拉米夫定治疗中乙型肝炎病毒基因序列变异特点及其准种研究

目的研究拉米夫定治疗中乙型肝炎病毒（HBV）逆转录酶区核苷酸序列变异、特点、含量及其与基因型和病毒载量的关系。方法普通DNA测序法检测拉米夫定治疗的117份慢乙肝患者血清HBV逆转录酶区基因序列及其基因型;其中99份用TaqMan法定量HBVDNA;64份用焦磷酸测序（Pyrosequencing）检测YMDD基序中碱基的频率。结果HBVYMDD变异组中C型43例,B型10例,A／B混合型1例;YMDD不变异组中C型54例,B型8例,D型1例,HBVDNA含量平均对数值在变异组和不变异组分别为：6．5699和6．6165;YMDD变异与其基因型及病毒载量无统计学意义;rtL180M位点变异与rtM204I／V位点变异高度相关;并且HBV野生株与变异株均同时存在。结论结合两种测序方法可以用来研究拉米夫定治疗中HBV基因序列变异情况及对YMDD耐药株进行定量。     

三种乙型肝炎病毒拉米夫定耐药突变的酶切分析方法及其应用

目的：建立三种乙型肝炎（乙肝）病毒拉米夫定耐药突变的酶切分析方法，了解这三种耐药突变在拉米夫定治疗患者中的发生情况。方法：设计3对HBV P基因引物进行聚合酶链反应（PCR）扩增，应用Nde I和Nla Ⅲ两种限制性内切酶对PCR产物进行酶切，酶切产物用电泳加以分析。应用此方法检测70例拉米夫定治疗的慢性乙肝患者中3种拉米夫定耐药突变的发生情况。结果：Nde I和NlaⅢ两种限制性内切酶对PCR产物进行酶切可有效区分HBV野株和YMDD（酪氨酸－蛋氨酸－天冬氨酸－天冬氨酸）变异株，并确定是YI（异亮氨酸）DD或YV（缬氨酸）DD变异。用Nla Ⅲ内切酶对PCR扩增产物酶切可有效区分HBV野株和L526M（第526位氨基酸由亮氨酸变为蛋氨酸）变异株。利用此方法发现11例患者在服用拉米夫定过程中出现耐药突变，其中YIDD变异6种，YVDD变异5例，后者有1例伴有L526M点突变。结论：本方法只需应用常规PCR和酶切技术，既可有效筛检出YMDD变异株标本，还可以确定属于YIDD还是YVDD变异，或是否伴有L526M点突变等，均可作为临床监测拉米夫定耐药性的参考。     

PCR-RFLP结合克隆测序监测乙肝病毒拉米夫定耐药突变株的产生

目的 建立PCR结合酶切的方法监测慢性乙型肝炎患者体内拉米夫定耐药突变株的产生,并与PCR产物克隆后测序相结合。了解此方法的可靠性和可行性,同时用此方法筛查50例应用拉米夫定治疗的慢性乙型肝炎患者中耐药株的发生情况。方法 拉米夫定治疗的慢性乙型肝炎患者50例,治疗时间9个月-24个月,设计错配PCR结合限制性片段长度多态性方法,快速检测患者体内乙型肝炎病毒（HBV）酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸（Tyrosine-Methionine-Aspartic acid-Aspartic acid,YMDD)变异株的发生情况,对筛检耐药株阳性的标本应用PCR产物克隆后测序加以证实。结果 在50例服用拉米夫定患者中发现9斧正患者在用药超过9个月时出现拉米夫定耐药突变株（YMDD变异株）,其中YIDD变异5例,YVDD变异4例,后者有3例合并有1526M突变。结论 本方法在检测YMDD变异方面具人快速简便的特点,经克隆后测序证实,具有较好的可靠性。     

