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.