各种恩替卡韦耐药HBV株鉴定及替诺福韦酯对其抑制力分析  

Identification of various entecavir-resistant HBV strains and analysis of the inhibitory efficacy of tenofovir disoproxil

作　　者：李艳乐[1] 刘妍[2] 许智慧[2] 王晓[2] 陈丽[2] 戴久增[2] 姚增涛[2] 江玲[2] 张连峰[1] 徐东平[2] 炎研究室 

机构地区：[1]郑州大学第一附属医院消化科,450000 [2]解放军第三○二医院全军传染病研究所/肝衰竭诊疗与研究中心病毒性肝炎研究室,北京100039

出　　处：《传染病信息》2013年第2期96-99,107,共5页Infectious Disease Information

基　　金：北京市自然科学基金面上项目(7132224);国家"十二五"科技重大专项(2012ZX10004503)

摘　　要：目的分析临床分离的不同形式恩替卡韦(ETV)耐药HBV株的病毒复制力并探讨替诺福韦酯(TDF)对其的抑制力。方法从4例ETV治疗失败的慢性乙型肝炎患者血清中提取并扩增HBV反转录酶(reverse transcriptase,RT)基因,克隆测序分析其基因型耐药变异特点。选取代表性野生型和ETV耐药型HBV克隆株,构建pTriEx-HBV(C)1.1倍重组表达载体。提纯质粒转染HepG2细胞系,5d后检测上清中HBVDNA产生量,分析不同变异形式ETV耐药变异株体外复制力水平及TDF对其抑制力。结果 4份血清中共得到16种含不同HBVRT区变异形式的克隆株,10种与ETV耐药有关,另有6种与拉米夫定耐药有关。表型分析显示:与野生株相比,ETV耐药变异株复制力水平均有所下降,其中rtT184I+M204I变异株复制力最低,是野生株的19.3%,rtM204I+M250L基础上出现补偿突变(rtL80I、rtV173L和rtL180M)的变异株复制力较高,存在相同补偿突变的前提下,rtM204I基础上的耐药变异较rtM204V基础上的复制力低。TDF对各种类型ETV耐药变异病毒的复制力均有较强抑制作用(96.11%～99.90%)。结论与野生株相比,不同变异形式的ETV耐药变异株体外复制力均下降,TDF可有效抑制ETV耐药变异株复制,将是治疗ETV耐药患者的一种很好的选择。Objective To analyze the replicative competence of HBV isolates with different entecavir-resistant （ETV-r） patterns and to investigate the inhibitory efficacy of tenofovir disoproxil （TDF） in vitro. Methods HBV DNA was extracted from the sera of 4 ETV-refractory patients with chronic hepatitis B. The reverse transcriptase （RT） gene was amplified by nested PCR. Geno- typic ETV-r mutation was analyzed by clonal sequencing, pTriEx-HBV （C） 1.1 expression plasmids containing HBV wild-type and ETV-r mutants were constructed and transfeeted into HepG2 cells. The replieative competence of ETV-r mutants and the inhibitory efficacy of TDF were analyzed by measuring the HBV DNA production in supernatant after 5 days of transfection. Results HBV mutants harboring 16 different mutational patterns in the RT region were identified from the 4 samples. Among them, l0 patterns were associated with ETV resistance, and 6 patterns with lamivudine resistance. Phenotypic analysis showed that all ETV-r strains exhibited reduced replieative competence compared to wild-type strains in vitro, rtT184I＋M204I strain showed the lowest replica- tive competence, only 19.3% that of wild-type strains, while the ETV-r mutants harboring complementary mutations（rtLSOI, rtV173L and rtL180M） to rtM204I＋M250L showed the relatively higher replicative competence. ETV-r mutants with rtM204I showed a lower replicative competence compared to rtM204V in conjunction with the same complementary mutations. In vitro phenotypic analysis showed that TDF could effectively suppress the replication of ETV-r strains with the inhibitory ratio of 96.11%-99.90%. Conclusions Various ETV-r strains show a lower replicative competence compared to wild-type strains in vitro. TDF can effec- tively suppress the replication of ETV-r strains and will be an optimal choice for ETV-r patients in clinical practice.

关 键 词：乙型肝炎病毒 突变 抗病毒药 抗药性 

分 类 号：R512.62[医药卫生—内科学]