乙型肝炎病毒rtN238H变异与阿德福韦酯耐药的相关性研究

Hepatitis B virus rtN238H variant infection with adefovir resistance in patients with chronic hepatitis B

目的阐明乙型肝炎病毒(HBV)多聚酶/逆转录酶区rtN238H变异对临床产生阿德福韦(ADV)耐药的影响。方法分析了1789例慢性乙型肝炎患者rtN238H变异的发生频率及其与ADV用药的关系;从典型病例患者血清中克隆获得HBV rtN238H变异株并通过回复定点突变获得对应的野生株,分别构建pTriEx-HBV1.1重组表达载体,瞬时转染HepG2细胞。给予转染细胞不同浓度ADV处理,用DNase消化游离DNA后,对上清中成熟病毒颗粒中的HBV DNA进行裂解定量以评价病毒的复制力。结果在1789例乙型肝炎患者中rtN238H变异为181例(10.1%),其中HBV B基因型感染为151例(83.4%),C基因型感染为30例(16.6%);ADV经治和未治患者分别为130例(71.8.%)和51例(28.2%),其中对ADV治疗产生临床应答(含部分应答)的占82.3%(107/130),无应答或出现病毒学反跳的占17.7%(23/130),但与野生株相比,rtN238H变异株对ADV的敏感性和病毒复制力无明显的改变。结论 rtN238H是HBV自然发生的多态性变异,对ADV敏感性和复制力无直接的影响。

Objective To elucidate the impact of hepatitis B virus rtN238H variant on adefovir resistance.Methods The rtN238H incidence and its relationship with adefovir（ADV）administration were analyzed in 1789 patients with chronic hepatitis B.The rtN238N variant from a representative case was cloned and its wild-type counterpart was acquired by reverse site-directed mutagenesis.pTriEx-HBV1.1 vetors harboring N238H variant or wild-type strain were respectively constructed and transfected into HepG2 cells.The cells were treated with serial concentration of ADV.After digesting uncoated DNA with DNase,HBV DNA in viral particles in culture supernatant was released and quantitated by real-time PCR.Results One hundred and eighty one（10.1%）patients were identified as having rtN238H,out of them,151（83.4%）were genotype B,and 30（16.6%）were genotype C;130 ADV-treated patients（71.8.%）had higher incidence of rtN238H variant than 51 ADV-untreated patients（28.2%）;However,compared to wild-type strain,rtN238H had similar sensitivity to ADV and viral replication competence in vitro.Conclusion The rtN238H is a naturally-developed polymorphism without direct impact on ADV sensitivity and viral replication.