96例急性乙型肝炎患者HBV多聚酶区的耐药变异分析  

Analysis of drug-resistant mutants in HBV polymerase domain in 96 patients with acute hepatitis B

作　　者：许智慧[1] 陈丽[1] 徐腾[2] 刘妍[1] 王耀[1] 韦柳婷[1] 任晓强[1] 貌盼勇[1] 徐东平[1] 

机构地区：[1]解放军302医院传染病研究所病毒性肝炎研究室,北京100039 [2]北京大学医学部公共卫生学院,北京100083

出　　处：《解放军医学杂志》2010年第6期639-641,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家"十一五"传染病重大专项子课题(2008ZX10002-011);国家重点基础研究发展计划课题(2007CB512803);北京市自然科学基金重点课题(7091006)

摘　　要：目的检测未接受过核苷(酸)类似物(NA)治疗的急性乙型肝炎患者感染的HBV是否存在耐药变异。方法收集96例急性乙型肝炎患者住院早期血清,提取HBV DNA,采用巢式PCR方法扩增HBV反转录酶(RT)全基因,对PCR产物进行DNA双向测序,对RT/S基因序列进行分子进化树分析反基因分型,对rt80、rt173、rt180、rt181、rt184、rt202、rt204、rt236和rt250等位点上的耐药相关变异进行分析,并用克隆测序法进行验证,每个样本测定10～20个克隆。结果用直接测序法检出NA耐药变异8例(8.3%),C型6例,B型2例。其中6例为拉米夫定(LAM)耐药变异,包括4例rtM204I、1例rtL80I+rtM204I和1例rtL180M+rtM204I;另2例检出与阿德福韦酯(ADV)耐药相关的rtA181V变异。变异株多数与野生型病毒株共存。克隆测序法的结果与直接测序法的结果大体相符,部分样本中有2种或多种耐药变异株共存,其中1例患者的样本中除LAM变异株外,还检出了ADV和恩替卡韦(ETV)耐药变异株,变异形式分别为rtA181T+rtN236T和rtL180M+rtS202G+rtM204V。结论未接受过NA治疗的急性乙型肝炎患者可以感染NA耐药株病毒,NA耐药病毒可以在人群中传播引起急性乙型肝炎,变异病毒的传播致病不只局限于LAM耐药株。Objective To investigate whether the resistant mutants exist or not in the patients with acute hepatitis B （AHB） untreated with nucleoside analog （NA）. Methods Sera were collected from 96 AHB patients early after their admission for HBV DNA extraction. The complete reverse-transcriptase （RT） genome region of HBV was amplified by nested PCR,and the PCR products were sequenced in bi-direction. HBV genotypes were classified by phylogenetic tree analysis based on RT/S gene sequences. Drug-resistance variations at positions of rt80,rt173,rt180,rt181,rt184,rt202,rt204,rt236 and rt250 were analyzed. The results of direct PCR sequencing were verified by clone sequencing with 10 to 20 isolates of each sample for screening. Results Direct PCR sequencing showed that 8 samples （8.3%） were positive for drug-resistant variants,among them 6 were with genotype C and 2 with genotype B viruses. Lamivudine-resistant variants were detected in 6 cases,including 4 harboring rtM204I,1 harboring rtL80I＋rtM204I and 1 harboring rtL180M＋rtM204I. The other 2 patients harbored rtA181V variant associated with adefovir resistance. Co-existence of the variants with wild-type virus was common. The results of clone sequencing were generally consistent with that of direct sequencing. The co-existence of two or more drug-resistant variants was found in some samples,including an isolate resistant to adefovir （rtA181T＋rtN236T） and an isolate resistant to entecavir （rtL180M＋rtS202G＋rtM204V） from one patient,except the lamivudine-resistant variant. Conclusions The NA-untreated patients with AHB may be infected by NA-resistant virus. NA-resistant HBV is transmissible to cause acute hepatitis B in Chinese population. The primary infection caused by viral transmission is not limited in lamivudine-resistant variants.

关 键 词：肝炎病毒 乙型 拉米夫定 恩替卡韦 药物耐受性 突变 

分 类 号：R512.62[医药卫生—内科学]