基于准种特点的乙型肝炎病毒基因型耐药和表型耐药的规律性研究

Hepatitis B virus (HBV) in an individual patient is a mixture of different viruses in genome, with characteristics of quasispecies. HBV's resistance to antiviral drugs is the result of dominant strains with mutations evolved under the selection pressures of antiviral drugs. The process of drug resistance has been ignored in the past time, while the focus is on "phenotypic drug resistance". This work will establish a practical RT-ARMS-qPCR method with high sensitivity and specificity based on the HBV quasispecies, the method will be used to monitor the YMDD mutation (rtM204I/V/S) and genotypic drug resistance; the phenotypic drug resistance detection method will be set up using viral vector and liver originated cell line. By analysing the relation of genotypic drug ressitance, phenotypic drug resistance and therapeutic efficacy, to investigate the molecular mechanisms, processes and its regularity of the develoment of drug resistance; to provide evidence for the prediction and prevention of drug resistance in practice; the establishment of in vitro drug sensitive analysis method is important for the selection of antiviral drug, it would be helpful for the prevention of drug resistance. YMDD mutation is essential for the drug resistance to nucleoside analogue, this study will provide experiences for the prediction and prevention of drug resistance caused by YMDD mutation alone or combined with other site muations.

每个患者体内的乙型肝炎病毒（HBV）都是由基因序列存在差异的病毒株组成的病毒群，具有准种( quasispecies)的特点。HBV耐药是在抗病毒药物的选择压力下突变株逐渐成为优势株的结果。以往的研究忽略了耐药发生的动态过程，只注意耐药这一"结局"。 本研究基于HBV的准种特点，在前期建立灵敏度高、特异性强、简便实用的RT-ARMS-qPCR方法的基础上，动态定量检测拉米夫定治疗过程中YMDD变异（rtM204I/V/S）的HBV DNA；利用质粒载体和肝细胞来源的细胞系建立实用的表型耐药的检测方法。通过分析基因型耐药－表型耐药－临床疗效三者间的关系，确定导致表型耐药的突变型HBV DNA的阈值。理论上探讨耐药发生发展的特点、机制和规律；实践中为预测和预防耐药、合理选择抗病毒药物提供依据。 YMDD变异是所有核苷类似物耐药的基础，本研究对单独该位点或合并其他位点耐药的预测和预防有重要意义。

HBV耐药分为基因型耐药和表型耐药，对二者的规律性研究具有重要意义。本研究建立了用于检测HBV YM(V/I)DD基因型耐药的RT-ARMS-qPCR和RT-AS-LNA-qPCR方法，并对其进行了详细的性能验证，该技术重复性好、敏感度高、特异性较强，检测范围宽；本研究用所建的方法监测了接受拉米夫定治疗的CHB患者YMDD野生及其耐药突变株的动态变化情况，初步探明了基于准种特点的基因型耐药的发生规律和过程，该研究对临床用药决策具有重要的指导意义。.另外，我们还成功建立了基于瞬时转染细胞模型的 HBV 表型耐药检测方法，用其检测了不同比例组成的 HBV 准种群在体外表型耐药的变化规律，并在此基础上评价了表型耐药检测技术在HBV准种群耐药检测的应用价值。.本项目理论上有望阐明HBV耐药发生发展的特点和机制；实践中可为预测和预防耐药、合理选择抗病毒药物提供依据。