单链构象多态性分析研究HCV NS5A区基因变异

用逆转录、套式不对称聚合酶链反应（ＰＣＲ）制备单链ＤＮＡ，经凝胶电泳，Ｓｏｕｔｈｅｒｎ转移后与地戈辛标记探针杂交。根据单链ＤＮＡ条带数目对５２例慢性肝病患者血清进行ＨＣＶ ＮＳ５Ａ区基因变异程度分析，结果９０％的慢迁肝、３５％的慢活肝、１６％的肝硬化与１０％的肝癌患者出现１条单链ＤＮＡ带，２０％的慢活肝、４２％的肝硬化、６０％的肝癌患者呈现多条单链ＤＮＡ带慢迁肝则未见多条单链ＤＮＡ带。临床参数分析     

Selection and application of serotypical synthetic peptides derived from hepatitis C virus NS5A region

Background   Numerous studies have reported a relationship between hepatitis C virus (HCV) genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay (ELISA) method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a (NS5A) region was developed.
Methods  Based on 45 sequences, representing HCV genotypes 1-6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182-2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1-6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis.
Results  The correspondence of amino acids in HCV NS5A region with amino acids in positions 2182-2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182-2211(R1), 2272-2301 (R7) and 2302-2331 (R9): the highly variable 2212-2241 (R3) and 2257-2286 (R6). Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera.  Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis.   
Conclusions  The major antigenic regions in HCV NS5A region were at 2212-2241(R3), 2272-2301(R7) and 2302-2331(R9).  Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.

     

丙型肝炎病毒5’非编码区PCR产物单链构象多态性分析及序列测定

目的：研究慢性肝炎病人血清、肝细胞肝癌组织中丙型肝炎病毒（ＨＣＶ）５＇非编码区变异情况．方法：收集１０例慢性肝炎病人及４例肝细胞肝癌病人血清和肝细胞肝癌组织，检测抗ＨＣＶ抗体阳性，进行ＨＣＶ５＇非编码区ＲＮＡ的逆转录ＰＣＲ扩增，ＰＣＲ产物进行单链构象多态性分析，并对部分病例序列进行测定．结果：６例慢性肝炎病人血清及３例肝细胞肝癌组织中检测到ＨＣＶＲＮＡ，单链构象多态性分析显示各例条带之间无明显差异，对１例肝细胞肝癌组织中ＨＣＶ５＇非编码区ｃＤＮＡＰＣＲ产物的序列测定表明它与ＨＣＶ－１的同源性为９７．３％，与ＨＣＶ－ＢＫ的同源性为９７．７％．结论：我们的结果提示在西安地区ＨＣＶ５＇非编码区几乎是一样的，并进一步证实不同地区ＨＣＶ５＇非编码区的高度保守性．     

丙型肝炎病毒2a型E2区基因cDNA的变异分析

目的 了解丙型肝炎病毒2a型基因组E2区序列的一级结构及变异特征，为进一步研究HCV包膜蛋白的生物学功能打下基础。方法 用逆转录套式多聚酶链反应(RT-nPR)法从1例NANB型肝炎患者血清中扩增出一条约1100bp的片段，以限制性内切酶EcoRI、HindⅢ双酶切后连入pUC19载体中，转化感受态细胞，经限制性内切酶长度多态性分析(RFLP)和PCR法证实为阳性克隆后，用全自动序列分析仪测序。结果 测得约1100bp的核苷酸序列，其核苷酸序列及其编码的氨基酸序列分别与HCV-1、HC-C2、HCV-BK、HC-J6、HC-J8相应序列作比较，显示分离株HCV在核苷酸水平上与以上分离株的同源性分别为63．1％、64．2％、64．1％、86．9％、69．3％，在氨基酸水平上的同源性分别为69．6％、70．7％、69．6％、86．2％、83．1％。结论 分离株(HCV-JF)与HC-J6同属2a型。     

