乙型肝炎病毒前C/C区基因变异检测的临床意义

目的:研究乙型肝炎病毒(HBV)前C?C区基因的一级结构及其变异特点。探讨前C区1896位基因突变与血清标志物,肝功能损害的关系。方法:收集慢性乙型病毒性肝炎患者血清388份,进行前C/C区基因测序,HBV标志物、HBV-DNA、ALT、AST测定。结果:HBVA1896突变毒株158例,A1899突变毒株57例。C区变异大部分集中在第5、27、60位氨基酸的序列中。结论:A1896突变与HBeAg分泌障碍有关。A1896突变可加重肝脏损害。     

乙型肝炎病毒前C区1896位点变异与肝细胞损伤的关系

慢性乙型肝炎病毒(HBV)感染患者出现HBeAg转阴是病毒复制减弱和病情好转的标志,我们认为这种观点不够全面.因为,随着抗病毒药物的治疗,乙型肝炎病毒基因位点极易发生变异,其中HBV前C区1896位点(G1896A)变异后形成终止密码是HBeAg阴性乙型肝炎的重要机理,我们对非母婴垂直传播(水平感染)感染HBV的慢性乙型肝炎患者127例的血清联检肝功能酶学指标:丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、胆碱脂酶(CHE),肝纤维化指标:透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原(PⅢ)、Ⅳ前胶原(Ⅳ.C),以及G1896A位点变异.分组比较前C区1896位点变异与肝细胞损伤的关系.     

PCR-RFLP检测拉米夫定抗HBV感染中聚合酶YMDD变异

目的 建立用PCR－限制性片段长度多态性技术（PCR－RFLP），分析拉米夫定抗HBV感染中聚合酶YMDD变异方法。方法 在拉米夫定抗HBV治疗过程中HBV DNA由阴性再次转为阳性患者及HBV DNA始终保持阳性的血清达1年或1年以上，用3对引物扩增HBV P基因的C区，扩增产物分别用3个限制性内切酶分析，酶切结果用8.4%聚丙烯酰胺凝胶电泳分析。检测HBV YMDD变异，同时血清用全自动测序仪检测YMDD变异。分析比较两者结果。结果 35例病人，包括33例HBV DNA再次阳性患者，2例用药1年HBV DNA未转阴。14例病人出现YMDD变异。PCR－RFLP结果为6例YVDD变异、4例YIDD、1例YI／MDD、21例YMDD与测序一致。另3例PCR－RFLP结果为YI／VDD，即混合变异，测序报告为2例YIDD、1例YVDD，分析相应的测序图，存在混合变异。并把YIDD与YVDD变异的血清混合，行PCR－RFLP，结果与YI／VDD一致。结论 用PCR－RFLP能快速、简便、灵敏地检测YMDD变异。     

HBV DNA的前C区1896 G/A变异与血清HBV DNA水平

为探讨乙型肝炎病毒（HBV）前C区1896G／A变异对血清HBV DNA水平复制及临床表现的影响，本研究报道采用酶联免疫吸附试验（EHSA）和荧光定量PCR方法（FQ-PCR）及突变特异引物PCR方法（AS-PCR）对95例诊断符合2000年修订的病毒性肝炎防治方案的HBV感染者进行了HBV血清免疫标志物和HBV DNA定量及HBV DNA1896位点G／A变异检测的结果。     

HBV感染孕妇体内病毒前C区G1896A变异与母婴垂直传播的关系

目的 分析孕妇体内乙型肝炎病毒(HBV)前C区G1896A变异对母婴垂直传播的影响.方法 收集40例本院妇产科HBeAg(-)/HBsAg( )的孕妇血标本,同时收集这些孕妇分娩时新生儿脐带血40例.荧光定量PCR(FQ-PCR)检测HBV孕妇血清和新生儿脐带血中HBV-DNA栽量,PCR-ELISA法检测孕妇血清中HBV-DNA前C区G1896A变异.分析HBV前C区G1896A变异及孕妇血清HBV-DNA载量对母婴垂直传播率的影响.结果 40例孕妇血清共检测到25例HBV前C区G1896A变异(62.5%);变异组母婴垂直传播发生率为44.0%(11/25),未变异组母婴垂直传播发生率为40.0%(6/15),二组比较无统计学差异(x2=0.0614,P＞0.05).孕妇血中HBV-DNA高载量组(≥1×105 copies/ml)母婴垂直传播发生率为62.5%(10/16),低栽量组(＜1 × 105 copies/ml)母婴垂直传播发生率为29.2%(7/24),二组相比有显著性差异(x2=4.3649,P＜0.05).结论 HBV前C区G1896A变异未增加母婴HBV垂直传播率,孕妇血清中HBV-DNA栽量升高是母婴HBV垂直传播危险因素.     

