Episomal and integrated hepatitis B transcriptome mapping uncovers heterogeneity with the potential for drug-resistance

Hepatitis B virus (HBV) is a small DNA virus that establishes chronic infection and drives progressive liver disease and cancer; presenting a global health problem with more than 250 million infections. HBV replicates via an episomal covalently-closed-circular DNA (cccDNA) and integrated viral DNA fragments are linked to carcinogenesis. Current treatments only suppress HBV replication and there is a global initiative to develop genome targeting therapies, including siRNAs, antisense oligonucleotides and epigenetic modifiers specific for HBV cccDNA. However, our knowledge of the cccDNA and integrant transcriptomes is confounded by overlapping viral RNAs. Using targeted long-read sequencing we mapped the HBV transcriptome in liver biopsies from eleven treatment naïve patients. Probe enrichment yielded robust sequencing libraries and identified cccDNA-derived genomic and sub-genomic transcripts, and a repertoire of previously uncharacterised spliced, truncated and chimeric viral RNAs. Assigning viral transcripts to their respective DNA templates revealed differential promoter activity in cccDNA and integrants, with implications for the efficacy of epigenetic modifiers. Integrant-derived transcripts showed vast diversity in the viral-host junctions, posing a challenge for current nucleotide-targeting therapies. cccDNA was a source of genetic polymorphism, with distinct viral lineages present in the surface antigen encoding region, providing an insight into hepadnavirus evolution during chronic infection.