乙型肝炎病毒Y(I/V)DD变异与前C及C基因启动子变异生物信息研究

目的 分析HBV反转录酶区(RT)rtM204位点Y(I/V)DD变异与前C及C基因启动子(PC-BCP)变异关系. 方法 应用MEGA4对来自GenBank/EMBL/DDBJ的人类2 849株HBV全基因序列序列重新排列,分析Y(I/V)DD变异与PC-BCP变异及模式相关性. 结果 rtM204(I/V)DD变异株217株(8.0％); PC-BCP变异株1 543株(54.2％),有(1)G1896A、(2)G 1899A、(3) G1896A+G1899A、(4) A1762T/G1764A、(5) A1762T/G1764A+G1896A、(6)A 1762T/G 1764A+G1899A、(7) A1762T/G1764A+G1896A+G1899A 7种变异模式; Y(I/V)DD与PC-BCP联合变异165株.YMDD与PC-BCP双变异率高于单纯YMDD变异率(76％比24.0％,x2=45.283,P=0.000);YIDD与PC-BCP双变异率高于YVDD与PC-BCP双变异率(85％比64.9％,x2=11.836,P=0.000)及使用LAM致YMDD与PC-BCP双变异率高于未使用LAM预存YMDD与PC-BCP双变异率(89.3％比58.9％,x2=27.084,P=0.000).二分类logistic回归分析仅对YIDD有影响而对YVDD无影响的PC-BCP变异模式有G1896A-G1899A(P=0.000,OR=7.573)、A1762T/G1764A-G1899A(P=0.000,OR=6.539)和A1762T/G 1764A-G 1896A-G1899A(P=0.000,OR=6.596). 结论 YMDD变异与PC-BCP变异相关;PC-BCP变异模式的差异与YI/VDD变异选择强度有一定的关系.     

拉米夫定治疗前后乙型肝炎病毒YMDD变异的相关因素分析

Objective To identify factors associated with YMDD mutation in patients with chronic hepatitis B before and after Iamivudine treatment in Zunyi region.Methods 53 patients with chronic hepatitis B were enrolled in this study,HBV DNA,HBV markers,ALT,AST,TBil,albumin in the serum were examined at 0,3,6,12,18 and 24 months after lamivudine treatment.HBV genotype and YMDD mutation were determined by sequencing before lamivudine treatment.YMDD mutation was checked again if serum HBV DNA rebound to more than 1×104 copies/ml after the initial decrease.Results HBV genotype in Zunyi region iS constitute of B.C and B+C genotype.YMDD mutation occurred in 18 cases after lamivudine treatment.the rate of YMDD mutation was 15.1%,and 34.0% after 1 year and 2 years treatment.There are four types of mutation:nL180M/M204V,rtL180M/M2041,rtM204I,nL180M.rtM204V mutation in C gene was always acompanied by rtL180M mutation (100%). The rate of rtL180M/M204V mutation in genotype C group was significantly higher than that in genotype B group (77.8% to 25.0%),the same was true for the rtL180M/M204I mutation (22.2% to 12.5%). There was no point mutation in genotype C group. The point mutation of rtM204I and rtL180M appeared only in genotype B group. Gender,nation,family history of hepatitis B and HBeAg were not associated with YMDD mutation (P ＞ 0.05),while the mutation rate was associated with the disease course and severity of disease. YMDD mutation did not occure in patients with low HBV DNA level (＜105 copies/ml). Conclusion YMDD mutation after lamivudine therapy is associated with HBV gnnotype and P gene mutation type,and prolonged treatment increases the the mutation rate. In order to reduce the incidence of YMDD mutation,patients with shorter disease course,lower HBV DNA level,more serious liver damage should be treated with iamivudine.     