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。     

三联叠加疗法对慢性乙型肝炎患者乙型肝炎病毒复制和变异的影响

目的观察疗效确切的抗病毒药物和免疫调节剂,叠加伍用对慢性乙型肝炎病人HBV复制和变异的影响.方法应用拉米夫定、干扰素α-2b、黄芪注射液三联叠加疗法,对慢性乙型肝炎病人进行抗病毒治疗,并与单独应用拉米夫定的抗HBV效果进行比较评价.检测HBVDNA阴转率和HBeAg-抗HBe血清转换率;血清HBVDNA浓度;HBVDNA的YMDD变异率和前C区变异率.结果A组(叠加用药组)与B组(拉米夫定单药组)比较,HBVDNA阴转率分别在第12周、36周及48周差异有显著性(P<0.05),HBeAg阴转率在第36周、48周差异有显著性(P<0.05),抗HBe阳转率仅在第48周差异有显著性(P<0.05);两组患者血清HBVDNA浓度比较,治疗12周时降低程度差异有显著性(P<0.05),治疗第36周和48周时差异有非常显著性(P<0.01);治疗后12周,A组出现前C区BCP变异;疗后24周、36周和48周,两组均出现YMDD和前C区变异.结论与拉米夫定单独用药比较,三联叠加疗法可增加慢性乙型肝炎患者HBVDNA阴转率和HBeAg-抗HBe血清转换率,更显著降低血清HBVDNA浓度,并减少YMDD变异,故其抗HBV疗效优于拉米夫定单独用药.     

基因芯片检测拉米夫定治疗慢性乙型肝炎过程中HBV变异技术的建立和应用

目的建立乙型肝炎病毒变异基因诊断芯片对拉米夫定治疗慢性乙型肝炎过程中出现的肝炎病毒P基因区YMDD变异进行快速准确的检测诊断。方法设计特异性寡核苷酸探针阵列,特殊处理芯片载体。用点样法制备乙型肝炎病毒变异基因诊断芯片。在本院住院治疗患者中选择30例服用拉米夫定后,HBV DNA转阴(<500 copies/ml)168周后又反跳≥5 000 copies/ml的患者进行基因芯片杂交检测分析。同时用PCR直接测序法对上述30例患者血清标本进行双盲HBVDNA聚合酶活性区域测序对照。结果 30例服药后HBVDNA反跳患者中基因芯片测得HBV YMDD变异21例,其中YVDD变异11例。YIDD变异10例。HBVDNA PCR直接序列测定结果,有核苷酸A741变为G,使氨基酸由蛋氨酸552变为缬氨酸(氨基酸基序由YMDD变为YVDD)11例,并有6例核苷酸A669变为C,使氨基酸由亮氨酸528变为蛋氨酸。核苷酸G743变为T,使氨基酸由蛋氨酸552变为异亮氨酸(氨基酸基序由YMDD变为YIDD)10例。其中有3例伴有核苷酸T781变为C,使氨基酸由亮氨酸565变为脯氨酸。标本阳性变异序号与基因芯片检测结果完全一致。结论 乙型肝炎病毒变异基因诊断芯片可以同时检测YVDD,YIDD变异,同PCR直接测序法比较,准确率达100％,无假阳性。     

拉米夫定疗程中YMDD变异前后血清IL-12和TH1/TH2型细胞因子变化及意义

拉米夫定疗程中出现HBV变异的慢性乙型肝炎患者 2 0例 ,HBVDNA阳性 ,治疗前HBeAg均阳性 ,给予拉米夫定 10 0mg d ,分别检测治疗前、治疗 3个月、治疗 6个月、治疗 9个月、治疗 12个月等 5个时相点血清HBVDNA水平 ,在HBVDNA转阴的基础条件下 ,如果某时相点出现了HBVDNA反跳 ,则认为在该时相点发生了YMDD变异。健康献血员作血清细胞因子水平健康对照。采用ELISA法检测 5个时相点及健康献血员血清IL 12、IL 2、IFN γ、IL 4、IL 10水平 ,检测结果以pg ml为单位。计算与健康对照校对后的IFN γ IL 4比值作为TH平衡指标 (…     

