乙型肝炎病毒多聚酶变异与拉米夫定治疗反应

目的 探讨乙型肝炎病毒（HBV）多聚酶酪氨酸－蛋氨酸－天氧氨酸－天冬氨酸（YMDD）基序变异对拉米夫定抗病毒疗效的影响。方法 对19例拉米夫定治疗（100mg/d）48周时血清病毒核酸阳性的慢性乙型肝炎患者,采用聚合酶链反应（ PCR）产物直接测序技术,检测其血清中HBV多取和酶（YMDD）变异情况,并观察其血清HBV DNA和丙氨酸转氨酶（ALT）水平。结果 在检出YMDD变异株的10 例患者（4例为YV^550DD型,均伴L^526→M突变,6例为YI^550DD型）,至52周为止,共5例出现HBV DNA突发（分枝DNA信号扩增法）,其中2例伴ALT再高。追踪其治疗前血清均未发生上述变异。而在未检出YMDD变异的9例患者中,1例出现DNA突发后又阴转,而另1例治疗过程中HBV DNA未阴转、ALT始终波动于正常值上下的患者,发现多聚酶其他部位的变异^473→R,D^477→N,D^480→N,L^491→M)治疗前后相同。结论 乙型肝炎病毒多聚酶（ YMDD）变异与药物诱导相关,发生变异者较无变异者疗效降低;在拉米夫定治疗过程中,该变异的检测将有助于其疗效监测。     

拉米夫定治疗过程中乙型肝炎病毒多聚酶变异的检测

目的建立拉米夫定治疗过程中乙型肝炎病毒多聚酶变异的检测方法,对该部位变异类型、发生率及其与病毒滴度的关系进行初步分析。方法拉米夫定治疗的慢性乙型肝炎患者164例,在其中治疗64周时HBVDNA阳性的101份血清中,选择9份血清进行分析。设计台成一对引物SI、A1,采用聚台酶链反应法扩增得到包括YMDD基序（motif）的部分P区基因,片断长926bp,对扩增片断的部分校苷酸（约500bp）进行序列分析。HBVDNA定量用分枝DNA信号扩增祛。结果在9例患者中,发现YMDD（酪氨酸－蛋氨酸－天门冬氨酸－天门冬氨酸）基序变异的5例,占56％,其中M（蛋氨酸）－I（异亮氨酸）3例,M（蛋氨酸）－V（缬氨酸）2例,后者合并D（天门冬氨酸）→G（甘氨酸）变异1例。在2例血清HBVDNA低滴度（＜100MEq／ml）患者中未发现YMDD基序变异,7例中、高滴度（＞300ME／ml）患者中5例发现YMDD基序变异,占71％。结论拉米夫定是一种新的治疗慢性乙肝较有潜力的抗病毒药物,若能在治疗过程中对HBVDNA滴度变化及病毒变异发生进行监测,可指导临床的合理用药及联台用药。     

乙型肝炎病毒耐药变异的研究近况

变异是生物适应环境谋求生存的重要方式。乙型肝炎病毒（HBV）是嗜肝DNA病毒的原型。在慢性感染过程中,为适应生存环境,HBV发生基因变异是较为常见的现象。     

靶向核糖核酸酶对乙型肝炎病毒复制的作用

通过定量检测上清液中的乙型肝炎e抗原(HBeAg)和乙型肝炎病毒脱氧核糖核酸(HBV DNA)，进一步研究靶向核糖核酸酶对HBV复制的影响。     

拉米夫定耐药患者乙型肝炎病毒多聚酶区基因突变的检测分析

目的建立以基因测序检测乙型肝炎病毒（HBV）多聚酶区基因突变的方法,并分析拉米夫定耐药者HBV多聚酶区基因主要耐药突变的分布情况。方法采用巢式PCR方法对174例拉米夫定耐药患者血清HBV多聚酶区进行扩增,对PCR产物进行直接测序,将YMDD突变阳性与阴性的病例分别进行HBV耐药基因突变的分析和比较。结果本研究154例YMDD突变阳性的患者中有10种突变类型,其中以rtM204I突变最多见,为46例（29．9％）,其次为rtL180M／M204V（26．6％）、rtL180M／M204I（17．5％）、rtV173／L180M／M204V（13．0％）、rtM204V（2．6％）、rtL180M／M204V／M204I（5．8％）、rtV173L／L180M／M204V／M204I（1．3％）、rtV173L／M204IrtM204I／M204V（1．3％）、rtM204I／M204V（1．3％）。另外,rtM204V联合rtL180M或rtV173L的突变率较rtM204I联合rtL180M或rtV173L显著增高。从rtL180M突变角度来看,rtL180M与rtM204V的联合突变率最高,其次为rtL180M联合rtM204I突变,rtL180M与rtL173M的联合突变率最低。从rtV173L突变角度来看,rtV173L联合rtM204V或rtL180M突变的发生率均高于rtV173L联合rtM204I的突变率,且rtV173L突变多联合rtM204V和rtL180M突变同时发生。结论拉米夫定耐药株的氨基酸突变复杂多样,需要对拉米夫定耐药多个相关位点进行检测。     

