乙肝病毒是一种严重影响人类健康的病原体，也是引发慢性乙肝的元凶。根据国际卫生组织2015年估计，全球约有2.4亿慢性乙肝患者，大多分布在低收入及中等收入国家，每年约有78万患者死于慢性乙肝感染所致的肝衰竭、肝硬化和癌。在中国，约有9300万慢性乙肝感染者，其中，由乙肝病毒引发的肝硬化和肝癌患者比例分别高达60%和80%。随着乙肝疫苗接种的推行，我国婴幼儿的乙肝阳性率有了大幅度的降低。但是，对于其他感染者，依然缺乏彻底治愈的疗法，并且给患者家庭带来了沉重的经济负担和健康威胁。
大量临床与基础研究表明，慢性乙肝长期迁延不愈的根本原因在于乙肝病毒共价闭合环状DNA(cccDNA)在肝脏内的长期存在及其造成的病毒表面抗原血症。由于cccDNA在慢性乙肝患者肝脏中的水平极低，传统的分子生物学方法（如Southern印记、cccDNA特异性聚合酶链反应）不仅检测困难，而且无法显示其在组织内的分布特点。
鉴于目前对乙肝cccDNA研究存在的问题，来自复旦大学上海医学院医学分子病毒学重点实验室的Zhangxiaonan等研究者在bDNA信号扩增技术基础上进行二次开发，成功建立了一种能够在组织水平显示cccDNA分布的原位杂交技术。该技术还能显示乙肝病毒的其他复制中间体，如前基因组RNA(pgRNA)、松弛环状DNA(rcDNA)在单细胞水平的分布情况。研究者将该原位杂交技术与乙肝病毒主要抗原的免疫组化染色相结合，获得了病毒核酸与蛋白同时存在的图像。令人意外的是，乙肝病毒抗原与核酸在单细胞水平呈现惊人的“马赛克状”分布，即病毒的ccc DNA、RNA与表面抗原均显示显著的负相关关系。本研究还进一步证实，经过一年以上抗病毒治疗的患者的肝细胞内仍存在乙肝病毒的cccDNA。综合前期研究，本研究基于大量患者样本的观察结果，提出在单细胞水平乙肝病毒存在“抗原富集期”、“DNA富集期”和“潜伏期”的“三阶段”假说，推测患者体内肝细胞生理水平和病毒复制活跃度的变化可以导致这三个阶段的相互转换。该假说从单细胞水平描述了病毒生活周期的复杂变化模式，丰富了学术界对乙肝病毒在肝内存活的认识，为进一步针对不同病情患者清除乙肝病毒cccDNA的新策略提供了理论基础。
主要实验结果如下：
Figure 1. Validation of the specificity of the strand-specific probe sets design. Tissue sections from CHB patients were pretreated with the indicated nuclease and hybridized with the designated probe sets: (A–C) probe set1(original magnification, ×200; insets, ×400); (D–F)probe set 2 (original magnification, ×200; insets, ×400); (G–I) probe set3 (original magnification, ×400; insets, ×600). After hybridization and signal amplification, NBT/BCIP was used for colorization, followed by counterstaining with nuclear fast red.
Figure 2. Subcellular localization of HBV DNA signal is highly related to its replication status. Representative cytoplasmic (A) and nuclear (B) staining pattern of HBV DNA. (C) Serum HBV DNA levels from cytoplasmic- (n = 26) and nuclear-localized (n = 21) groups were compared. The P value was determined by a Mann-Whitney U test. (D) Comparison of the percentage of cytoplasmic- and nuclear-localized samples from HBeAg-positive and -negative patients.
Figure 3. Spatial relationship between intrahepatic viral nucleic acids (HBV DNA, RNA, and cccDNA) and surface antigen. Probe set 2 or 3 was hybridized with HBV DNA (A and C) or cccDNA (B and D), in conjunction with HBsAg IHC using adjacent sections from the same liver specimen. Images in C and D are higher magnifications of the boxed areas in A and B, respectively, which show similar regions in adjacent slices. HBV RNA was visualized with either probe set 1 (E) or probe set 3 (F) in the context of HBsAg expression using adjacent sections from the same liver specimen. Blue-purple, DNA, cccDNA, or RNA; brown, HBsAg. Original magnification, ×100 (A and B), ×400 (C and D), ×600 (C and D insets), ×200 (E and F), ×400 (E and F insets).
Figure 4. Highly complex distribution of HBsAg, HBcAg, and viral DNA. Liver sections from CHB patients were double stained with anti-HBsAg and anti-HBcAg, followed by visualization with DAB and permanent red, respectively. Results from a representative specimen are shown. Original magnification, ×200 (A), ×400 (B).
The image in B is a magnification of the boxed area in A. Double immunofluorescence staining of HBsAg and HBcAg from the same specimen was performed and analyzed with TissueQuest. (C) Scatterplot showing the expression of HBsAg and HBcAg. (D) Colocalization indices from 25 HBsAg and HBcAg double-positive specimens were calculated. (E and F ) ISH of HBV DNA with probe set 2, in conjunction with HBsAg and HBcAg double IHC. Original magnification, ×400 and ×600 (insets).
Figure 5. Relationship between HBV DNA and cccDNA. Whole-tissue scan of HBV DNA (A) and cccDNA (B) ISH from adjacent sections of 5 CHB liver specimens. One pair of representative images is shown. The selected regions were serially magnified (original magnification, ×200 and ×600) in the insets.
Figure 6. Distribution of HBV DNA and cccDNA before and after adeforvir therapy. Serial liver sections from 9 CHB patients before and 48 weeks after adefovir therapy were hybridized with probe set 2 or 3 to visualize HBV DNA (A and B) or cccDNA (C and D). Results from 1 representative patient (no. 3) are shown. Original magnification, ×200.
其中，HBsAg 和 HBcAg的免疫组化及免疫荧光实验由奥地利TG公司的TissueFAX系统采图，TissueQuest软件分析，该研究建立的乙肝病毒cccDNA原位杂交技术，不仅对基础研究具有较大意义，同时也具有广阔的应用前景。该方法可以用来监测慢性乙肝患者进行抗病毒治疗后cccDNA的清除情况，从而为评价抗病毒药物清除病毒的效果提供了重要的技术平台。目前，研究者正在对该方法进一步开发，将其作为乙肝病毒分子病理学检测方法进行推广，为临床医师综合评价患者肝脏内病毒学状态提供参考。