Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as a reservoir for viral selection under antiviral treatment and the immune response, leading to the acute exacerbation and subsequent development of liver failure. However, there is no clear experimental evidence for a significant role of HBV quasispecies in viral pathogenesis. In the present study, HBV sequences were amplified from a patient with severe liver disease and constructed into HBV replication-competent plasmids. Western blot, Enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining were performed to analyze the expression, secretion and subcellular localization of viral proteins in vitro. Viral replication intermediates were detected by Southern blot. HBV gene expression and replication and the induction of specific immune responses in an HBV hydrodynamic injection (HI) mouse model were investigated. The results demonstrated that two naturally occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS expressed only a non-exportable hepatitis B surface antigen (HBsAg) with abnormal intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4 (SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg were induced in mice by the HBV variants when co-applied by HI. These findings uncovered an unexpected aspect of HBV quasispecies: that the coexistence of different variants can significantly modulate specific host immune responses, representing a novel mechanism for the immunopathogenesis of HBV infection.
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