江西地区儿童感染的乙型肝炎病毒"a"抗原变异分析

目的:初步分析江西省乙型肝炎疫苗免疫儿童感染的乙型肝炎病毒(Hepatitis B Virus,HBV)"a"抗原决定簇的变异。方法:收集在江西省儿童医院就医的13 117名乙肝疫苗免疫儿童(7.39±3.66岁)血清标本,酶联免疫吸附方法检测HBV-M,抽提HBV表面抗原(HBsAg)阳性血清标本中的HBV DNA,扩增HBV S基因,PCR产物测序并与标准序列对比,分析"a"抗原决定簇的变异与血清型;利用在线Genotyping软件对儿童感染的HBV进行分型。同时用荧光定量PCR检测血清HBV DNA含量。结果:从13 117份血清样品中,检测出HBsAg阳性标本230份(1.75%),扩增HBV S基因并成功测序118份。检测出24份标本有"a"抗原决定簇变异(变异率20.34%),"a"抗原决定簇两茎环间的变异率和男女儿童感染的HBV"a"抗原决定簇的变异率无显著性差异。Q129H、G145A突变后,血清HBV DNA水平较未变异株降低(P<0.05)其他各组则无显著变化。118份测序标本利用Genotyping比对后,112份属于B型,其中adw血清型105份,ayw血清型7份;6份属于C型,均为adr血清型。结论:在江西省乙肝疫苗免疫儿童人群检测出"a"抗原决定簇变异的HBV感染,未发现有明显优势的变异株类型。HBV"a"抗原决定簇区突变对血清HBV DNA含量有一定的影响。     

免疫疗法治疗慢性乙肝病毒感染者疗效观察

目的 探讨抗HBe阳性的HBV慢性感染者前C区变异株的发生率及治疗方法。方法 选择抗HBe阳性的HBV慢性感染者，应用DNA序列分析方法检测其前C区的变异的发生率，然后用免疫疗法(左旋咪唑搽剂 乙肝疫苗 潘生丁)治疗，观察其对HBV前C区变异株患者的疗效，并以HBeAg阳性患者为对照。结果 (1)抗HBe阳性的HBV慢性感染者前C区变异发生率为10／12；10例均为A1896终止变异，其中5例与野生株混合，6例伴BCP变异。(2)本试验的免疫疗法对前C区变异株的疗效(5，7)优于HBeAg阳性者(2，11)的疗效。结论 (1)本研究对象中抗HBe阳性的HBV慢性感染者前C区变异发生率较高；(2)本研究所试用的免疫疗法对前C区变异株的疗效优于对HBeAg阳性者的疗效。但均因例数太少难于肯定，需进行更大样本的研究。     

HBV拉米夫定耐药株及BCP、Pre-C突变株芯片检测方法的应用研究

目的 构建针对HBV拉米夫定耐药株及c区启动子(basal core promoter,BCP)、前C区(Pre-C)突变株,多位点突变的基因芯片检测方法.并与DNA测序法比较,以了解该芯片的灵敏度、特异性、稳定性等性能并初步应用于临床实践.方法 该基因芯片能对HBV DNA及P区(DNA多聚酶区):180、204、207三个拉米夫定耐药突变位点;C区启动子(basal core promoter,BCP)及前C区(Pre-C):nt1896、nt1899、nt1862、nt1764、nt1762 5个突变热点,共8个HBV突变位点进行检测,并用测序法对该基因芯片进行验证.结果 ①检测HBV DNA方面,两种方法检测结果100%相符.②检测突变位点方面:总体统计32份阳性血清共有256(32×8)个突变位点.两种方法检测结果有251个突变位点完全相符;5个突变位点不完全相符,基因芯片法检测为混合型,测序法检测为野生型或突变型之一.结论 结果提示该基因芯片和DNA测序法检测结果阳性率无差异,特异性与DNA测序法相当,检测混合株有更大优势.     