白细胞介素12B基因多态性与丙型肝炎病毒感染相关性的Meta分析

目的 探讨白细胞介素12B（interleukin 12B,IL-12B）基因3＇UTR A＞C多态性与丙型肝炎病毒（hepatitis C virus,HCV）感染的相关性.方法 计算机检索PubMed、Cochrane Library、CBM、万方、知网、维普等数据库,收集1988年11月-2014年4月关于IL-12B基因3＇UTR A＞C多态性与HCV感染相关性的病例对照研究.由两名评价者独立评价纳入研究的质量并提取资料,并用Stata12.0软件进行Meta分析.结果 6项病例对照研究符合纳入标准,包括1 396例患者和1 254例对照者.Meta分析结果显示,人群中携带3＇UTR C等位基因者发生HCV感染的风险是非携带者的1.12倍（OR=1.12,95％ CI：0.90 ～ 1.39）,差异无统计学意义（Z=0.10,P＞0.05）.基于种族的亚组分析显示,亚裔人群中携带3＆#39;UTR C等位基因者发生HCV感染的风险是非携带者的1.04倍（OR=1.04,95％ CI：0.67～1.61）,差异无统计学意义（Z=0.18,P＞0.05）;非亚裔人群中亦未发现相关性（Z=1.56,P＞0.05）.结论 基于目前研究结果显示,IL-12B基因3＇UTR A＞C多态性与HCV感染风险无明显相关性.     

RFLP基因分型在动态监测乙型肝炎病毒基因变异中的应用

目的探讨基因分型结合克隆后测序方法在动态监测ＨＢＶ基因变异中的价值。方法分析１例重型肝炎患者发 病后血清,用 ＰＣＲ扩增血清中 ＨＢＶ ｐｒｅＳ／Ｓ基因片段并克隆,２份标本各随机挑取 ３个阳性克隆进行测序;同时用 ＲＦＬＰ方法分析２份血清中ＨＢＶ毒株的基因型。结果ＰＦＬＰ分析２份血清为Ｂ基因型为主的Ｂ／Ｃ基因型混合毒株感 染;６个克隆中仅１株为Ｃ基因型。比较２份血清ＨＢＶ ｐｒｅＳ／Ｓ克隆核酸序列,有１７０个点位的差异,剔除Ｃ基因型株 后,仅５４个位点差异。结论克隆后测序结合ＲＦＬＰ对ＨＢＶ基因型测定有助于更准确地对ＨＢＶ基因变异进行动态分 析。     

丙型肝炎病毒NS5A基因真核表达载体的构建

目的：构建HCV NS5A/pCI-neo真核表达载体,转染Huh-7细胞系,为体外高效表达NS5A蛋白奠定基础。方法：用PCR方法从带有丙型肝炎病毒NS5A基因的pC DNA 3.1（+）/HCV NS345质粒中扩增出HCV NS5A基因,然后TA克隆于pGEM-T载体中,转化大肠杆菌JM109。阳性克隆经测序鉴定,再经双酶切消化后插入真核表达载体pCI-neo上,经酶切鉴定与测序证实。结果：用PCR方法扩增出HCV NS5A基因全序列,因其 cDNA的G+C含量高直接测序未成功,后经TA克隆,筛选得到的阳性克隆经测序鉴定,与设计的HCV NS5A基因序列完全一致。经酶切连接并成功插入pCI-neo上。结论：成功构建了高效表达丙型肝炎病毒NS5A基因的pCI-neo真核表达载体。     

人乙型肝炎病毒包膜蛋白多态性的生物信息学分析及其意义

目的 构建乙肝病毒生物数据库（Bio-HBV Database）,针对HBV包膜蛋白序列进行多态性分析。方法 构建Bio-HBV生物数据库获得国际基因序列库中所有完整的包膜蛋白并进行比对,采用信息熵评价序列位点的保守性,结合BLOSUM90评分系统和PAML（phylogenetic analyses by maximum likelihood）软件包寻找选择压力下的异常氨基酸替换模式。结果包膜蛋白中ps35,ps132,s49和s127等氨基酸替换具有高度的统计学意义。此外包膜蛋白内有多个重要区段,直接影响HBV生物学功能。我们对这些区段逐一分析了功能与结构的关系。结论 通过Bio-HBV生物数据库,用生物信息学方法不仅验证了已知生物学特性的功能域的保守性,更提出了潜在的可以作为研究切入点的新功能域。     

