Visualizing in situ viral replication across the natural history of chronic HBV infection

Background and Aims: Chronic HBV infection evolves through different phases. Interactions between viral replication and the host immune response in the liver underlie the pathogenesis of this disease. The aim of this study was to directly visualize the HBV replication intermediates at a single-cell resolution inscribed on morphological changes corresponding to disease activity. Methods: A set of archived formalin-fixed paraffin-embedded liver needle biopsies from treatment-naive patients were collected and categorized into phases according to the American Association for the Study of the Liver Diseases (AASLD) guidelines. HBV RNA and DNA were detected using in situ hybridization assays. Results: The hepatocytes were ubiquitously infected in subjects with immune tolerance, and their percentage was gradually decreased in immune-active and inactive chronic hepatitis B phases. HBV-infected hepatocytes were prone to localize close to fibrous septa. The subcellular distribution of signals was able to distinguish hepatocytes with productive infection from those harboring HBV integrants and transcriptionally inactive covalently closed circular DNAs. A smaller number of hepatocytes with productive infection and more harboring transcriptionally inactive covalently closed circular DNA or HBV integrants became apparent in the inactive chronic hepatitis B phase. Conclusion: An atlas of in situ characteristics of viral-host interactions for each phase is described, which sheds light on the nature of viral replication and disease pathogenesis among the phases of chronic HBV infection.