Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, USA.
Methods Mol Biol. 2024;2837:99-111. doi: 10.1007/978-1-0716-4027-2_9.
Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294 million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment. However, drug development targeting cccDNA formation and maintenance is hindered by the lack of sufficient biological knowledge on cccDNA, and of its reliable detection due to its low abundance and the presence of high levels of HBV DNA species similar to cccDNA. Here, we describe a Southern blot method for reliably detecting the HBV cccDNA even in the presence of high levels of plasmid DNA and other HBV DNA species, based on the efficient removal of plasmid DNA and all DNA species with free 3' ends. This approach also allows the detection of certain potential intermediates during cccDNA formation.
乙型肝炎病毒（HBV）感染仍然是一个全球性的公共卫生问题，全球约有 2.94 亿人慢性感染 HBV。由于其无法消除感染肝细胞内的 HBV 共价闭合环状 DNA（cccDNA），即病毒附加体，已批准的抗病毒药物很少能治愈慢性 HBV 感染。cccDNA 的持续存在是 HBV 感染慢性的基础，也是抗病毒治疗停止后频繁复发的原因。然而，由于对 cccDNA 的形成和维持缺乏足够的生物学知识，以及由于其含量低，并且存在与 cccDNA 相似的高水平 HBV DNA 物种，因此针对 cccDNA 的药物开发受到阻碍。在这里，我们描述了一种 Southern blot 方法，即使在存在高水平质粒 DNA 和其他 HBV DNA 物种的情况下，也能可靠地检测 HBV cccDNA，该方法基于有效去除质粒 DNA 和所有具有游离 3' 末端的 DNA 物种。该方法还允许检测 cccDNA 形成过程中的某些潜在中间体。