乙型肝炎病毒全基因组研究的新方法

目的为从全基因水平研究乙型肝炎病毒变异与致病性的关系，建立血清标本经聚合酶链反应（ＰＣＲ）加酶切方法扩增及克隆ＨＢＶ全基因组的新方法。方法设计了含ＳｐｅⅠ，ＳａｌⅠ及ＳａｐⅠ酶切点的引物，分别扩增１．１５ｋｂ和２．０５ｋｂ的单链及双链ＤＮＡ区。经酶切及连接后获得ＨＢＶ全基因克隆。结果用新建立的方法直接从少量血清中克隆了ＨＢＶ全基因，转染ＨｅｐＧ２细胞可表达抗原并有胞内复制。结论该法可用于对临床上ＨＢＶ毒株的基因结构和功能的研究     

乙型肝炎病毒聚合酶变异及其耐药的产生和监测

乙型肝炎病毒(HBV)以逆转录方式进行基因组的复制,易发生病毒突变;同时,在宿主体内特定的压力下,包括内源性免疫清除、外源疫苗和抗病毒药物等,各种逃逸突变株被选择产生,其中在核苷(酸)类似物治疗中出现的耐药株可导致HBV DNA水平再次升高、丙氨酸转氨酶(ALT)反弹和HBV e抗原(HBeAg)血清学转换的逆转,已成为目     

乙型肝炎病毒基因自然变异耐药性的研究

我们用DNA测序法对102例未经核苷(酸)类似物治疗的慢性乙型肝炎(CHB)患者HBV逆转录酶基因的10个突变位点进行了检测分析,现报道如下.一、资料与方法1.研究对象:收集2008年3月-2010年3月天津塘沽传染病医院住院治疗的CHB患者的血清标本.所有患者均符合2000年西安病毒性肝炎学术会议修订的《病毒性肝炎防治方案》诊断标准[1].     

乙型肝炎病毒耐药变异研究的回顾与展望

核苷(酸)类似物长期抗HBV治疗面临的最重要问题之一是HBV的耐药性问题。目前所有口服核苷(酸)类似物,包括拉米夫定、阿德福韦、恩替卡韦和替比夫定,治疗:过程中均可出现耐药性。特别是最近报道发现未用过阿德福韦的患者对阿德福韦原发耐药,序贯联合孩苷(酸)类似物治疗的患者出现多重耐药性等更引起临床医师的高度关注。有     

Evolution of hepatitis B virus polymerase gene mutations in hepatitis B e antigen-negative patients receiving lamivudine therapy

Lamivudine has been shown to be effective in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, but its long-term efficacy and the rate of resistant mutations in patients with HBeAg-negative chronic hepatitis B is less clear. Twenty-nine patients with HBeAg-negative chronic hepatitis B, who have received lamivudine for at least 1 year were studied to determine the antiviral response, the rate and pattern of lamivudine-resistant mutations, and the effect of lamivudine-resistant mutations on HBeAg status. The mean duration of treatment was 21 +/- 7 months. Before treatment, core promoter variant was detected in 16 (55%) patients and precore stop codon variant in 18 (62%) patients. Serum hepatitis B virus (HBV) DNA was detected by solution hybridization assay in 62%, 4%, and 24% and by polymerase chain reaction (PCR) assay in 100%, 31%, and 40% at months 0, 6, and 24, respectively. The cumulative rates of detection of lamivudine-resistant mutations after 1 and 2 years of treatment were 10% and 56%, respectively. In addition to the duration of treatment, core promoter mutation was associated with the selection of lamivudine-resistant mutants. Three patients with lamivudine-resistant mutations had reversion of the precore stop codon mutation; in 2 patients this was accompanied by the reappearance of HBeAg. We found that lamivudine-resistant mutants were detected at similar rates in patients with HBeAg-negative as in patients with HBeAg-positive chronic hepatitis B. Additional changes in other parts of the HBV genome may restore the replication fitness of lamivudine-resistant mutants.     