慢性乙型肝炎患者HBV耐药模式及rtA181变异准种分析

目的分析慢性乙型肝炎患者HBV逆转录酶区耐药变异情况及rtA181变异准种的分布。方法提取患者血清中HBVDNA,PCR扩增逆转录酶区域,产物测序后经软件比对分析耐药变异情况和基因型;对部分rtA181变异标本进行克隆后测序分析准种分布。结果 489例标本中,检出明确耐药变异265例,在B、C基因型中分布比例存在差异（56.6%vs43.0%,P=0.022）。拉米夫定相关耐药138例（52.1%）,以M204I、M204I/V＋L180M±L80I/V变异为主;阿德福韦相关耐药35例（13.2%）,以N236T＋A181T/V较为多见;拉米夫定＋阿德福韦相关耐药70例（26.4%）,几乎都和A181有关。rtA181准种分析发现1例位于同一病毒株的多耐药组合,且未发现单一A181T变异的病毒准种。结论 HBV耐药变异主要表现为M204和A181相关变异,耐药模式复杂;检测HBV逆转录酶区的变异有助于临床及时发现和确认耐药情况,指导临床合理进行抗病毒用药。     

乙型肝炎病毒耐拉米夫定多聚酶基因变异检测方法研究

目的 建立简便、准确、实用的检测乙型肝炎病毒耐拉米夫定多聚酶(P)基因变异的方法。方法 根据HBV基因序列，设计5只寡核苷酸引物，用巢式聚合酶链反应(nested PCR)分别扩增HBVP基因B区和C区片段，产物用NdeⅠ或NIa Ⅲ酶切，琼脂糖胶电泳，分析酶切产物长度多态性(RFLP)，建立检测P基因变异的方法。对30例长期服用拉米夫定的慢性乙型肝炎(慢乙肝)患者检测YMDD基序及526位点变异，16例未用拉米夫定的慢乙肝患者为对照。4份PCR产物作克隆测序以验证方法的准确、可靠。结果 所建的巢式PCR-RFLP方法操作简便、快速，从模板提取到酶切后电泳分析仅需11h；灵敏度高，可检测到10^3拷贝／ml的HBVDNA；结果准确，4份经酶切分别判断为野毒株或变异株的标本经测序证实。30例用拉米夫定的慢乙肝患者中，发现单纯YMDD变异8例(26．7％)，YMDD联合L526M变异3例(10．0％)，16例对照未检出上述位点的变异。结论 本方法简便、准确，适合较大样本检测。可用于临床筛检常见拉米夫定耐药性HBVP基因变异。     

锁核酸捕获TaqMan探针实时PCR和PCR-RFLP检测乙型肝炎病毒基因变异的研究

目的评价锁核酸捕获TaqMan探针实时聚合酶链反应（PCR）和聚合酶链反应-限制性片段长度多态性（PCR-RFLP）检测乙型肝炎病毒（HBV）基因变异的特点。方法分别用直接测序法、锁核酸（LNA）捕获TaqMan探针实时PCR和PCR-RFLP检测77例接受拉米夫定（LAU）治疗6个月以上的慢性乙型肝炎患者的血清样本。结果锁核酸捕获TaqMan探针实时PCR和PCR-RFLP都能检测HBV野生型YMDD及其变异型YIDD和YVDD。此外，两种方法尚能鉴定HBV野生型和变异型混合种群。更为重要的是，在检测HBV野生型和变异型共生变异方面，锁核酸捕获TaqMan探针实时PCR比PCR-RFLP更敏感、更省时。结论锁核酸捕获TaqMan探针实时PCR是一种检测HBV YMDD变异的灵敏度和特异性都高的快速法，在监测HBV患者LAM抗病毒治疗的疗效及发现新耐药病毒基因型等方面具有广泛的应用前景。     

Line probe assay for monitoring drug resistance in hepatitis B virus-infected patients during antiviral therapy

Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.     

