乙型肝炎病毒rtA181T、rtN238T、基因型及预存耐药变异对临床核苷（酸）类药物耐药影响的研究

发布时间：2018-01-21 23:03

本文关键词： 乙型肝炎病毒基因变异耐药表型耐药分析　出处：《中国人民解放军医学院》2015年博士论文　论文类型：学位论文

【摘要】：背景我国目前有9300万慢性乙型肝炎病毒(hepatitis B virus, HB V)感染者,每年有30万人死于乙肝相关肝病。目前中国已上市的抗HBV药核苷(酸)类(nucleos(t)itde analogs, NA)类药物包括核苷类的拉米夫定(Lamivudine, LAM)、替比夫定(Telbivudine, LdT)、恩替卡韦(Entecavir, ETV)和核苷酸类的阿德福韦(Adefovir, ADV)、替诺福韦(Tenfovir, TDF)。NA药物能够有效抑制病毒,副作用小,但长期治疗过程中病毒易产生耐药变异。HBV耐药变异可引起病毒学突破和生化学突破,是抗HBV治疗失败或疗效不佳的重要原因,可以造成肝炎再发和肝病进展,甚至可引起暴发性肝衰竭导致患者死亡。本研究针对临床上HBV耐药变异中存在的难点和热点,综合利用临床大样本测序、动态随访、克隆测序、质粒瞬时转染细胞评价复制力和表型耐药特性等多种研究手段,重点对以往研究结果不够明确的HBV rtA181T、rtN238T 和 HB V基因型对耐药的影响进行了分析；同时,对以往争议较大的NA未治疗患者的预存耐药变异进行了研究。结果可以加深对我国临床实践中的HBV耐药变异情况的了解,向临床优化管理耐药变异提供重要依据。目的本研究旨在：①研究较大样本量NA经治慢性HBV感染患者rtA181T/sW172的临床流行情况,明确rtA181T变异的发生特点与临床意义。② 分析ADV非经典耐药变异的临床流行情况,对其中检出率较高的HBV变异毒株进行动态克隆分析和表型耐药检测。③ 阐明我国流行的HBV B基因型和C基因型病毒是否与耐药变异发生相关。④ 明确NA未治疗患者预存耐药变异的临床检出率,揭示预存耐药变异毒株的表型耐药特性和临床演变规律。方法(1)通过聚合酶链式反应(polymerase chain reaction, PCR)直接测序法对18419例慢性HBV感染患者进行逆转录酶(reverse transcriptase, RT)基因测序,明确rtA181T/sW172各种变异形式所占比例,分析不同rtA181T/sW172变异形式患者HBV DNA和表面抗原(hepatitis B surface antigen, HBsAg)水平的差异。(2)从第一部分病例中选取11143例单独应用NA药物治疗或未应用NA治疗的慢性HBV感染者,分析其7种ADV非经典耐药位点的变异情况,即rtV84M, rtS85A, rtV214A, rtQ215S, rt1233V, rtP237H和rtN238T：对1例rtN238T患者进行动态克隆测序分析;构建含有rtN238T+A181V、rtN238T、rtA181V和无耐药变异野生株的pTriEx-1.1-HBV载体,瞬时转染HepG2细胞,检测上清中的病毒复制中单位本以评价各病毒株的复制力和对核苷(酸)类似物的耐药性。(3)从第一部分病例中选取13847例经历过NA治疗的非肝癌(hepatocacinoma, HCC)患者,应用Mega 6软件构建系统进化树确定病毒基因型,分析不同基因型病毒中各种耐药变异的分布情况。(4)从第一部分病例中选取845例未经NA治疗的住院患者,确定原发性耐药变异的检出率；构建rtL80I+M204I、rtL180M+M204I 和 rtA181V+N236T3种变异株的pTriEx-1.1-HBV表达质粒,并进行复制力和表型耐药分析；克隆测序分析1例预耐药变异患者体内病毒的动态演变情况。结果(1)共检出750例(4.1%)rtA181T变异,其发生率呈逐年升高的趋势。发现rtA181T对应sW172共有9种变异形式,其中166例(22.1%)为sW172*单独存在,525例(69.9%)为sW172终止变异与野生株共存；不同sW172变异形式患者的HBV DNA水平和HBsAg水平并无差异。(2)在7种ADV非经典耐药变异中,rtN238T变异检出率在ADV治疗患者中显著高于应用其它NA药物治疗的患者。对1例患者动态克隆分析发现,发生rtN238T+A181V变异后,应用ETV治疗33个月,患者体内病毒全部转变为野生株。病毒株复制力检测结果显示rtN238T+A181V显著高于rtA181V (P0.01)。表型耐药分析显示,与野生株比较,rtN238T+A181V 和 rtA181V变异株对ADV敏感性显著下降,rtN238T变异株对ADV敏感,但1tN238T+A181V变异株较rtA181V株的复制力显著提高。(3)LAM相关耐药变异发生率为HBV/C型显著高于HBV/B型(31.67% vs.25.26%)。整体来看ADV耐药变异在HBV/C和HBV/B型之间并无差异(8.96%vs.9.87%)；具体来看,rtA181V的发生率为HBV/C型高于HBV/B型(5.29% vs.1.36%),但rtN236T变异则相反(2.70% vs. HBV/B 6.54%)。ETV药变异也有类似的现象,整体来看ETV耐药变异在HBV/C和HBV/B型之间并无差异(4.32%vs.3.62%);具体来看,rtM204V/I+rt184/202的发生率为HBV/C型高于H1BV/B型(3.66%vs.2.16%),但rtM204V/I+rt250变异则相反(0.67% vs.1.46%)。多重耐药变异发生率也为HBV/C型显著高于HBV/B型(0.83%vs.0.35%),主要变异形式是rtM204V/I+rtA181V(0.72% vs.0.30%)。(4)共有17例(2.01%)NA未用药患者检出原发耐药变异,直序法和克隆测序法结果显示所有患者均为耐药变异株与野生株共存。通过克隆测序共发现了13种耐药变异株,包括rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T, and rtN236T。表型耐药分析显示2种LAM预存耐药变异株rtL80I+M204I 和 rtL180M+M204V与野生株相比,对LAM的耐药倍数都大于1000;1种ADV预存耐药变异株 rtA181V+N236T 对 ADV的耐药倍数为15.4。动态随访发现,预存耐药变异rtM204I在ETV治疗后在病毒池中的比例上升(20%→85%)。结论rtA181T变异株在临床患者中常与rt181非变异株共存,单独出现该变异未对HBsAg和HBV DN A水平有明显影响;rtN238T变异可以增加rtA181V经典耐药变异株的复制力,是1种ADV耐药相关的复制力补偿变异;HBV/C基因型患者更易发生LAM和多重耐药变异;在NA未治疗患者中,预存耐药变异的检出率并不高,该类患者开始NA治疗时耐药检测并非必要。
[Abstract]:The background of our country at present has 93 million chronic hepatitis B virus (hepatitis B virus, HB V) infection, 300 thousand people died of HBV related liver disease each year. At present, China has listed the anti HBV drug class of nucleoside (acid) (nucleos (T) itDe analogs, NA) drugs include nucleoside Rami J Ding (Lamivudine, LAM) telbivudine, (Telbivudine, LdT), entecavir (Entecavir, ETV) and nucleotides (Adefovir, ADV), A Duff Vee Nuo Fuwei (Tenfovir, TDF).NA drugs can effectively inhibit the virus, the side effect is small, but the treatment process of long period