乙型肝炎病毒B/C基因型重组新方式的探讨

目的 研究HBV B/C基因型重组方式.方法 采用聚合酶链反应扩增4例景洪僾伲族慢性乙型肝炎患者血清中的HBV全基因并与pMD18-T载体相连,转化入大肠杆菌E.coli JM109中,经限制性内切酶酶切鉴定,阳性克隆DNA序列测定后进行HBV基因分型及重组部位的鉴定.结果 4名患者16条HBV全基因均为有不同程度的C基因型毒株重组的B基因型,重组方式有两种:群1与C基因型毒株重组部位只有1个,位于HBV前C/C区nt1825～nt2320,共496 bp;群2与C基因型毒株重组部位有2个,位于P基因区nt822～nt1020和前C/C基因区nt1825～nt2320,共695 bp.结论 这种B/C重组方式尚未见报道,Bj亚型与C基因型毒株除了在前C/C基因区重组外,同时还伴有少部分P基因区重组.     

乙型肝炎病毒C基因启动子区准种与变异特点的研究

目的 研究乙型肝炎病毒(HBV)C基因启动子区难种与变异的特点。方法 以中国株HBV基因序列为依据，设计特异性聚合酶链反应(PCR)引物，自3例慢性HBV感染患者外周血血清中扩增HBVCP序列，克隆入pGEM Teasy质粒，随机挑选克隆进行DNA测序以确定病毒的变异程度。结果 测序结果发现HBV CP序列高度保守，但在TATA样盒(1—3)可发生多种突变，其中184nt(T→C)位替换突变员为常见；在直接重复序列(DR)I上游存有一缺失突变高发区33．3％(5／15)。结论 CP区内有一缺失高变区，TATA样盒3的变异可能影响前C蛋白的表达。结果 提示HBV长期携带者体内有HBV准种共存。     

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.     

Genotype F prevails in HBV infected patients of Hispanic origin in Central America and may carry the precore stop mutant

The distribution of HBV genotypes and the presence of the precore stop mutation were investigated in HBV strains from Central America. 333 HBsAg positive sera from chronic HBsAg carriers and acute hepatitis B cases from five different countries (Costa Rica, Nicaragua, Honduras, El Salvador and Guatemala) were tested for HBV DNA by nested PCR. Genotyping by limited sequencing within the S gene was performed on 90 strains, 66 from sera with a high level of HBV DNA, and another 24 from sera positive for HBV DNA only after nested PCR. 23 of the samples were anti-HBe positive. Genotype F was found in 71 (79%), A in 13 (14%), D in 5 (6%) and C in one of the 90 sera. 18 patients with genotype F infection had anti-HBe and HBV DNA in serum. Since the three published precore sequences of genotype F strains have a C¹⁸⁵⁸, which is known to prevent the precore stop mutation from G to A at position 1896, the precore and part of the core genes were sequenced from 19 anti-HBe positive sera with HBV DNA, 17 with genotype F and 2 with genotype A. The A¹⁸⁹⁶ mutation was found in 11 of the 17 genotype F strains. All these had a T¹⁸⁵⁸, which was also present in 5 of the 6 genotype F strains with G¹⁸⁹⁶. The precore region was therefore sequenced from genotype F strains from 5 HBeAg positive sera from the five different Central American countries. These also had a T¹⁸⁵⁸, which thus is the wild type substitution in genotype F in Central America. A number of mutations were recorded between residues 57 and 68 in the core protein corresponding to a unique clustering region of the genotype F strains. The predominance of genotype F in Central American populations of Hispanic origin was not anticipated since this genotype is regarded as indigenous to the Amerindian populations of the New World. J. Med. Virol. 51:305–312, 1997. © 1997 Wiley-Liss, Inc.     

