Detection and characterization of Hepatitis B virus double-stranded linear DNA-derived covalently closed circular DNA in chronic hepatitis B patients

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Background and Aims Hepatitis B virus (HBV) generates a double-stranded linear DNA (dslDNA) byproduct during replication. This dslDNA can undergo intermolecular and intramolecular nonhomologous end-joining (NHEJ) recombination, resulting in viral integration and dslDNA derived covalently closed circular DNAs (dsl-cccDNAs), respectively. The insertions and deletions (INDELs) at the end-joining site around the direct repeat (DR) 1 motif have been used to differentiate dsl-cccDNA from the authentic cccDNA. The frequency and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear. Methods HBV-targeted next-generation sequencing (NGS) was used to identify 32 dsl-cccDNA positive candidates, 22 HBeAg(+) and 10 HBeAg(-), from 56 liver biopsies of antiviral treatment-naive CHB patients for dsl-cccDNA confirmation and characterization by cccDNA-PCR NGS. INDELs within the DR2-1 region (nt 1600–1840) of the cccDNA were analyzed. Results Various clonally expanded, heterogenous ∼22-nt deletions in the X-gene were detected in all 32 samples, which are likely quasi-species from the authentic cccDNA. We, therefore, defined dsl-cccDNA only by the presence of INDELs clustered at the DR1 surrounding region (nt 1800–1840). The percentage of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples [11.32 (3.24–26.94)% vs. 7.72 (2.16–28.23)%, p=0.01]. The diversity of dsl-cccDNA species correlated with cccDNA levels (log-transformed; r=0.82, p<0.001), HBeAg(+) CHB (p<0.001), and serum HBV DNA (p<0.001). Conclusions dsl-cccDNA is more prevalent and heterogenous among the HBeAg(+) CHB subjects, which is likely due to the active viral cccDNA transcription and reverse transcription at the HBeAg(+) phase. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis. The potential impact of dsl-cccDNA in HBV therapeutic response deserves further assessment.