102例慢性HBV感染者病毒基因变异情况分析

目的探讨乙型肝炎病毒感染者病毒基因分型和变异情况及其临床意义。方法我院住院和门诊诊治的慢性HBV携带者53例和慢性乙型肝炎患者49例,取血清,送往北京地坛医院肝病研究所进行HBV DNA基因测序。结果在102例慢性HBV感染者中,HBV B型36例(35.29%),C型59例(57.84%),B/C混合型4例,未定型3例。慢性乙型肝炎组B型17例,C型28例,B/C混合型3例,未定型1例,携带者组B型19例、C型31例、B/C混合型1例、未定型2例(χ~2=0.32 P=0.57);在53例携带者中,检出耐药位点5例(9.4%),其中3例为rtA181T,1例为rtL180M+rtM204V,1例为rtM/V207I突变,在49例慢性乙型肝炎患者中,共检出耐药位点13例(26.5%),其中5例为rtA181T,1例为rtL180M+rtM204V,2例为rtM/V207I,2例为rtN236T,1例为rtA181T+rtM204I+rts202N,1例为rtI169V+rtV173D,1例为rtM204I+rtM204L。结论在HBV携带者和慢性乙型肝炎患者均检出耐药位点变异,其意义有待于进一步探讨。     

拉米夫定治疗无良好应答患者乙型肝炎病毒P区突变与基因型的关系

目的 观察拉米夫定治疗后无良好应答的慢性乙型肝炎患者HBV P区变异情况与基因型的关系.方法 对631例拉米夫定治疗后无良好应答的慢性乙型肝炎患者进行研究.通过荧光定量PCR或核酸测序确定HBV基因型,直接测序观察P区突变,实时荧光定量PCR方法检测患者病毒载量,比较不同基因型患者的HBV DNA水平及HBV P区变异情况.计量资料采用成组设计资料t检验,计数资料采用x~2检验或Fisher精确检验.结果 631例慢性乙型肝炎患者中,B基因型HBV感染者272例,C基因型感染者359例,C基因型感染者患者年龄为(39.1±11.4)岁,明显大于B基因型感染患者的(33.7±9.7)岁(t=-6.55,P<0.01).C基因患者病毒载量为(5.96±1.22)log_(10)拷贝/ml,高于B基因型患者的(5.58±1.21)log_(10)拷贝/ml,t=-2.01,P<0.05.A181V/T变异在C基因型的发生率高于B基因型(0.4%比5.3%,χ~2=12.23,P<0.01),M204I/V,L180M、T184A/G/I/S、S202G/I和V173L变异发生率在B、C基因型之间差异无统计学意义(P值均>0.05).M204I在B基因型的发生率为20.6%,高于C基因型的13.9%(χ~2=4.91,P<0.05);M204V和M201Ⅳ变异在B、C基因型中的发生率差异无统计学意义(χ~2值分别为1.70和2.21,P值均>0.05).拉米夫定耐药发生率在B、C基因型间差异无统计学意义(χ~2=0.00,P>0.05).结论 拉米夫定常见耐药位点在B、C基因型之间无明显差异,但是C基因HBV感染患者病毒载量高于B基因型HBV感染患者;M204I变异在B基因型中出现频率高于C基因型,拉米夫定加用或改用阿德福韦酯后可能会使A181V/T变异在C基因型出现的概率高于B基因型;年龄、免疫因素和非常见位点的变异或许是影响拉米夫定疗效的重要因素.     