Genetic heterogeneity in the precore region of hepatitis B virus in hepatitis B e antigen-negative chronic hepatitis B Patients: Spontaneous seroconversion and interferon-induced seroconversion

To elucidate the relationship between the clinical severity of chronic liver disease and the precore mutations in hepatitis B e antigen (HBeAg)-nega-tive hepatitis B virus (HBV) carriers, mutations were investigated in the precore region of HBV DNA in 20 chronic hepatitis B patients who sero-converted either spontaneously or after the administration of α-interferon (IFN), and 5 asymptomatic carriers. The precore mutation with a stop codon at nucleotide 1896 was found in all patients, irrespective of the histology and in all asymptomatic carriers. The second mutation at nucleotide 1899 was found in 40% of cases studied but always followed by the first mutation at nucleotide 1896. The mixed viral infection of precore mutant and wild-type HBV virus was found in 40% of seroconverted cases after IFN treatment and in sera of HBV carriers obtained within a year after the spontaneous Seroconversion. These data suggest that the precore mutants prevail over wild-type HBV in all HBeAg-negative HBV carriers within several years after the sero-conversion, but their prevalence could not confine the clinical severity of chronic liver disease. © 1995 Wiley-Liss, Inc.     

The precore mutation is associated with severity of liver damage in Brazilian patients with chronic hepatitis B.

BACKGROUND: The clinical relevance of the G1896A precore mutation in chronic hepatitis B is still poorly understood. OBJECTIVES: To assess the frequency of G1896A precore mutation in Brazilian patients with chronic hepatitis B, as well as its relation to the viral genotype, serum HBV-DNA levels and liver damage. STUDY DESIGN: Fifty chronic hepatitis B patients (29 HBeAg-negative and 21 HBeAg-positive) were studied. HBV-DNA was quantified by the Amplicor HBV Monitor test and precore region and S gene were amplified and submitted to automatic sequencing. The histological activity index (HAI), degrees of hepatic fibrosis and distribution of core antigen (HBcAg) in hepatocytes were determined. RESULTS: Precore mutation occurred in 1/21 (4.8%) HBeAg-positive patients and in 17/29 (58.6%) HBeAg-negative (p < 0.0001). Genotype D was identified in 56.5%, genotype A in 41.3%, and genotype F in 2.2%. The frequency of genotypes D and A, as well as serum levels of ALT and HBV-DNA were similar in patients infected with wild type and with precore mutant. Patients infected with precore mutant presented a higher frequency of moderate/severe HAI (p: 0.03) and moderate/severe fibrosis and cirrhosis (p: 0.03) than those infected with wild type. There was no association between G1896A mutation and cytoplasmic expression of HBcAg. CONCLUSIONS: Precore mutation was frequent among Brazilian subjects with chronic hepatitis B and its presence was associated with greater severity of liver disease.     

Analysis of the precore and core promoter DNA sequence in liver tissues from patients with hepatocellular carcinoma.

To investigate the role of mutant hepatitis B virus (HBV) in the development of hepatocellular carcinoma (HCC), 20 patients with HCC were studied for precore and core promoter mutations in tumorous and nontumorous tissues. The precore and core promoter region was amplified and analyzed by direct sequencing. Among the 20 tumorous and nontumorous tissues, precore mutant HBV was found in 12 (60%) and 18 (90%), respectively. Of the 12 tumorous tissues with precore mutant, nine tissues had a single mutation (1896) and one tissue had another single mutation (1899). The remaining two tissues had a double mutation (1896 and 1899). A single mutation (1896) and a single mutation (1899) were found in 11 and two of the 18 nontumorous tissues with precore mutant, respectively. Among 20 tumorous and nontumorous tissues, HBV with a C to T mutation at nucleotide (nt) 1846 was detected in six and eight, respectively, and was associated with the virus carrying a mutation (1896 or 1899) except in two tumorous tissues. Mutations at nt 1762 and 1764 in core promoter were observed in 16 (80%) tumorous tissues and 18 (90%) nontumorous tissues. Mutations in the precore and core promoter region were found frequently in nontumorous tissue and in tumorous tissue (18/20 and 12/20 in precore region, 18/20 and 16/20 in core promoter respectively). The high prevalence of precore and core promoter mutations in liver tissue from patients with HCC suggests that these mutations may contribute to the development of HCC.     

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.     

