Phenotypic Assay of a Hepatitis B Virus Strain Carrying an rtS246T Variant Using a New Strategy
Phenotypic Assay of a Hepatitis B Virus Strain Carrying an rtS246T Variant Using a New Strategy
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使用新策略对携带 rtS246T 变体的乙型肝炎病毒株进行表型分析
DOI：
10.1002/jmv.22260
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发表时间：
2012-01-01
影响因子：
12.7
通讯作者：
Huang, Ai-long
中科院分区：
文献类型：
--
作者：
Hu, Jie-li;Cui, Jing;Huang, Ai-long
B型肝炎病毒（HBV）的表型检测在慢性B型肝炎患者长期核苷（酸）治疗过程中出现的耐药问题的研究中起着重要作用。目前可用的大多数表型测定系统依赖于将重组复制能力的HBV DNA转染到肝癌细胞系中。由于HBV基因组的序列异质性和独特结构，使用常规消化-连接技术克隆临床HBV分离株以产生可复制重组体可能非常困难。在这项研究中，开发了一种新的策略，用于构建HBV 1.1 x重组体。该策略的核心是“片段取代反应”（FSR）。FSR允许PCR片段不经消化或连接而被克隆，为从血清HBV DNA扩增的片段或基因组的克隆提供了新的工具，从而使得HBV表型的测定更加方便。使用这种策略，进行了一个表型分析的HBV菌株携带的rtS 246 T变异体分离的慢性乙型肝炎B，只有部分响应恩替卡韦治疗的患者。结果表明，该菌株在体外对恩替卡韦敏感。医学病毒学杂志84：34-43，2012. (C)2011 Wiley Periodicals，Inc.
Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long-term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication-competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion-and-ligation techniques to generate replication-competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1 x recombinant was developed. The core of this strategy is the "fragment substitution reaction'' (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro. J. Med. Virol. 84: 34-43, 2012. (C) 2011 Wiley Periodicals, Inc.