virus resistance mutation can cause.HBV mutations and virological breakthrough biochemical breakthrough, is the failure of anti HBV treatment or poor efficacy of the important reasons, can cause hepatitis recurrence and progress of liver disease, even can cause fulminant hepatic failure leads to death of the patient. This study for HBV mutations in clinically difficult And hot spots, the comprehensive utilization of a large clinical sample sequencing, dynamic follow-up, cloning and sequencing of plasmid transfected cell evaluation replication and phenotypic resistance characteristics of a variety of research methods, research results are not clear HBV rtA181T focus on the past, the effects of rtN238T and HB genotype of V gene on resistance are analyzed; at the same time, the resistance variation of deposit the controversial NA pre untreated patients were studied. The results can enhance the resistance to HBV variation in clinical practice in China to provide an important basis to clinical optimization management of drug resistance. The purpose of this study aims at: 1. Study of larger sample size NA by clinical epidemiological treatment of chronic HBV infection in patients with rtA181T/sW172. Clear the characteristics and clinical significance of rtA181T mutation. The clinical analysis of the prevalence of drug-resistant mutations in non classical ADV, of which HBV variation strain high detection rate Dynamic analysis and cloning of resistant phenotype detection. HBV genotype B and genotype C virus illuminate epidemic in China is related to drug resistance mutations. The clinical definite NA patients with pre-existing resistance mutation detection rate, phenotypic resistance characteristics reveal pre-existing resistance variation of strains and clinical evolution method (1. Polymerase chain reaction (polymerase) by chain reaction, PCR) direct sequencing method in 18419 cases of patients with chronic HBV infection by reverse transcriptase (reverse transcriptase RT) gene sequencing, clear rtA181T/sW172 variation form of the proportion of cases, analysis of different rtA181T/sW172 variants in patients with HBV DNA (hepatitis B surface and surface antigen antigen, HBsAg) level the difference. (2) infection selected by NA alone in the treatment of 11143 cases of drug cases from the first part or application of NA for the treatment of chronic HBV, analysis of the 7 kinds of non classical ADV resistance Mutation sites, namely rtV84M, rtS85A, rtV214A, rtQ215S, rt1233V, rtP237H and rtN238T: dynamic analysis of cloning and sequencing of 1 rtN238T patients; to construct rtN238T+A181V rtN238T, rtA181V, and pTriEx-1.1-HBV carrier no resistance of wild strains, transient transfection of HepG2 cells detected in the supernatant of virus replication in this unit evaluation of the virus replication and drug resistance to nucleoside (acid) analogues. (3) a total of 13847 patients with non HCC experienced NA treatment from the first part of the cases in (hepatocacinoma, HCC) patients, viral genotypes should be determined by the Mega 6 software to construct phylogenetic tree analysis, distribution of various resistance variation genotype of the virus. (4) from the first part were selected in 845 cases without NA patients, to determine the detection rate of primary drug resistance; construction of rtL80I+ M204I, rtL180M+M204I and rtA181V+N The 236T3 mutant pTriEx-1.1-HBV expression plasmid, and analyzed replication and phenotypic resistance; dynamic cloning and sequencing analysis of 1 cases of drug resistance in patients with