拉米夫定治疗前后乙型肝炎病毒YMDD变异的相关因素分析

目的 了解遵义地区HBV基因型以及拉米夫定治疗前后发生YMDD变异的相关因素,及早进行拉米夫定疗效及耐药的预测. 方法 53例慢性乙型肝炎患者分别在口服拉米夫定前及治疗后3、6、12、18、24个月进行血清HBV DNA定量、乙型肝炎标志物、ALT、AST、总胆红素,白蛋白的检测.同时在接受拉米夫定治疗前采用基因测序法检测HBV基因型及YMDD变异株,治疗后HBV DNA定量下降又反弹升高,且血清HBV DNA＞1×104拷贝/ml时,再次进行YMDD变异株检测.率的比较用卡方检验及确切概率法,两组均数之间比较采用独立样本t检验,有序变量之间的比较采用秩和检验.结果 遵义地区的HBV基因型由B、C及B+C基因型构成.拉米夫定治疗后18例检出YMDD变异株,用药1年和2年的变异率分别为15.1%和34.0%.HBV突变类型有rtL180M/M204V、rtL180M/M204I、rtM204I和rtL180M四种,其中C区rtM204V全部合并rtL180M突变(100%),C基因型中rtL180M/M204V联合突变及rtL180M/M204I联合突变明显高于B基因型(77.8%比25.0%及22.2%比12.5%);C基因型中未见点突变,而rtM204I、rtL180M的点突变仅见于B基因型.YMDD变异与未变异组性别、民族、乙型肝炎家族史及HBeAg情况差异无统计学意义(P＞0.05),病程≥2年组和年龄＜35岁组变异率明显升高(X2值分别为4.707和5.853,P值均＜0.05).不同HBV DNA滴度患者YMDD变异率差异无统计学意义(X2=0.801,P＞0.05),但HBV DNA＜105拷贝/ml者未发现YMDD变异.结论 拉米夫定治疗后YMDD变异可能与HBV基因型及P基因突变类型有关,并随治疗时间的延长而增加.为了减少YMDD变异的发生,应选用病程短、HBV DNA水平较低、肝损害较重的患者进行拉米夫定治疗,有条件的应检测HBV基因型.     

乙型肝炎病毒RT区原发性耐药变异的研究

目的探讨核苷(酸)类似物(NA)治疗前乙型肝炎病毒(HBV)RT区原发性耐药变异的发生情况。方法采用PCR产物直接测序技术对61例未接受NA治疗的慢性HBV感染者进行HBV RT区序列测定,分析NA治疗前HBV RT区原发性耐药变异的发生率。结果通过HBV基因扩增、PCR产物直接测序后,共获得61个HBV RT区基因序列,采用HBV RT区基因进化树基因分型共分出B型42例(42/61,68.85%),C型19例(19/61,31.15%),未检出其他基因型。通过核苷酸序列及氨基酸序列分析,对NA常见的10个耐药变异位点(rtI169T、rtV173L、rtL180M、rtA181V/T、rtT184G/S/A/I、rtA194T、rtS202I/G、rtM204I/V、rtN236T、rtM250V)进行比对后,61例患者中共检出HBV RT区变异3例(rtM204I 2例,rtM204I+L180M 1例),自然耐药变异的检出率为4.92%。结论慢性HBV感染者存在少量RT区原发性耐药变异位点,与原发性无应答有关。     

核苷类似物治疗慢性乙型肝炎患者的HBV序列测定及结果分析

目的 调查行核苷类似物治疗的慢乙肝患者HBV基因型及耐药位点的存在状况.方法 选择29例慢乙肝患者,均采用拉米夫定、阿德福韦、恩替卡韦、替比夫定等核苷类似物药物治疗6个月～2年.采用直接测序法检测患者血浆中的HBV基因分型及耐药位点,并对结果进行分析.结果 29例患者中,HBV基因C型26例、B型3例.HBV变异位点有6个,其中rtL180M 7例,rtM204I/V/S 11例,rtA181V/I/S 7例,rtN236T、rtT184A/G/I/S、rtM250V/L各1例.变异模式包括rtA181V/I/S 5例,rtM204L/V/S、rtL180M+rtM204I/V/S各4例,rtN236T、rtM204I/V/S+rtA181V/I/S、rtL180M+ rtA181V/I/S、rtL180M+ rtM204I/V/S+ rtM250V/L、rtL180M+ rtM204I/V/S+ rtT184A/G/I/S各1例.结论 29例慢乙肝患者的HBV基因多为C型,经核苷类似物治疗后,HBV的主要突变位点有rtM204L/V/S、rtL180M及rtA181V/I/S.除rtA181V/I/S、rtM204L/V/S单位点突变外,还存在rtL180M+rtM204L/V/S、rtL180M+ rtA181V/I/S、rtL180M+ rtM204I/V/S+rtT184A/G/I/S等突变模式,给治疗带来困难.     

