Abstract
Hepatitis B virus (HBV) remains a major global health threat because covalently closed circular DNA (cccDNA) persists in hepatocytes and limits the efficacy of current antiviral therapies. Effective HBV research and drug screening require culture models that recapitulate the complete viral life cycle and allow for quantitative monitoring of replication. In this study, an 11-amino acid luminescent reporter, HiBiT, was inserted at multiple sites within the preS1 region of a genotype D HBV genome, and the C terminus of preS1 was identified as optimal for maintaining robust replication. We then established HepG2-B4 cells stably replicating HiBiT-HBV with HiBiT at the preS1 C terminus. Extracellular HiBiT activity and supernatant levels of HBV-DNA, HBsAg, and HBcAg increased continuously until day 42 and were reduced by nucleos(t)ide analog treatment, and cccDNA was confirmed by Southern blot analysis. Supernatants from HepG2-B4 cells infected naïve HepG2-NTCP cells and primary human hepatocytes, as shown by extracellular HiBiT activity. Transcriptome analysis revealed distinct gene expression changes in HepG2-B4 cells compared with parental HepG2 cells. These findings indicate that the HepG2-B4 system provides a rapid, quantitative, and scalable platform for HBV replication and infection studies and is suitable for mechanistic investigations and high-throughput antiviral screening.