丙型肝炎病毒结构区基因变异的研究

目的：探讨丙型肝炎病毒（HCV）结构区主变异在HCV感染慢性化中的作用。方法：采用逆转录－聚合酶链反应（RT－PCR）扩增HCV的C、E1及E2／NS1区核苷酸片断（1kb）,扩增产物进行克隆,以单链构象多态性（SSCP）／异质性双体分析（HDA）方法对每份血清中30个克隆的C／E1和E2／NS1区准种（Quasispecies）进行优化筛选,分析HCV急性感染者和持续性感染者血清种复杂性。结果：HCV的C／E1区和E2／NS1区增片段形成的SSCP条带间差异明显,但E2区形成的异质性双体与同源双体之间差距准种复杂性的增加与病毒持续性感染有关。     

母乳喂养与婴儿HCV感染关系的META分析

目的:探讨母乳喂养与婴儿HCV感染的关系.方法:为了获取适于META分析的文献,对中国生物医学数据库、维普数据库、方正数据库、PUBMED数据库和MEDLINE进行检索. 纳入标准依据Abdolmaleky HM方法[1]. 用RevMan4.2软件对纳入文献采用固定效应模型或随机效应模型计算OR值. χ2检验每组OR值异质性. 联系的强度采用OR值进行评价.结果:对中国生物医学数据库和MEDLINE进行检索后共有120篇文献,37篇为综述,被舍弃. 对其余文献进行仔细阅读后,只有6篇文献符合本次研究标准. META分析OR值为0.6 (95%CI=0.22-1.60),证实母乳喂养与婴儿HCV感染无关.结论:母乳喂养不是婴儿感染HCV的危险因素.     

乙型肝炎病毒C基因多态性及其蛋白特性分析

目的:调查温州医学院大学生人群乙型肝炎病毒(HBV)C基因多态性及其HBc蛋白特性变化,为了解乙肝病毒核心抗原(HBcAg)变异提供理论依据。方法:采用HBV-C基因特异性引物对21份温州医学院大学生HBV携带者(血清学检测为大三阳患者)血清标本进行PCR扩增鉴定,并取19份PCR检测阳性产物直接测序;进一步用Vector NTI 8.0和DNA Star软件分析HBV-C基因多态性及HBc蛋白特性的变化。结果:21份标本中,HBV-C DNA阳性率达90.48%(19/21),与标准基因序列(Gen Bank X01587)比较,HBV-C基因同源性为93.3%～95.1%。HBV-C基因分别形成51种突变模式,其中一处由于核苷酸的改变,翻译的氨基酸也发生了相应的改变,此处氨基酸序列的第130处脯氨酸(Pro)突变为苏氨酸(Thr),其余标本均为同义突变;但突变处编码蛋白在亲水性、表面可及性及抗原性与标准株相比较无明显变化。结论:温州医学院大学生HBV携带者的HBV C基因具有多态性,但是由C基因决定的核心蛋白序列相对保守。     

Trans-cleaving activity of hepatitis delta virus genomic ribozymes

It is ~rted lhat hepatitis delta virus (HDV)genondc HNA can self-cleave intramolecularly in the sitebetween Ugh, and G@, ~rted by Makino et .ill]. ThisPIDCess is catalyZed by HDV genondc RNA itself whichbelongs to the fthely of catalytic RNAs named riboZymes.The intramolecular self-cleaVage is also called ets-cleavage, and the self-cleaving HDV genondc riboZyme(g. Rz)is defined as ets-g. Rz. SOme reseaxches have shown lhatfoe genondc riboZyme which possesses the shortest natoalseq…     

丙型肝炎病毒NS5B在Hep3B细胞的表达及活性分析

目的丙型肝炎病毒NS5B基因转染人肝癌细胞系Hep3B细胞,检测NS5B的表达与细胞内RNA聚合酶活性。方法使用脂质体把包含全长丙型肝炎病毒NS5B基因的重组载体pcDNA-5B转染到Hep3B细胞。RT-PCR和Wes-tern blot鉴定NS5B基因的表达,荧光素酶检测NS5B的细胞内RNA依赖的RNA聚合酶活性。结果RT-PCR和Westernblot发现转染NS5B基因的Hep3B细胞产生NS5B mRNA和NS5B蛋白,荧光素酶分析表明Hep3B细胞表达的NS5B蛋白具有细胞内RNA依赖的RNA聚合酶活性。结论人肝癌细胞系Hep3B细胞可以成功表达NS5B基因,且表达的NS5B蛋白具备细胞内RNA依赖的RNA聚合酶活性。     