乙型肝炎患者拉米夫定耐药株变异率快速检测

HBV感染是导致肝硬化和肝癌的重要因素,拉米夫定是抑制HBV的有效药物之一.但是,长时间应用拉米夫定会导致HBV出现YMDD区段发生变异从而产生耐药[1-2].HBV反转录酶的保守序列YMDD中蛋氨酸(M)被缬氨酸(V)或异亮氨酸(Ⅰ)所取代,变异后成为YVDD(rtM204V)或YIDD(rtM204I),使患者体内病毒复制反弹[3-4].     

Hepatitis B virus subgenotypes and basal core promoter mutations in Indonesia

AIM： To identify the distribution of hepatitis B virus （HBV） subgenotype and basal core promoter （BCP） mutations among patients with HBV-associated liver disease in Indonesia.
METHODS： Patients with chronic hepatitis （CH, n =61）, liver cirrhosis （LC, n = 62）, and hepatocellular carcinoma （HCC, n = 48） were included in this study. HBV subgenotype was identified based on S or preS gene sequence, and mutations in the HBx gene including the overlapping BCP region were examined by direct sequencing.
RESULTS： HBV genotype B （subgenotypes B2, B3, B4, 85 and B7） the major genotype in the samples, accounted for 75.4%, 71.0% and 75.0% of CH, LC and HCC patients, respectively, while the genotype C （subgenotypes C1, C2 and C3） was detected in 24.6%, 29.0%, and 25.0% of CH, LC, and HCC patients, respectively. Subgenotypes B3 （84.9%） and C1 （82.2%） were the main subgenotype in HBV genotype B and C, respectively. Serotype adw2 （84.9%） and adrq＋ （89.4%） were the most prevalent in HBV genotype B and C, respectively. Double mutation （A1762T/G1764A） in the BCP was significantly higher in LC （59.7%） and HCC （54.2%） than in CH （19.7%）, suggesting that this mutation was associated with severity of liver disease. The T1753V was also higher in LC （46.8%）, but lower in HCC （22.9%） and CH （18.0%）, suggesting that this mutation may be an indicator of cirrhosis.
CONCLUSION： HBV genotype B/B3 and C/C1 are the major genotypes in Indonesia. Mutations in BCP, such as A1762T/G1764A and T1753V, might have an association with manifestations of liver disease.     

HBeAg阳性患者中乙型肝炎病毒基因型分布及YMDD变异位点分析

目的通过研究乙型肝炎患者HBV基因型的分布及YMDD变异位点的分布特点,了解两者之间的关系,探讨临床意义。方法选取在我院门诊和住院部就诊的HBeAg阳性并经拉米夫定治疗两年以上的慢性乙型肝炎患者137例,用实时荧光PCR方法检测HBV DNA分型情况,用LDR方法检测YMDD位点变异情况。结果 137例患者中21例未能检测出基因型,其中男性17例,女性4例;其余116例标本确定了感染病毒的基因型,包括男性94例,女性22例;其中A型2例(1.72%)、B型19例(16.38%)、C型91例(78.45%)、D型4例(3.45%);分型成功率为84.67%(116/137)。116例标本中有41例患者的感染病毒中检测到了YMDD变异位点,变异发生率为35.35%。其中B基因型中检出7例(36.84%);C基因型中检出33例(36.26%);D基因型中检出1例(25.0%);A基因型中未检测出变异位点。B、C基因型中变异位点的检出率高于D型,但各基因型之间差异无统计学意义(χ2=1.392,P>0.05)。在41例变异中,其中I突变占60.9%,V突变占31.7%,I/V共生突变占7.4%。结论兰州大学第一医院所在地区患者中HBV基因型主要以C型为主,其次是B型,也有少量D、A型存在;B、C基因型的YMDD位点发生突变的几率相近。突变几率与基因型无关。     