基因芯片检测拉米夫定治疗乙肝病毒感染耐药分析

目的 分析拉米夫定治疗乙肝病毒感染耐药性,旨在为指导慢性乙肝患者临床药物治疗提供新方法.方法 应用基因芯片检测255份51名拉米夫定抗乙肝病毒治疗自愿者不同阶段服药的临床标本及145份HBV DNA阴性血清耐药突变,并对突变进行分析.结果 经一年药物治疗10名患者产生耐药突变,耐药率19.6%（10/51）,产生突变阳性标本病毒载量＞105拷贝/ml.药物治疗后产生突变＞6个月.10例突变病毒株中,552密码子处突变9例,528密码子处突变3例,密码子555中未发现突变.5例血清存在混合感染,其中4例为野生型病毒与突变型的混合,野生型病毒所占比例皆在40%以下.1例血清出现在（4,4）位点的密码子ATT与（3,3）位点密码子TTG两种突变,两种突变类型病毒并行产生同步增长.（3,3）突变类型首次报道.结论 拉米夫定抗乙肝病毒治疗6个月以上易产生耐药,产生耐药病毒载量＞105拷贝/ml.主要发生在YMDD区,且存在两种突变类型病毒的混合感染,以上结果对慢性乙肝患者用药具有指导作用.     

Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.     

Monitoring drug resistance in chronic hepatitis B virus (HBV)-infected patients during lamivudine therapy: evaluation of performance of INNO-LiPA HBV DR assay

Sensitive and early detection of emerging hepatitis B virus (HBV) drug resistance may not only help monitor the viral dynamics associated with lamivudine treatment but could also improve therapeutic decision making. This is especially important when new antivirals effective against lamivudine-resistant HBV become available. A total of 159 serum samples from 33 chronic HBV patients receiving lamivudine treatment were analyzed at four centers for the presence of lamivudine-resistant mutations at codons 528 [180] (proposed revised nomenclature according to Stuyver et al. [Hepatology 33:751-757, 2001] shown in brackets), 552 [204], and 555 [207] of the HBV polymerase. Sequencing data were compared with results generated by the INNO-LiPA HBV DR line probe assay (LiPA), an assay based on reverse hybridization of amplified HBV DNA fragments with specific nucleotide probes immobilized on nitrocellulose strips. LiPA provided at least the same information as sequencing for 97.5% of all codons analyzed for codon 528 [180], 95% for codon 552 [204], and 100% for codon 555 [207]. The most common reason for discrepant or indeterminate results (0.4% and 1.5%, respectively) in a small percentage of the population tested could be attributed to polymorphisms not yet covered by LiPA probes. In at least five patients, a mutant could be detected earlier by LiPA than by sequencing. In 15 patients, LiPA detected mixed wild-type and mutant virus populations before viral breakthrough. These results demonstrate that INNO-LiPA HBV DR is a highly sensitive and easily applicable assay for the detection and monitoring of lamivudine-resistant mutations in chronic hepatitis B patients and that the assay is more sensitive than sequencing in detecting mixed mutant and wild-type sequences.     

Prevalence and profile of mutations associated with lamivudine therapy in Indian patients with chronic hepatitis B in the surface and polymerase genes of hepatitis B virus

Lamivudine results in the selection of resistant hepatitis B virus (HBV) variants. Because the surface gene of HBV overlaps completely the polymerase gene, the incidence and profile of surface and polymerase gene mutations were investigated prospectively in chronic HBV patients who were on lamivudine therapy. Twenty-six patients with chronic liver disease confirmed histologically were included in this study. Extracted HBV DNA from sera samples were subjected to PCR amplification for the mutation prone regions of the surface and polymerase genes of the HBV genome. The emergence of mutant forms and biochemical derangements were studied carefully during the course of the therapy. In six of 26 (23%) patients, mutations emerged on lamivudine therapy. YM552I/VDD resistant mutants were observed in one (6%) and five (29%) patients at Month 12 and 18, respectively, out of 17 patients, who had completed more than 9 months of therapy. The mean time of emergence of resistance was 16.4 +/- 6.8 months. In three of the five patients, emergence of YM552I/VDD mutation was accompanied with a rise in HBV DNA levels. In two patients, mutations were noticed at the end of the viral breakthrough; when the DNA level went down to undetectable levels (<0.5 pg/mL). In two patients, normal ALT levels were found at the time of emergence of the YMDD mutation. YM552I/VDD mutations were observed in 43% of HBeAg positive and 20% of anti-HBe positive patients (P = ns). Although the 'a'-determinant region was found to be unaffected; in one patient, a novel pattern due to emergence of YIDD mutant was observed; the corresponding aa in the S-ORF turned to a stop codon. In summary, the frequency of emergence of YM552I/VDD mutations was 29% at Month 18 in the Indian patients. The presence of normal ALT and low levels of HBV DNA do not exclude the existence of resistant mutants. Novel mutations in the S-ORF, which lead to premature surface gene termination might affect the production of HBsAg and need further study.     

