of sick children were inoculated into HEp-2 and HeLa culture tubes, and the cultures were identified by neutralization assay with type-specific reference rabbit antiserum. Type-specific primers were also utilized to confirm the serotype. The restriction fragments of HAdV genome DNA were cloned into pBlueScript SK （ ＋ ） vectors and sequenced, and the 5＇ and 3＇ ends of the linear HAdV-3 genome were directly sequenced with double purified genomic DNA as templates. General features of the HAdV-3 genome sequences were explored by using several bio-software. Phylogenetic analysis was done with MEGA 3.0 software. The genomic sequences of Guangzhou01 and Guangzhou02 possess the same 4 early regions and 5 late regions and have 39 coding sequences and two RNA coding sequences. Other non-coding regions are conservative. Inverted repeats and palindromes were identified in the genome sequences. The genomes of group B human adenovirus as well as HAdV-3 have close phylogenetic relationship with that of chimpanzee adenovirus type 21. The genomic lengths of these two isolated strains are 35 273 bp and 35 269 bp, respectively. The phylogenetic analysis showed that HAdV-B species has some relationship with certain types of chimpanzee adenovirus.     

Epitope mapping and characterization of a neutralizing monoclonal antibody against human adenovirus type 3

Human adenovirus serotype 3 (HAdV-3) has occurred as a global epidemic in recent years causing serious diseases such as pneumonia in pediatric and adult patients. Development of reliable diagnostic reagents and identification of neutralizing epitopes is important for the surveillance and control of infection. In this study, a neutralizing monoclonal antibody (MAb) MAb 1B6 was generated using the HAdV-3 virion. MAb 1B6 specially recognized the HAdV-3 virus particles and the HAdV-3 hexon protein, but not the virus particles or the hexon protein of HAdV-7 and HAdV-4 by western-blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). Analysis using a series of peptides from the hexon protein and chimeric adenovirus (Ad) particles of epitope mutants revealed that MAb 1B6 bound to the exposed region (amino acid positions 414–424 of hexon) in hypervariable region 7 (HVR7). ELISA demonstrated that MAb 1B6 could recognize the corresponding regions of other HAdV-3 genotypes that have some residues substituted. The identification of the neutralizing epitope and the generation of MAb 1B6 may be useful for clinical serotype-specific diagnosis, subunit vaccine construction for HAdV-3 infection, and virion structural analysis.     

广州地区人腺病毒5型血清中和抗体阳性率初步调查

目的评估广州地区人群中人腺病毒5型（HAdV-5）中和抗体阳性率情况。方法采用以β-半乳糖苷酶（LacZ）为报告基因,结合CMV启动子的人重组腺病毒5型载体,运用化学发光法,检测209份免疫功能正常的成人血清样本中HAdV-5的中和抗体。结果 HAdV-5中和抗体的阳性率为82.3%（172/209）。HAdV-5中和抗体在〈1：20（低滴度）、1：40-1：160（中滴度）、1：320-1：1280（高滴度）、〉1：1280（超高滴度）时均能检测到,而且随着滴度的增加,阳性率逐渐升高。在20～40岁时HAdV-5中和抗体阳性率最高,在〈20岁时HAdV-5阳性率最低。结论广州地区人群中针对HAdV-5的先存中和抗体阳性率高,应用基于该种DNA病毒作为载体进行基因疫苗及基因治疗的研究时,具有一定的局限性。     

The complete genomic sequence analysis of genotype 4 human astrovirus HASTVgz01 strain in Guangzhou