Evolution of wild-type and precore mutant HBV infection after liver transplantation

Recurrence of hepatitis B virus (HBV) infection after liver transplantation is associated with varying degree of graft damage. The aim of the study was to investigate longitudinally the changes of wild-type and precore A₁₈₉₆HBV mutant viral populations after reinfection and their impact on liver graft damage. The wild-type HBV and A₁₈₉₆HBV strains were quantitated before and serially after orthotopic liver transplantation (OLT) in 14 hepatitis B surface antigen (HBsAg)-positive liver graft recipients (4 hepatitis B e antigen [HBeAg]+; 10 anti-HBe+). Before OLT, the wild-type precore HBV was present in all 4 HBeAg-positive patients and in 2/10 anti-HBe-positive patients; a mixed virus population was present in 6 patients; and A₁₈₉₆HBV mutant alone in 2 patients. After OLT, A₁₈₉₆HBV mutant appeared and gradually accumulated in 5/6 patients who had the wild-type HBV before OLT and 1 of these patients seroconverted from HBeAg to anti-HBe 52 months after transplantation. A mixed HBV population was present continuously in 6 patients before and after OLT. Of the 2 patients with A₁₈₉₆HBV only pre-OLT, the wild type appeared in one patient and the other patient retained persistently the A₁₈₉₆HBV mutant. There was no relationship between liver graft histology and the type of viral population at reinfection or at the end of follow up. Changes in the HBV population occur during follow up of recurrent hepatitis B in liver transplant recipients with frequent accumulation of precore A₁₈₉₆HBV mutants, but the type of viral population does not determine the severity of hepatitis B in the graft. J. Med. Virol. 59:5–13, 1999. © 1999 Wiley-Liss, Inc.     

Prevalence of precore-defective mutant of hepatitis B virus in HBV carriers

Two hundred and seventy-three serum specimens from hepatitis B virus (HBV) carriers were examined for the presence of a characteristic one point mutation at nucleotide (nt) 1896 from the EcoRI site of the HBV genome in the precore region (the preC mutant) using restriction fragment length polymorphism (RFLP) analysis. This assay approach could detect preC mutants or wild-type sequences when either form constituted more than 10% of the total sample. Overall, 65.5% (76/116) of HBeAg-positive carriers had only the preC wild-type. All HBeAg-positive asymptomatic carriers (n = 14) had only the preC wild-type. In patients with chronic hepatitis B and in anti-HBe-positive asymptomatic carriers, increased prevalence of the preC mutant was associated with the development of anti-HBe antibodies and normalization of the serum alanine aminotransferase concentration. Furthermore, 27 (29.0%) of 93 HBeAg-negative carriers had unexpectedly preC wild-type sequences only. Direct sequencing of the HBV precore region of HBV specimens from 24 patients revealed no mutation at nt 1896, supporting the specificity of the RFLP analysis. These results suggest that RFLP analysis was accurate for the detection of the preC mutation and that the absence of serum HBeAg cannot be explained solely by the dominance of the preC mutant. © 1995 Wiley-Liss, Inc.     

乙型肝炎病毒基因型C亚型和核心启动子、前C/核心区基因变异的关系

目的 研究慢性乙型肝炎病毒（HBV）感染者中HBV基因型C亚型（HBV／C）的核心启动子、前C／核心区基因变异情况，分析HBV／C亚型的病毒学特征。方法 用酶联免疫法（ELISA）筛选出79例HBV／C，再用聚合酶链反应．限制性酶长度多态性分析方法（PCR-RFLP）进行HBV／C亚型分析；同时针对HBV核心启动子、DreC／核心区基因进行半巢式PCR及PCR产物直接测序。结果 ①79例HBV／C中，33例（41．8％）为HBV／C1亚型，46例（58．2％）为HBV／C2亚型。②HBV／C1亚型仅见于来自中国南方的患者（P〈0．0001）。③A1898位点变异仅见于HBV／C1亚型（P=0．056），V1753位点变异在HBV／C1亚型中多见（P〈0．05）；HBV／C2以T1858（90％）、A1896（40％）位点变异多见（P〈0．008）。T1762／A1764位点变异在HBV／C两种亚型中均常见。④肝细胞癌（HCC）患者中，V1753和T1762／A1764变异最常见（P〈0．05）。结论 HBV／CI和HBV／C2在中国有明显的地区差异；V1753合并T1762／A1764双变异与发展为HCC相关，尤其在HBV／C1患者。