In vivo dynamics and pathogenicity of wild-type and resistant Hepatitis B virus during long-term lamivudine monotherapy - a clinical note.

BACKGROUND: Genotypic resistance of Hepatitis B virus (HBV) against lamivudine evolves within months after onset of therapy. OBJECTIVES: To determine the longitudinal order in which resistance mutations appear and to compare the kinetics and pathogenicity of wild-type and resistant HBV. STUDY DESIGN: In a longitudinal study, consecutive samples were drawn over a period of 28 months from a patient with chronic hepatitis B, and resistance mutations were followed by sequencing a part of the polymerase region of HBV. These data were compared with HBV copy numbers, HBsAg and ALT levels, and results of consecutive liver biopsies. RESULTS: After 21 weeks of treatment, a silent mutation at codon 528 (CTG to TTG) occurred. Significant genotypic resistance was detectable after 68 weeks, indicated by a substitution of isoleucine for methionine at residue 552 (M552I). Nineteen weeks later, the virus exhibited additional resistance-associated mutations (L528M and I552V). The resulting high-level resistance was reflected by an increase of serum HBV copies of 4.7 log(10). The turnover of wild-type and resistant HBV was 2.6x10(6) and 1.8x10(6) virions/day, respectively. HBsAg and ALT levels were lower within the period when resistant HBV was detectable. During treatment the progress of liver fibrosis was arrested. CONCLUSIONS: The in vivo replicative capacities and dynamics of wild-type and resistant HBV were similar. However, resistant HBV seemed to exhibit reduced pathogenicity.     

Two Sensitive PCR-Based Methods for Detection of Hepatitis B Virus Variants Associated with Reduced Susceptibility to Lamivudine

Two novel assays, a restriction fragment length polymorphism (RFLP) assay and an assay based on the 5'-nuclease activity of Taq DNA polymerase, were developed for screening viral variants in lamivudine-treated patients' sera containing <1,000 copies of the hepatitis B virus (HBV) genome per ml. Both assays were designed to detect single-nucleotide changes within the HBV DNA polymerase gene that are associated with lamivudine resistance in vitro and have been used to screen a number of patients' sera for variant virus. Results obtained with these assays and standard sequencing technology were compared with regard to throughput, ability to detect individual virus species present at low concentrations, and ability to detect, distinguish, and quantitate wild-type (wt) and HBV tyrosine methionine(552) aspartate aspartate motif variants in mixed viral populations. Unlike DNA sequencing, both assays are amenable to high-throughput screening and were shown to be able to quantitatively detect variant virus in the presence of a background of wt virus. As with DNA sequencing, both new assays incorporate a PCR amplification step and are able to detect the relatively low amounts of virus found in lamivudine-treated patients' sera. However, these assays are far less labor intensive than the DNA-sequencing techniques presently in use. Overall, the RFLP assay was more sensitive than DNA sequencing in detecting and determining the ratios of wt to variant virus. Furthermore, the RFLP assay and 5'-nuclease assay were equally sensitive in the detection of mixed viral species, but the RFLP assay was superior to the 5'-nuclease assay in the quantitation of mixed viral species. These assays should prove useful for further understanding of virological response to therapy and disease progression.