Adaptive Evolution and Distinct Mutation Signatures of Full‐Length HBV Quasispecies in HBeAg‐Negative Chronic Hepatitis B

The evolutionary profile of hepatitis B virus (HBV) quasispecies may influence the clinical course of chronic hepatitis B (CHB), but few studies have characterized quasispecies according to hepatitis B e antigen (HBeAg) status. In this study, we analyzed 289 full-length HBV clones from 19 treatment-naïve CHB patients with long-term infection (> 10 years), comprising nine HBeAg-positive and ten HBeAg-negative, using molecular cloning and Sanger sequencing. Compared with HBeAg-positive patients, HBeAg-negative patients displayed higher quasispecies diversity (mean intrapatient sequence divergence 1.09% vs. 0.44%) and more complex phylogenetic structures. They also exhibited a greater number of positively selected sites, with 70.8% located within known T- or B-cell epitope regions, predominantly in the surface (S), polymerase (Pol), and X regions. Classical basal core promoter (BCP) and precore (PreC) mutations were detected in 52.8% of HBeAg-negative clones, often coexisting with wild-type strains. In patients lacking these classical BCP/preC mutations but showing sustained viremia, intrahost recombination was observed. Moreover, overlapping reading frames, particularly +1 frameshifts in Pol/S region, demonstrated asymmetric distribution patterns. In patients harboring deletion mutations, intact quasispecies were also maintained. Collectively, these findings reveal multiple adaptive strategies that sustain HBV replication and immune escape in HBeAg-negative patients, providing mechanistic insights for disease monitoring and therapeutic interventions.