Zhang Xinxin, Liu Chuanmiao, Gong Qiming, Zhang Shenying, Zhang Donghua, Lu Zhimeng, Wang Yuan
Department of Infectious Diseases, Rui Jin Hospital, Shanghai Second Medical University, Shanghai, China.
J Gastroenterol Hepatol. 2003 Dec;18(12):1353-7. doi: 10.1046/j.1440-1746.2003.03176.x.
Long-term lamivudine treatment for chronic hepatitis B virus (HBV) infection induces the emergence of lamivudine resistant HBV YMDD mutant strains. The aim of the present study was to observe the clone evolution of YMDD wild type and mutant strains in pretreatment and post-treatment samples during lamivudine therapy and analyze their clinical significance.
Ten serum samples (five before and five after 48 weeks of therapy) obtained from five patients chronically infected with HBV and treated with lamivudine were studied. Part of the HBV polymerase gene flanking the YMDD motif was sequenced after polymerase chain reaction (PCR) amplification. Meanwhile, 20-24 clones were selected at random from each sample and YMDD wild type and mutant strains were detected by real-time fluorimetry PCR established in our laboratory.
The YMDD mutants were not detectable in all five patients before treatment and were found in four patients after 48 weeks of therapy by sequencing directly on PCR products. Analysis of individual clones showed that the ratios of mutant strains in each of the five patients were 0, 9.5, 0, 4.5 and 5.6%, respectively, before therapy and 100, 100, 65, 100 and 0%, respectively, after 48 weeks of therapy. M552I was detected before therapy in one patient but M552V became the domain strain after therapy. Until 52 weeks of therapy, serum HBV DNA and alanine aminotransferase (ALT) breakthrough were found in two of the four patients with YMDD mutations. The fifth patient experienced breakthrough of ALT but HBV DNA remained undetectable.
The mutant strains of YMDD motif of HBV polymerase could be found in patients before lamivudine treatment, indicating that antiviral therapy allows the rapid selection of resistant strains. The replication ability of the M552V mutant strain might be stronger than that of the M552I mutant strain.
长期使用拉米夫定治疗慢性乙型肝炎病毒（HBV）感染会导致拉米夫定耐药的HBV YMDD突变株出现。本研究的目的是观察拉米夫定治疗期间治疗前和治疗后样本中YMDD野生型和突变株的克隆演变，并分析其临床意义。
研究了从5例慢性HBV感染且接受拉米夫定治疗的患者中获取的10份血清样本（治疗48周前5份，治疗48周后5份）。聚合酶链反应（PCR）扩增后，对YMDD基序侧翼的部分HBV聚合酶基因进行测序。同时，从每个样本中随机选择20 - 24个克隆，并用本实验室建立的实时荧光定量PCR检测YMDD野生型和突变株。
直接对PCR产物进行测序发现，所有5例患者治疗前均未检测到YMDD突变株，治疗48周后4例患者检测到YMDD突变株。对单个克隆的分析显示，5例患者治疗前突变株比例分别为0、9.5、0、4.5和5.6%，治疗48周后分别为100、100、65、100和0%。1例患者治疗前检测到M552I，但治疗后M552V成为优势株。治疗至52周时，4例YMDD突变患者中有2例出现血清HBV DNA和丙氨酸氨基转移酶（ALT）突破。第5例患者出现ALT突破，但HBV DNA仍未检测到。
在拉米夫定治疗前患者中可发现HBV聚合酶YMDD基序的突变株，表明抗病毒治疗可快速选择耐药株。M552V突变株的复制能力可能强于M552I突变株。
