Characterization of hepatitis B virus genotypes in different stages of infection regarding their molecular virology in vitro and in vivo

Hepatitis B virus (HBV) infection is a major driver of liver disease, leading to hepatocellular carcinoma (HCC) and cirrhosis. Chronic HBV infection progresses through four distinct phases, with correct phase classification being critical for clinical decision-making. The presence of diverse HBV genotypes (Gt) and naturally occurring mutations influences disease progression, treatment responses, and viral protein expression. Notably, genotype G (GtG) has been associated with increased fibrosis risk in co-infected patients, necessitating further investigation into its molecular and clinical impact. This study analyzed chronic HBV infection phases using a genotype-stratified approach, with a focus on the benign phase 3 (HBeAg-negative infection) and the prevalence of GtG co-infections. While standard PCR genotyping failed to detect GtG in the German Albatros trial, targeted PCR identified a 4-8% prevalence in patients initially classified as infected with GtA or GtE. In vitro, co-expression of GtG/A and GtG/E enhanced G-specific core protein synthesis, reducing the core protein expression of the second genotype. Viral replication remained unaffected, but HBsAg expression and release were diminished for GtG/E. Patient-derived HBeAg-negative isolates (GtA, B, and D) exhibited reduced viral replication, intracellular and extracellular HBsAg levels, and endoplasmic reticulum retention of surface proteins compared to wildtype genomes. Reversion of the precore mutation (G1896A) increased intracellular HBsAg levels but did not enhance release. Additionally, patient isolates induced cytoprotective gene expression and displayed altered proliferative signaling, independent of HBeAg presence or specific mutations. No genotype-dependent differences in HBsAg composition or particle density were observed across infection phases. In conclusion, GtG co-infections predominantly occurred alongside GtA and E, with standard PCR methods failing to detect them adequately. GtG may exert a regulatory role through core protein interactions, potentially influencing disease severity. The implementation of GtG-specific screening could enhance clinical care for affected patients. HBeAg-negative isolates exhibited an antiproliferative phenotype, suggesting a selective advantage in later infection stages that may reduce disease progression and HCC risk.