乙型肝炎病毒多重耐药的发生演变机制与优化治疗方法研究

Multi-drug resistant hepatitis B virus (MDR HBV) infection is a new challenge for hepatitis B management. This study is to clarify its developmental and evolutionary mechanism and explore optimal antiviral strategy for treating MDR HBV infection, on the base of the previous work. The study design is as follow. 1) MDR HBV strains are screened for large-size of clinical samples, and their evolution in viral pool is investigated by clonal sequence analysis of samples from long-term follow-up patients. The influence of different anti-HBV schedule on growth and decline of MDR HBV strains is analyzed. 2) Replication-competent amplicons harboring genes of MDR HBV strains and their wild-type counterparts are constructed for measuring viral replication capacity and drug susceptibility. Inhibitory effect of the combination use of nucleoside plus nucleotide analog on MDR HBV strains is determined by measuring viral replication capacity in the presence or absence of serially-diluted nucleos(t)ide analogs, i.e., adefovir plus lamivudine, adefovir plus entecavir, tenofovir plus lamivudine, and tenofovir plus entecavir. 3) Stable HBV-replicating cell line HepG2.A64 (entecavir-resistant) is long-termly treated with inadequate dose of adefovir, and cell line HepG2.B111 (adefovir-resistant) is long-termly treated with inadequate dose of lamivudine for inducing emergence of MDR HBV strains. HBV cccDNA is dynamically sequenced to monitor novel-emerged drug-resistant mutants to verify the developmental mechanism of MDR HBV. 4) Adeno-associated virus-mediated MDR HBV persistent infection model is established in mice for in vivo evaluating the inhibitory effect of different antiviral schedules on MDR HBV replication. The applicants have a solid accumulation in relevant studies, with advantage of key technique and abundant sample resources. Results will provide an important basis for optimal management of MDR HBV infection.

HBV多重耐药是乙型肝炎防治面临的新挑战。本研究拟在前期工作基础上阐明HBV多重耐药发生演变机制，探索最优化治疗方法。方案：1)大样本筛查多重耐药病例并长期随访，克隆测序分析多重耐药毒株演变规律；分析临床治疗方案对HBV多重耐药消长的影响。2)构建各种多重耐药毒株复制子，测定复制力和耐药性；测试药物联合(阿德福韦+拉米夫定或+恩替卡韦、替诺福韦+拉米夫定或+恩替卡韦)对多重耐药毒株的抑制效果。3)用亚有效剂量阿德褔韦、拉米夫定分别对HBV稳定复制细胞系HepG2.A64(恩替卡韦耐药)、HepG2.B111(阿德福韦耐药)长期加压诱导，对cccDNA动态测序，监测多重耐药变异株的发生演变，验证多重耐药发生机制。4)建立AAV介导的多重耐药HBV慢性感染小鼠模型，评价各方案对多重耐药HBV的作用。申报者研究基础扎实，具有关键技术和样本资源优势。结果将对科学防治HBV多重耐药感染提供重要依据。

多重耐药乙型肝炎病毒（MDR HBV）感染，给乙型肝炎防治带来新挑战。本研究拟在前期工作基础上，通过较大样本病例随访，结合临床、体外细胞与体内动物水平研究，明确MDR HBV发生演变机制，探索最优化治疗方法，结果具有重要的科学意义和实际应用价值。.重要成果：1. 首次鉴定了多种新型复杂耐药变异及表型特点，发现rtM204Q为拉米夫定（LAM）原发耐药变异，rtN236V、rtA181S为阿德福韦（ADV）原发耐药变异，rtI233V为ADV补偿耐药变异，为临床及时诊断ADV或LAM应答不佳患者的耐药变异病毒感染提供帮助。2. 在29639个真实临床HBV感染样本中首次鉴定了多种新型MDR HBV并明确其检出率，动态样本克隆测序率先揭示了MDR HBV的演变规律。表型结果显示多重耐药HBV株的体外复制力低于野生株，原发多重耐药突变位点越多病毒复制力越低而耐药性越强，TDF对MDR-ETVr(-)突变株的抑制效果优于ADV+ETV，TDF+ETV对MDR-ETVr(+)突变株的抑制效果优于TDF。首次明确体外用药抑制MDR病毒支持体内补救治疗策略，在国内外HBV防治研究领域产生了较大影响。3. 通过监测HepG2.2.15细胞内cccDNA逆转录酶基因序列，用含适量浓度ADV培养基持续培养110代成功诱导经典耐药突变rtA181V和rtN236T。4. 利用重组8型腺相关病毒载体携带1.3倍 C基因型HBV基因组体内转导C57BL/6小鼠，成功建立了稳定病毒复制并持续表达抗原的C基因型野生和多重耐药HBV小鼠复制模型。.本课题共发表学术论文15篇（其中8篇标注本基金第一资助号），包括SCI论文5篇（项目负责人均为第一或共同第一作者），中文核心期刊论文10篇（项目负责人为共同通讯或第二作者）；参加国内外学术交流16篇；培养研究生毕业3名；获专利授权1项。总体上完成了课题任务书指标。.