To analyze the genomic molecular structure and genotype of human astrovirus isolated from infant in Guangzhou of China, the primers were designed based on the genomic sequence of astrovirus from the GenBank and the target sequence were amplified by RT-PCR. Then the PCR-products were cloned to T vector and sequenced. The genomic nucleotide sequences were analyzed by the programs CLUSTAL W and DNASTAR. It was found that the full genomic length of HASTVgz01 strain was 6721 bp and the ORFs were 6558 bp. The 5' and 3' UTR were 82 and 81 nucleotides. The genome included 3 open reading frames (ORFs): ORF1a, ORF1b and ORF2. The 5'-terminal ORF1a started at nucleotide 83 and extended to nucleotide 2845. ORFlb (nt 2785 to nt 4332) overlaped ORFla by 61 nucleotides. The 3'-terminal ORF2 began at nucleotide 4325 and terminated at nucleotide 6640. ORF2 had 2316 nucleotides. Compared with other astrovirus sequences in GenBank, the homology of the amino acid sequence of ORF2 of HASTVgz01 strain with that of serotype 4 was 93% . Homology with other serotypes ranged from 61% to 70% . The complete nucleotide sequence of astrovirus HASTVgz01 strain isolated from Guangzhou in China was 6721 bp in length, GenBank accession NO. DQ344027. Comparing the ORF2 of astrovirus HASTVgz01 with the known sequences of types 1-8 the highest homology was serotype 4 (93%). Comparative sequence analysis of the HASTVgz01 ORF2 with the reported human astrovirus sequences revealed that the isolated astrovirus belongs to genotype (serotype) 4.     

Phylogenetic analysis of human coronavirus NL63 circulating in Italy

BACKGROUND: Five known human coronaviruses infect the human respiratory tract: HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63 and HCoV-HKU1. OBJECTIVES: To evaluate the prevalence of HCoV-NL63 in hospitalized adult patients and to perform molecular characterization of Italian strains. STUDY DESIGN: HCoV-NL63 was sought by RT-PCR in 510 consecutive lower respiratory tract (LRT) samples, collected from 433 Central-Southern Italy patients over a 1-year period. Phylogenetic analysis was performed by partial sequencing of S and ORF1a. Additional S sequences from Northern Italy were included in the phylogenetic trees. RESULTS: HCoV-NL63 was detected in 10 patients (2.0%) with symptomatic respiratory diseases, mainly during winter. Phylogenetic analysis indicated a certain degree of heterogeneity in Italian isolates. The ORF1a gene clustering in phylogenetic trees did not match with that of the S gene. CONCLUSIONS: As observed by others, HCoV-NL63 is often associated with another virus. Phylogenetic characterization of HCoV-NL63 circulating in Italy indicates that this virus circulates as a mixture of variant strains, as observed in other countries.     

Computational analysis of human adenovirus serotype 18

The genome of the sole remaining unsequenced member of species A, human adenovirus type 18 (HAdV-A18), has been sequenced and analyzed. Members of species A are implicated as gastrointestinal pathogens and were shown to be tumorigenic in rodents. These whole genome and in silico proteome data are important as references for reexamining and integrating earlier work and observations based on lower resolution techniques, such as restriction enzyme digestion patterns, particularly for hypotheses based on pre-genomics data. Additionally, the genome of HAdV-A18 will also serve as reference for current studies examining the molecular evolution and origins of human and simian adenoviruses, particularly genome recombination studies. Applications of this virus as a potential vector for gene delivery protocols may be practical as data accumulate on this and other adenovirus genomes.     

人腺病毒3型感染对H9c2细胞microRNA表达谱的影响

目的研究腺病毒感染对大鼠心肌来源的H9c2细胞microRNA表达谱的影响。方法用人腺病毒3型（Ad3）攻击成层和未成层的H9c2细胞,6h后收获总RNA,基因芯片检测microRNA表达谱。结果比较未成层与成层的H9c2细胞microRNA的表达谱,H9c2细胞成层后有54种microRNA表达显著下调,7种microRNA表达显著上调。与未感染病毒的细胞比较,Ad3感染的未成层H9c2细胞有41种microRNA表达显著下调,12种microRNA表达显著上调。与未感染病毒的细胞比较,Ad3感染成层H9c2细胞后有8种microRNA的表达显著下调,62种microRNA的表达显著上调。结论细胞不同的生长状态及Ad3感染显著改变了H9c2细胞的microRNA表达谱。