趋化因子CXC家族基因多态性与丙型肝炎抗病毒治疗效果的相关性分析

目的: 探索趋化因子CXC家族基因多态性与慢性丙型肝炎治疗效果的关系,以便在治疗过程及时干预和提高疗效。方法: 对确诊的282例慢性丙型肝炎患者采用聚乙二醇干扰素α联合利巴韦林抗病毒治疗48周并随访24周,观察持续病毒学应答（sustained virological response,SVR）结果,运用TaqMan MGB荧光探针PCR法对所有患者进行基因分型,采用Logistic回归分析SVR的影响因素。结果： 治疗48周后,180例 (63.83%)患者达到SVR。多因素Logistic回归分析结果表明,促甲状腺激素水平高（OR=1.22,95% CI=1.04～1.42）和空腹血糖浓度低（OR=0.85,95% CI=0.72～0.99）有利于提高SVR率;而基线HCV RNA高（OR=0.72, 95%,CI=0.56～0.99）不利于提高SVR率。但并未发现趋化因子CXCL9 rs10336（相加模型: OR=0.97,95% CI=0.40～2.33）、rs3733236（相加模型: OR=0.89, 95% CI = 0.43～1.87）和CXCL12 rs1801157（相加模型: OR=1.31, 95% CI=0.80～1.86）与SVR存在显著关联。 结论： 未发现丙肝患者抗病毒治疗效果与趋化因子CXC家族基因多态性存在显著相关,但基线HCV RNA、血清促甲状腺激素和空腹血糖是影响慢性丙肝患者SVR的独立影响因素。     

丙肝病毒核心抗原检测在丙肝病毒感染诊断中的作用研究

目的探讨丙肝核心抗原检测在丙肝感染诊断中的作用。方法收集175例丙肝抗体检测标本同时检测HCVAb、HCV游离抗原、HCV总抗原、HCVRNA。结果 75例HCVAb阳性反应标本,检出HCV游离抗原阳性17例,HCV总抗原阳性54例,HCVRNA阳性50例;100例HCVAb阴性反应标本,检出HCV游离抗原阳性1例,HCV总抗原阳性2例,HCVRNA阳性1例。结论 HCV核心抗原是HCV早期感染的标志之一,检测HCV核心抗原有利于HCV感染的早期诊断。     

丙型肝炎病毒C区的DNA改组

目的：利用DNA改组技术进行不同基因型别丙型肝炎病毒（HCV）基因组C区的人工进化。方法：首先利用PCR扩增了三段具有较高序列同源性的460bp基因片段，然后将其等量混合，在Mg^2＋存在的条件下，用脱氧核糖核酸酶（DNase Ⅰ）切割成50bp左右的小片段。这些小片段在不外加引物的条件下，利用PCR反应进行重聚，再将重聚物经过一轮正常的PCR扩增。结果：获得了与原片段大小相当的基因片段。结论：这一技术为进一步筛选高活性的HCVC区基因打下基础，有利于从一组序列同源性程度较高的基因库构建随机嵌合基因，并为改组其他基因家族提供了借鉴。     

Detection of hepatitis G virus RNA in hepatitis C patients and hepatitis C virus infected hemodialysis patients

AlthoughsensitiveandspecificimmunoassayandmolecularbiologicaltechniquesforthedetectionofthehepatitisA--Evirusesareavailable,theetiologyofasubstantialfractionofpost--transfusionandcommunity--acquiredhepatitiscasesremainsundefined[1],suggestingtheexistenceofadditionalcausativeagents.Anewhumanhepatitisviruswasisolatedbytwoindependentgroups.ThenewvirusisprovisionallydesignatedashepatitisGvirus(HGV)[ZJorGBvirusC(GBV~C)[3'4),whichwasthoughttobetheagentofpartofthenon--A--Ehepatitispatients.Fro…     