Clonal evolution of hepatitis B virus polymerase gene mutations during lamivudine-adefovir combination treatment

AIM: To identify hepatitis B virus polymerase gene mutations during antiviral therapy using lamivudine-adefovir sequential monotherapy followed by lamivudine-adefovir combination therapy.METHODS: The patient cohort included four adult chronic hepatitis B patients who had undergone sequential monotherapy, first with lamivudine (LMV) and then, after developing viral breakthrough, with adefovir (ADV) therapy. All of the patients had non-response or viral breakthrough after LMV-ADV sequential monotherapy, which resulted in the switching of their antiviral regimen to LMV-ADV combination therapy. Eleven serum samples from the four patients who showed non-response to rescue LMV-ADV combination therapy were collected sequentially at a time before the antiviral treatment and then during the LMV monotherapy, ADV monotherapy, and LMV-ADV combination therapy. For the genotypic analysis, the whole 1310-bp polymerase gene region was amplified, cloned and sequenced.RESULTS: All patients had been previously treated with 100 mg of LMV once daily for a 15- to 26-mo period. The emergence of resistance mutations to LMV, such as rtM204V/I and/or rtL180M, were found in all patients. Their antiviral regimens were switched to ADV monotherapy as the second line treatment. All patients had viral breakthrough or non-response after the LMV-ADV sequential monotherapy. ADV-resistant mutations were detected after 13 to 19 mo of LMV-ADV sequential monotherapy. The rtA181V/T mutations were predominantly identified during the ADV treatment in the LMV-resistant patients. Twenty-seven of 38 clones were combined with an amino acid change at rt181; three clones had mutations in rt236 and one clone had a combined mutation. The rtA181V/T mutations were not suppressed by the LMV-ADV combination therapy. Thirty-nine of 64 clones showed an rtA181V/T mutation and six clones showed combined mutations in rt181 and rt236. Mutations in rt204 re-emerged during the combination treatment. The rt181 and rt204 mutations did not co-exist in one clone.CONCLUSION: Add-on lamivudine therapy with adefovir for adefovir resistance may not suppress the pre-existing adefovir-resistant mutation that develops during lamivudine-adefovir sequential monotherapy.     

拉米夫定治疗前后乙型肝炎病毒P区基因的动态变化

目的研究拉米夫定治疗前后慢性乙型肝炎患者体内乙型肝炎病毒P基因区的变异情况。方法从5例慢性乙型肝炎患者拉米夫定治疗前和治疗12个月的血清标本中，扩增目的片段，阳性结果双酶切后克隆至JM105感受态细胞。每份标本随机挑取20个阳性克隆，以错配聚合酶链反应一限制性片段长度多态性分析法检测YMDD基因序列，出现变异者进行双向测序。结果5例慢性乙型肝炎患者在拉米夫定治疗12个月后，其中2例HBVDNA为阴性，2例HBVDNA转阴后又转阳，测序结果提示出现M5521变异；1例HBVDNA始终阳性，和治疗前一样存在D553G的变异。结论D553G变异可能是慢性乙型肝炎患者拉米夫定治疗无效的原因之一，拉米夫定治疗后出现的乙型肝炎病毒基因组P区YMDD变异是抗病毒药物诱导的结果。     

Long-term follow-up of chronic hepatitis B after the emergence of mutations in the hepatitis B virus polymerase region

Treatment of chronic hepatitis B has been greatly improved by the use of lamivudine, but mutations occur in the polymerase region of hepatitis B virus (HBV) and lamivudine-resistant mutants frequently develop. The emergence of lamivudine-resistant strains of HBV is a problem for treating chronic hepatitis B using lamivudine. We observed biochemical and virological changes in 15 patients with chronic hepatitis B for a median period of 29 months (range: 4-42 months) after the emergence of lamivudine-resistant mutants of HBV. Patterns of mutation of the polymerase gene were examined by sequencing the LLAQ motif in domain B and the YMDD motif in domain C. Exacerbation of liver dysfunction occurred in 14 (93.3%) of the 15 patients at a median of 4 months after the emergence of mutations. However, exacerbation of liver dysfunction was observed only in four patients (26.7%) at the time of appearance of the first mutations and in 80.0% of the patients at the time of appearance of the second mutations. Increase in serum alanine aminotransferase (ALT) levels was significantly greater at the time of appearance of second mutations (P = 0.0096). In most cases, wild-type HBV was mutated with the substitution of only rtM204I at first, and rtL180M/M204I mutations and then rtL180M/M204V mutations subsequently appeared. Further mutations of the polymerase region caused clinical deterioration. Thus as mutations emerge in the polymerase region, the clinical outcome deteriorates. Thus, monitoring the patterns of mutation of the polymerase gene is useful when using lamivudine for treating HBV.     