pre virus evolution. Results (1) were detected in 750 cases (4.1%) rtA181T mutation, its incidence was the trend of increased year by year. RtA181T sW172 has found the corresponding 9 kinds of variation form, of which 166 cases (22.1%) are separately for sW172*, 525 cases (69.9%) for the sW172 mutation and wild strains of HBV DNA coexist; and HBsAg level had no difference in different sW172 variant patients. (2) in the 7 kinds of non classical ADV mutation, rtN238T mutation detection rate in ADV treatment group was significantly higher than that of other NA drugs in the treatment of patients. In 1 patients with dynamic clonal analysis showed that the rtN238T+A181V mutation occurred after the application of ETV for 33 months, the virus in patients with completely converted to wild strains. Disease strain replication detection The results showed that rtN238T+A181V was significantly higher than that of rtA181V (P0.01). Phenotypic resistance analysis showed that compared with wild-type, rtN238T+A181V mutant and rtA181V sensitivity to ADV decreased significantly, rtN238T mutants were sensitive to ADV, but the 1tN238T+A181V mutant than rtA181V replication strains increased significantly. (3) LAM mutation incidence of HBV/C significantly higher than HBV/B (31.67% vs.25.26%). The whole ADV mutations between HBV/C and HBV/B had no difference (8.96%vs.9.87%); specifically, the incidence of rtA181V was higher than that of the HBV/B type HBV/C (5.29% vs.1.36%), but the rtN236T variation is opposite (2.70% vs. 6.54% HBV/B).ETV drug variant is also a similar phenomenon, the overall the ETV mutation between HBV/C and HBV/B had no difference (4.32%vs.3.62%); specifically, the incidence of rtM204V/I+rt184/202 was higher than that of the H1BV/B type HBV/C (3.66%vs.2.16%), but rtM204V The /I+rt250 variation is opposite (0.67% vs.1.46%). Multidrug resistant mutation rate for type HBV/C was significantly higher than that of HBV/B (0.83%vs.0.35%), the main form of variation is rtM204V/I+rtA181V (0.72% vs.0.30%). (4) a total of 17 cases (2.01%) of NA were detected in untreated primary resistance variation, direct sequence and clone sequencing method the results showed that all patients were mutant and wild strains coexist. By cloning and sequencing found a total of 13 resistant strains, including rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+, L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T, and rtN236T. analysis showed that the phenotypic resistance of 2 kinds of LAM pre existing resistance rtL80I+M204I and rtL180M+M204V strains and wild strains compared to multiple drug resistance of LAM are greater than 1000; 1 ADV pre existing resistance mutant rtA181V+N236T of A DV multiple drug resistance for the discovery of 15.4. dynamic follow-up, pre-existing resistance variation of rtM204I after ETV treatment in the virus pool increased (20% - 85%). Conclusion rtA181T mutation in patients with rt181 and non mutant often coexist, it appears alone did not have obvious influence on the variation of HBsAg and HBV DN A levels; rtN238T mutation can increase the replication capacity of classic rtA181V resistant mutant, replication is 1 ADV resistance compensation variation; HBV/C genotype were more susceptible to LAM and multidrug resistance variation; in untreated NA patients, the detection rate of pre-existing resistance variation is not high, the patients began treatment resistant NA the test is not necessary.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R512.62

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