YMDD mutations and genotypes of hepatitis B virus in northern China

The objective of this research was to determine the relationship between YMDD mutations and the genotypes of hepatitis B virus (HBV) during lamivudine treatment. HBV genotypes were determined by nested PCR with 6 pairs of HBV genotype-specific primers (A to F) in serum specimens from 142 hepatitis B patients receiving lamivudine antiviral therapy. YMDD mutations were detected by fluorescent hybridization bioprobe PCR and melting curve assay (FH-PCR-MC). Among 142 serum specimens, 13 samples were genotype B (9.2%), 125 samples were genotype C (88%), 4 samples were genotype D (2.8%), and 80 YMDD mutations were found. The YMDD mutation rates were 69.2 and 54.4% in genotype B and genotype C, respectively. There was no significant difference in the YMDD mutation rate between genotypes B and C. Nine genotype B sera with YMDD mutations were found, including 2 YIDD mutations and 7 YVDD (M + V) mutations. Sixty-eight genotype C sera with YMDD mutations were found, including 34 mutations I (M + I) and 17 mutations V (M + V). There was a significant difference in the YMDD mutation types between genotypes B and C. Our results suggested that the YMDD mutation rate was 56.3% in patients treated with lamivudine for 2-4 years. YIDD was the main mutation type. The YMDD mutation rate showed no significant difference between HBV types B and C (P > 0.05), while the YMDD mutation types showed a significant difference between HBV types B and C in Northern China (chi2 test = 4.6, P < 0.05).     

Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment

INTRODUCTIONHepatitis B virus (HBV) is one of the most common infectious diseases in the world. More than 300 million people worldwide are estimated to have chronic HBV infection. Ten percent of these patients will die as a direct consequence of persistent viral infection[1]. Nucleoside analogue therapy allows safe, long-term suppression of HBV and is a major milestone in the treatment of chronic hepatitis B. Lamivudine, the f irst of these agents approved worldwide, effectively supp…     

慢性乙型肝炎患者HBV基因型分布及其耐药基因突变分析*

目的 探讨慢性乙型肝炎（CHB）患者HBV基因型及其耐药突变发生情况。方法 纳入240例接受核苷（酸）类似物单药或联合或序贯治疗的CHB患者,采用PCR扩增HBV逆转录（RT）区和序列测定鉴定耐药基因突变,采用HBV S基因测序法鉴定基因型。结果 在35例单用拉米夫定治疗的CHB患者中,发生耐药突变14例（40.0%）,突变位点为rtL80I/V、rtVl73L、rtLl80M、rtM204V/I和rtV207I,23例单用阿德福韦治疗者发生耐药突变11例（47.8%）,突变位点为rtAl81T/V、rtS213T/N、rtV214A、rtQ215S/H/P、rtl233V、rtN236T、rtP237H和rtN/H238A/K/D/S,70例单用恩替卡韦治疗者发生耐药突变10例（14.3%）,突变位点为rtM204I,12例单用替比夫定治疗者发生耐药突变5例（41.7%）,突变位点为rtI169T、rtL180M、rtT184G/S/A/I/L/F、rtS202I/G、rtM204V和rtM250V/I/L,100例接受联合或序贯治疗者发生耐药突变51例（51.0%）,突变位点为rtA194T,恩替卡韦治疗患者耐药突变发生率最低（P<0.05）;240例CHB患者中,HBV基因B型21例（8.8%）、C型216例（90.0）和D型3例（1.2%）;在发生耐药突变的91例患者中,B型6例（6.6%）、C型83例（91.2%）和D型2例（2.2%,x²=1.22,P>0.05）;在发生耐药突变的6例B型感染者中有2例（33.3%）和83例C型感染者中有15例（18.1%）发生了多重耐药突变。结论 检测CHB患者感染HBV基因型并及时获得耐药突变基因分布,将有助于指导临床治疗。     