Mutations in the hepatitis B virus polymerase gene associated with antiviral treatment for hepatitis B

Significant advances have been made, during the last 5 years, in the treatment of chronic hepatitis B. Several new antiviral agents: lamivudine, famciclovir, lobucavir and adefovir, have been shown to be safe and effective in inhibiting hepatitis B virus (HBV) replication. These compounds can be administered orally and are well tolerated. However, virus clearance is uncommon after short courses (<6 months) of therapy. Lamivudine and famciclovir have been evaluated in Phase III clinical trials in patients with chronic hepatitis B as well as in liver transplant recipients. Unfortunately, drug-resistant mutants involving the HBV polymerase gene, leading to breakthrough infection, have been reported in some patients who have received long courses (≥ 12 months) of treatment. The incidence, clinical outcome and biological significance of these mutants will be reviewed.     

Concentrating missense mutations in core gene of hepatitis B virus

To elucidate the temporal relationship between liver damage and mutation(s) in hepatitis B virus core gene, serial sera from a progressive liver disease patient and an asymptomatic carrier were studied. By direct sequencing, missense mutations in the core gene were only found in serum from the progressive liver disease patient during the period with frequent exacerbation. Using methods of cloning and sequencing, missense mutations were also found in clones derived from the progressive liver disease patient at a relatively early phase, but strains with a missense mutation from earlier sera did not exist in sera of a later period. Furthermore, there was a tendency of concentrating missense mutations in clones derived from the progressive liver disease patient. These data suggested that missense mutations in the core gene that occurred at an earlier phase might evoke an immune response to eliminate mutated virus and that concentrating missense mutations during a phase of exacerbation might be a result of adaptive mutation.     

乙型肝炎病毒三种基因分型方法的比较分析

随着乙型肝炎病毒（HBV）全基因序列测定病毒株的增多，近年人们开始根据生物系统发生学中的基因分类法建立HBV的基因分型技术和标准。对HBV进行基因分型有利于对HBV感染的流行病学、病因学和临床诊断与治疗进行更加深入细致的研究，现有研究表明不同亚型HBV感染的临床病程和对治疗的反应都存在一定的差异。我们分别应用自行建立的实时荧光聚合酶链反应（PCR）法、HBVS基因测序法和聚合酶链反应-限制性片段长度多态性（PCR—RFLP）法对75例慢性乙型肝炎患者的血清基因型进行检测，并对检测结果进行分析比较。     

Early detection of viral resistance by determination of hepatitis B virus polymerase mutations in patients treated by lamivudine for chronic hepatitis B

We have analyzed the molecular dynamics of emergence of drug-resistant strains in patients receiving lamivudine therapy for chronic hepatitis B. Twenty consecutive patients with lamivudine resistance were studied (13 hepatitis B e antigen [HBeAg]-positive patients and 7 HBe antibody [anti-HBe]-positive patients). Determination of viral genotype, precore mutants, and polymerase gene mutants (L528M, M552V, M552I) was performed using the research version of Lipa-HBV. Quantitative analysis of HBV DNA was performed using both branched DNA (bDNA) and polymerase chain reaction (PCR) assays. Polymerase mutants (genotypic resistance) were found in 16 of 20 patients. Genotypic resistance was detected earlier than the phenotypic resistance (P =.004). Quantitative PCR allowed detection of viral DNA throughout the entire study period in 16 of 20 patients. Analysis of pretreatment variables showed that high alanine transaminase (ALT) levels (>3 x the upper limit of normal [ULN]) was associated with a more rapid selection of drug-resistant mutants (P =.027) and a high hepatitis B virus (HBV) DNA level (>1,497 Meq/mL, bDNA) with a more rapid occurrence of phenotypic resistance (P =.04). At the time of viral breakthrough, the mean serum HBV-DNA values were not different from the pretreatment values (P =.37). ALT levels were higher in anti-HBe-positive patients compared with pretreatment values and to HBeAg-positive patients (P =.01). In 8 patients, antiviral therapy was modified after viral breakthrough, with the introduction of famciclovir and/or interferon alfa. Viral DNA became undetectable by bDNA in 3 patients who received interferon. Our results suggest that genotypic assays for polymerase mutant detection and quantitative determination of viremia with highly sensitive assay are warranted for an optimal monitoring of antiviral therapy of chronic hepatitis B.