检测体外乙型肝炎感染形成的低拷贝数整合病毒DNA。

StephenW

J Vis Exp. 2018 Nov 7;(141). doi: 10.3791/58202.
Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection.
Tu T1, Urban S2.
Author information

1
    Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital; Gastroenterology and Liver Research Laboratory, Ingham Institute; [email protected].
2
    Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital; German Center for Infection Research (DZIF).

Abstract

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

PMID:
    30474630
DOI:
    10.3791/58202

StephenW

J Vis Exp。 2018年11月7日;（141）。 doi：10.3791 / 58202。
检测体外乙型肝炎感染形成的低拷贝数整合病毒DNA。
Tu T1，Urban S2。
作者信息

1
    海德堡大学医院分子病毒学传染病系;英厄姆研究所消化内科和肝脏研究实验室; [email protected]。
2
    海德堡大学医院分子病毒学传染病系;德国感染研究中心（DZIF）。

抽象

乙型肝炎病毒（HBV）是一种常见的血源性病原体，可引起慢性感染引起的肝癌和肝硬化。病毒通常通过附加型DNA中间体复制;然而，已经观察到HBV DNA片段整合到宿主基因组中，即使这种形式对于病毒复制不是必需的。 HBV DNA整合的确切目的，时间和机制尚不清楚，但最近的数据显示它在感染后很早就发生了。在此，详细描述了HBV DNA整合的体外产生和检测。我们的方案专门扩增病毒 - 细胞DNA整合的单拷贝，并允许结合序列的绝对定量和单碱基对分辨率。该技术已应用于各种HBV易感细胞类型（包括原代人肝细胞），各种HBV突变体，以及各种药物暴露。我们预见这种技术将成为确定这种临床相关现象的潜在分子机制的关键分析。

结论：
    30474630
DOI：
    10.3791 / 58202

newchinabok

StephenW 发表于 2018-11-27 18:33
J Vis Exp。 2018年11月7日;（141）。 doi：10.3791 / 58202。
检测体外乙型肝炎感染形成的低拷贝数整合病 ...

绝大多数有整合，如果整合碰到癌基因就会致癌，闻玉梅的观点