乙型肝炎病毒基因型、YMDD变异与拉米夫定治疗后HBV DNA反弹关系的研究

目的探讨HBV基因型、YMDD变异与拉米夫定抗病毒治疗后HBV DNA反弹的关系。方法应用多引物对巢式PCR法、PCR-序列分析法检测拉米夫定治疗的27例乙型肝炎患者和19例从未用过抗病毒治疗的患者HBV基因型和P区（YMDD）的突变位点。结果在27例HBV DNA反弹的患者中，13例（48．15％）检出YMDD变异，而对照人群无YMDD变异（P〈0．05）。YMDD变异的位点为rtM204V／I（C区）±rtL180M（B区）；在治疗组YMDD变异的患者中，B、C基因型构成比（46．15％和59．26％）与对照组（53．85％和68．42％）比较无显著性差异（P〉0．05）。结论YMDD变异是拉米夫定治疗后出现耐药导致HBV DNA反弹的主要原因；YMDD变异的常见位点依然为rtM204V／I（C区）±rtL180M（B区）；YMDD变异在B、C基因型病人中无差别。     

拉米夫定耐药HBV毒株RT区变异分析

目的研究拉米夫定(LAM)治疗后出现HBV病毒学突破患者HBVRT区变异位点和变异类型。方法研究对象选自2004年4月至2007年3月在苏州大学附属第一医院门诊或住院治疗的慢性乙型肝炎患者,用聚合酶链式反应(PCR)方法扩增LAM治疗后出现HBV病毒学突破患者的血清HBVRT区基因,对PCR产物直接测序,用Chromas2.0软件分析HBVRT区基因的核苷酸和氨基酸差异、变异类型。结果109例患者在拉米夫定耐药后出现病毒学突破,其中94例出现拉米夫定耐药相关性变异,包括YMDD变异93例,单独rtA181T变异1例。13例(11.93%)患者经测序分析未发现YMDD变异,但用限制性内切酶片段长度多态性(RFLP)方法检测,均发现有YMDD变异。测序结果发生变异位点和出现频率:rtM204V/I93例(85.3%)、rtL180M51例(46.9%)、rtV173L/M7例(6.4%)、rtV207M/L/I4例(3.7%)、rtA181T4例(3.7%)、rtT184I/S/M2例(1.8%)、rtM250L2例(1.8%)。变异类型:rtM204V/I、rtA181T、rtM204V/I rtL180M、rtM204V/I rtL180M rtV173M等。结论拉米夫定耐药主要变异类型为rtM204V/I变异,常伴随rtL180M和rtV173L/M变异;少数拉米夫定耐药患者在阿德福韦和恩替卡韦治疗前即已产生阿德福韦和恩替卡韦耐药相关性变异。     

Hepatitis B virus with the rtL80V/I mutation is associated with a poor response to adefovir dipivoxil therapy

Background/Aims: Lamivudine (LAM) resistance is frequently associated with various types of genomic changes in hepatitis B virus (HBV)‐DNA including YMDD mutations (rtM204V/I). We intended to examine the effects of these genotypic variants on the antiviral efficacy of adefovir dipivoxil (ADV) therapy. Methods: A total of 97 chronic hepatitis B (CHB) patients with YMDD mutants who had been treated with ADV for >12 months were analysed. Mutations of the entire polymerase domain of HBV were determined by direct sequencing. Results: All the 97 patients had genotype C HBV associated with rtM204V/I mutations; 63 (65%) rtM204I, 27 (28%) rtM204V and seven (7%) both. The rtL80V/I and rtL180M variants were identified in 66 (68%) and 67 (69%) patients respectively. The rtM204I and rtM204V variants were strongly associated with rtL80V/I and rtL180M respectively (P<0.01). There was no difference in antiviral response at 12 months after ADV therapy between patients in relation to the type of YMDD mutation or the presence of rtL180M. However, interestingly, after ADV therapy for 12 months, patients with rtL80V/I achieved a much smaller reduction in serum HBV‐DNA titre than those without it (mean, ?3.43 vs. ?4.43 log₁₀ copies/ml; P=0.018). In addition, patients with rtL80V/I had lower rates of undetectable HBV‐DNA (20 vs. 26%), alanine aminotransferase normalization (70 vs. 81%) and HBeAg loss (16 vs. 26%) than those without it, although none of these differences was statistically significant. Conclusions: These results provide evidence that rtL80V/I variants of HBV may be associated with a poor antiviral response to ADV in CHB patients with YMDD mutants.