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

BACKGROUND AND AIMS: Hepatitis B virus (HBV) replication generates a double-stranded linear DNA (dslDNA) byproduct. 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 have been used to differentiate dsl-cccDNA from the authentic cccDNA. The prevalence and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear. APPROACH AND RESULTS: 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-naïve CHB patients for dsl-cccDNA confirmation and characterization by PSAD-cccDNA PCR NGS. INDELs within the DR2-1 region (nt 1600-1840) of the cccDNA were analyzed. Various clonally expanded, heterogenous ~22-nt deletions in the X gene region around nt 1760 were discovered in all 32 samples. The dsl-cccDNA species were then defined and characterized by the INDELs clustered at the DR1 surrounding region (nt 1800-1840). The proportion of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples. The diversity of dsl-cccDNA species positively correlated with cccDNA levels and serum viral load, and was higher in HBeAg(+) CHB. CONCLUSIONS: dsl-cccDNA is more abundant and diverse among the HBeAg(+) CHB subjects. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis.