In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection
In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection
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慢性乙型肝炎病毒感染肝内病毒学事件的原位分析
DOI：
10.1172/jci83339
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发表时间：
2016-03-01
影响因子：
15.9
通讯作者：
Yuan, Zhenghong
中科院分区：
文献类型：
--
作者：
Zhang, Xiaonan;Lu, Wei;Yuan, Zhenghong
持续性B型肝炎病毒（HBV）感染是通过在肝脏中形成共价闭合环状DNA（cccDNA）的核内池而建立的。关于HBV cccDNA在感染患者中的肝内分布，特别是在单细胞水平上的分布知之甚少。在这里，我们建立了一种高度敏感和特异性的ISH检测HBV RNA，DNA和cccDNA。我们的cccDNA探针组的特异性通过其严格的核内信号和一系列Southern印迹分析得到证实。使用我们的原位测定结合IHC或免疫荧光法发现了令人惊讶的病毒抗原和核酸的镶嵌分布。最引人注目的是，在HBV表面抗原阳性（IgA阳性）与HBV DNA和cccDNA阳性细胞之间发现了一种相互排斥的模式。对接受阿德福韦治疗1年的患者进行纵向观察，证实了病毒DNA的核库持续存在，尽管这些个体的细胞质DNA被有效耗尽。总之，我们用单细胞分辨率检测病毒核酸（包括cccDNA）的方法为监测慢性HBV感染中的肝内病毒学事件提供了一种手段。更重要的是，我们的观察揭示了HBV体内生命周期的复杂性。
Persistent hepatitis B virus (HBV) infection is established by the formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver. Very little is known about the intrahepatic distribution of HBV cccDNA in infected patients, particularly at the single-cell level. Here, we established a highly sensitive and specific ISH assay for the detection of HBV RNA, DNA, and cccDNA. The specificity of our cccDNA probe set was confirmed by its strict intranuclear signal and by a series of Southern blot analyses. Use of our in situ assay in conjunction with IHC or immunofluorescence uncovered a surprisingly mosaic distribution of viral antigens and nucleic acids. Most strikingly, a mutually exclusive pattern was found between HBV surface antigen-positive (HBsA-positive) and HBV DNA- and cccDNA-positive cells. A longitudinal observation of patients over a 1-year period of adeforvir therapy confirmed the persistence of a nuclear reservoir of viral DNA, although cytoplasmic DNA was effectively depleted in these individuals. In conclusion, our method for detecting viral nucleic acids, including cccDNA, with single-cell resolution provides a means for monitoring intrahepatic virological events in chronic HBV infection. More important, our observations unravel the complexity of the HBV life cycle in vivo.