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.

Background: Although immune complex formation is widely acknowledged as the etiological agent for the development of systemic lupus erythematosus, polyarteritis nodosa, reactive arthritis, etc., its roles in chronic hepatitis are less understood. This study aims to compare the immunohistochemistry profile of immune complex deposition in patients with chronic hepatitis B (CHB) and autoimmune hepatitis (AIH). Methods: Immunohistochemistry of C4d, a widely used marker for complement deposition was employed on liver biopsies from 72 and 15 patients with CHB and AIH, respectively. Statistical analysis was performed to analyze its prevalence and its association with a range of clinical and histological parameters. Results: Among the 15 AIH biopsies examined, C4d deposition was observed in 11 cases (73.3%), the majority of which showed a periportal staining pattern (10/11). In CHB, 61 (84.7%) of 72 cases tested positive for C4d, which did not differ significantly with that of AIH. While the periportal pattern was predominantly observed in CHB cases, positive staining in central veins, sinusoids, and hepatic parenchyma were also documented. In particular, C4d deposition is significantly associated with elevated serum ALT and liver inflammation in CHB. Of note, in specimens with a patchy parenchymal C4d staining pattern, a spatially correlated HBsAg IHC signal was observed in adjacent sections from the same tissue. Conclusions: These data suggest an involvement of immune complex-mediated immunopathy in autoimmune hepatitis and HBV-induced hepatitis. The positive intrahepatic C4d signal was associated with heightened liver inflammation. The colocalization of the C4d signal on hepatocytes with HBsAg strongly suggests a causal relationship between viral activity and complement deposition. These observations align with our recent evidence implicating the contribution of capsid–antibody complexes in the pathogenesis of CHB.