Single-Cell Hepatitis B Sequencing Reveals Distinct Viral Infection Events Consistent With Superinfection

BACKGROUND: The high error rate of the hepatitis B virus (HBV) polymerase leads to a genetically diverse quasispecies in individuals with chronic hepatitis B (CHB). Data regarding the propagation of these variants in individual hepatocytes may provide insight into viral replication and diversity of covalently closed circular DNA (cccDNA), which is the template for HBV replication. METHODS: We developed sequencing protocols to characterize HBV diversity between and within hepatocytes using RNA extracted from individually isolated hepatocytes. We sequenced HBV in >200 hepatocytes in four liver biopsies from people with HIV/HBV (HB1, HB2, HB3, HB6). RESULTS: We found that two biopsies (HB1 and HB6) showed HBV diversity between hepatocytes that met an experimentally identified threshold. Specifically, in HB1, HBV sequences from 86 individual cells were from two different haplotypes of genotype D: 70.2% of cells with haplotype a, 5.8% with haplotype b, and 24% with both haplotypes in the same cell. Furthermore, within single hepatocytes, up to three different HBV sequences were present per cell, including some cells with both genotypes A and D. HB6, who received years of lamivudine monotherapy, had evidence of drug-resistance (DR) mutations distributed among hepatocytes and demonstrated up to three different sequences, including wild-type and drug-resistant sequences, in the same hepatocyte. Modeling of infected hepatocytes did not reveal evidence of local HBV spread based on spatial proximity. CONCLUSIONS: Taken together, our findings demonstrate that individual hepatocytes may harbor multiple, transcriptionally active HBV cccDNA molecules, which likely arose from distinct infection events.