"异质性"是高等生物的最根本特性之一,此特性在人类肝脏中也有充分体现。为探讨乙肝病毒核酸在感染患者肝脏中的分布特点，本研究组成功建立一种能够在单细胞水平显示病毒pgRNA,rcDNA和cccDNA的原位杂交方法。将该方法与病毒抗原免疫组化结合后，意外发现抗原与核酸呈现“马赛克状”分布。在单细胞水平存在抗原富集期,DNA富集期和潜伏期三个阶段。基于上述发现,本项目拟：进一步建立完善HBV核酸原位杂交与抗原免疫组化定量分析方案，进而探讨处于自然史各时期患者肝组织内核酸与抗原在单细胞水平的分布差异; 分析在抗病毒治疗前后肝组织中病毒核酸与抗原的变化趋势;利用激光显微切割技术分离不同阶段的肝细胞，二代测序获得表达谱，分析不同阶段肝细胞的信号通路差异，并进一步在组织水平进行验证。本研究将深化对HBV肝内生活周期复杂变化模式的理解，为最终清除cccDNA，治愈乙肝提供理论基础与分子靶标。

“Heterogeneity” is one of the inherent features of all multi-cellular organisms, which also holds true for the human liver. In order to examine the intrahepatic distribution of HBV nucleic acids, we developed an in situ hybridization (ISH) assay which was capable of visualizing HBV pregenomic RNA (pgRNA), relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA) at single-cell level. Unexpectedly, combination of HBV DNA ISH and viral antigen immunohistochemistry revealed a “mosaic” distribution. It was found that three stages, i.e., antigen-rich stage, DNA-rich stage and latent stage, co-existed inside the human liver. Based on these observations, the current project plans : 1. To Improve and establish a quantitative work-flow for in situ detection of HBV nucleic acids and antigens. Based on this methodology, a comprehensive survey on distrubution of each stages during the natural course of chronic hepatitis B infction will be donducted. 2. To evaluate the longitudinal changes in the positive rate of HBV nucleic aicds and antigens before and after antiviral therapy. 3. To enrich hepatocytes in different stages using laser capture and microdissection and to obtain global expression profile using next-generation RNA sequencing. The differential signaling network deduced from expression profiles will be further confirmed using in situ RNA and protein analysis. Collectively, the data generated in this project would be instrumental for a deeper understanding of the complex intrahepatic life style of HBV and for identification of novel molecular targets aiming to eliminate cccDNA and hence a final cure of HBV infection.