HIV-1感染者合并HBV、HCV感染情况分析

目的 分析人类免疫缺陷病毒（HIV）感染者合并乙型肝炎病毒(HBV)或/和丙型肝炎病毒(HCV）感染的情况。方法 对260例HIV感染者的HBV或/和HCV合并感染情况进行回顾性分析,对HIV单独感染者、HIV/HBV合并感染者、HIV/HCV合并感染者及HIV/HBV/HCV合并感染者的CD4+T细胞数、CD4+/CD8+细胞比值及HIV-RNA载量水平进行比较分析。结果 在260例HIV感染者中,60例为HIV/HCV合并感染（23.1%）,29例为HIV/HBV合并感染（11.2%）,8例为HIV /HBV/HCV合并感染（3.1%）;男性的HIV/HCV合并感染率显著高于女性 （P<0.01）; HIV-1/HBV和HIV-1/HCV合并感染者的CD4+/CD8+ 细胞比值均显著低于HIV单独感染者（P<0.05）;HIV-1/HCV合并感染者的CD4+细胞数显著低于HIV单独感染者（P<0.05）;三组合并感染者的HIV-RNA载量与HIV单独感染者之间差异不显著。结论 HIV/HCV合并感染率显著高于HIV/HBV合并感染率;HIV感染者合并HBV、HCV感染可能进一步加重HIV/AIDS患者的细胞免疫功能损伤,以HIV/HCV合并感染尤甚。     

Prevalence of human immunodeficiency virus,hepatitis C virus,and hepatitis B virus infection among heroin injectors in the central region of Saudi Arabia

Objectives:

To calculate the prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infections among inpatient heroin users, and to study the relationships between these infections and patient demographics.

Methods:

In this retrospective study, heroin users’ inpatient records from the Alamal Hospital, Riyadh, Saudi Arabia were reviewed for HIV, HBV, and HCV screening results, age, number of admissions, education, and marital and occupational status. The study took place between January 2006 and November 2012. The prevalences of HIV, HBV, and HCV and their associations to demographics were evaluated.

Results:

A sample of 357 inpatients Saudi male heroin users (all injectors) aged 40 (±8.6) years with lifetime admissions averaged 5.8 (±5) times were studied. Screening results revealed that 20.1% of subjects were infection-free, 56.6% had a single infection, 13.2% were infected by 2 viruses, and 1.1% were infected by 3 viruses. Prevalence of HBV surface antigen was 7.7%, antibodies for HCV 77.8%, and HIV 9.8%. A significant association was found between positive HCV and positive HIV tests. Furthermore, HCV was more common among patients aged 20-29 years, those who were unemployed, and who had primary, secondary, or postsecondary education. Finally, HBV was associated in patients aged 30-39 years and those with secondary educations.

Conclusion:

Contracting serious contagious viral infections is very common among Saudi heroin injectors at rates similar to those seen among injectors in Western countries. Infection control, education, and harm reduction programs are of paramount importance.Substance users are more vulnerable to human immunodeficiency virus (HIV) and viral hepatitis than the general population.1-3 Specifically, those who inject drugs are at a higher risk for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections.1 Considering these viruses are blood borne infections, they are transmitted efficiently through sharing of contaminated needles and other injection paraphernalia.4-7 In the United States, approximately 9-12% of new HIV cases8,9 and 50% of new HCV cases10 are associated with illicit injection of drugs. The prevalence of HCV among injecting drug users in the industrialized world is greater than 60%.1-5 In Saudi Arabia, very few studies addressed HIV, HBV, and HCV infections in Saudi heroin users who inject drugs. Njoh and Zimm11 described more than 2000 drug users admitted to an addiction treatment center in Jeddah between January 1995 and May 1996 and found a 0.1% prevalence of HIV. In 1996,12 they found a prevalence of HCV seropositivity of 74.6%. In 1995, at the same treatment center, the HBV surface antigen was positive in 12.6% of 349 screened patients, 80% of whom injected drugs.13 Another screening of 344 Saudi injecting drug users in the eastern region, reported a 38% HCV RNA detection rate, and a 12% HBV DNA detection rates.14 No studies were performed to estimate the prevalence of these viral infections in the central region, and those performed in Jeddah (western Saudi Arabia) are old and may not correctly reflect the current status. In this study, we aim to estimate the prevalences of HBV, HCV, and HIV infections among heroin injectors in the Saudi central region and to explore any significant relationships related to patient demographics.