Background and Aims: Hepatitis B virus (HBV) infection exhibits marked cellular heterogeneity, which conventional poly(A)-based single-cell RNA sequencing fails to resolve this heterogeneity owing to the overwhelming host transcript background. To overcome this, we developed B-BEST (HBV can BE Seen on host Transcriptome), a targeted sc/snRNA seq approach using custom beads conjugated with HBV specific probes to simultaneously quantify five viral genomic regions (S, X, pgRNA, rcDNA, cccDNA). Approach and Results: We validated B-BEST in HepAD38 cells and integrated it with long-read sequencing, spatial transcriptomics, and in situ hybridization in liver tissues from treatment-naïve patients and antiviral treated humanized liver chimeric (Hu-URG) mice. B-BEST revealed significant heterogeneity among HBV-positive hepatocytes. In HBeAg-positive patients, HBV-positive subpopulations enriched for hepatic synthesis/metabolism and mitochondrial function were linked to active viral replication and transcription, with only a mild type I interferon response. Severe inflammation correlated with suppressed HBV replication. Long-read sequencing indicated that integrated HBV transcripts preferentially used host promoters and contributed to HBsAg persistence in HBeAg-negative patients. In Hu URG mice, entecavir upregulated metabolic pathways, while peginterferon alfa-2b induced broad spectrum antiviral programs. Notably, clonal expansion of hepatocytes diluted the intrahepatic viral reservoir when viral replication was inhibited, suggesting a proliferative dilution mechanism that may contribute to functional cure. Conclusions: In summary, our B-BEST platform provides resources for delineating the heterogeneous landscape of HBV infection, identifying host determinants and microenvironmental factors that govern viral replication and persistence, and highlighting hepatocyte proliferation as a potential clearance mechanism for antiviral therapy.
Huang et al. (Mon,) studied this question.