乙型肝炎病毒非经典阿德福韦耐药相关突变rtN238T的临床分析与表型鉴定

Chinical and phenotipic analyses of untypical adefovir-resistanence-associated mutation rtN238T of hepatitis B virus

目的 本研究旨在分析乙型肝炎病毒（HBV）阿德福韦（ADV）非经典耐药相关突变的临床流行特点和表型耐药特征.方法 选取1741例慢性HBV感染者,PCR直序法检测7种非经典ADV耐药突变.对rtN238T突变患者进行动态克隆测序;构建rtN238T、rtA181V和rtA181V＋ rtN238T突变株的pTriEx-HBV 1.1重组载体,瞬时转染HepG2细胞,评价各病毒株的表型耐药特性.结果 rtN238T突变在应用ADV治疗患者中的检出率高于未治疗患者和其他核苷类药物治疗患者（χ^2=17.10,P＜0.01）.随访患者在接受ADV治疗47个月后发生病毒学反弹和生化学突破,并检出rtN238T＋ A181V和rtN238T克隆株;随后应用ETV治疗33个月,病毒载量和ALT恢复正常,患者体内病毒全部转变为野生株.rtN238T＋ A181V病毒株复制力显著高于rtA181V（t=9.54,P＜0.01）.与野生株相比,rtN238T＋ A181V突变株对ADV敏感性下降.结论 rtN238T突变可以增加ADV经典耐药突变株rtA181V的复制力,是一种ADV耐药相关的复制力补偿突变.

Objective To identify clinical prevalence of untypical adefovir-resistant mutations of hepatitis B virus （HBV）,and to analyze their phenotypic characteristics.Methods 1741 patients with chronic HBV infection were evolved.Untypical adefovir-resistant mutations were analyzed by direct sequencing.Longitudinal analysis was performed by clonal sequencing.Wild-type and mutant HBV genomic amplicons were constructed into pTriEx-HBV 1.1 vector and transfected into HepG2 cells.The replication capacity and the 50% effective concentration of drugs （EC50） were calculated.Results Patients treated with adefovir alone were more likely to develop rtN238T mutation than those treated with other nucleos（t） ide drugs （χ^2 =17.10,P 〈 0.01）.The patient received adefovir for 47 months,and then viral rebound and biochemical breakthrough occurred with detection of rtN238T ＋ A181V and rtN238T mutation.Switching-to entecavir therapy suppressed HBV DNA and ALT to an undetectable level and converted all viruses into wild type ones.The reulsts of viral replication capacity showed that rtN238T ＋ A181V strain was higher than rtA181V strain （t =9.54,P 〈 0.01）.Compared to the wild type virus,rtN238T ＋ A181V variant was relatively less susceptible to adefovir.Conclusions rtN238T mutation conferred no resistance to ADV but enhanced natural replication capacity,hence it might represent a novel compensatory drug-resistant mutation for adefovir.