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

## TL;DR

This study identifies and characterizes a specific type of viral DNA in hepatitis B patients, which may contribute to immune evasion and disease progression.

## Contribution

The first characterization of dsl-cccDNA in liver samples from both HBeAg(+) and HBeAg(-) chronic hepatitis B patients.

## Key findings

- dsl-cccDNA is more abundant and diverse in HBeAg(+) patients compared to HBeAg(-) patients.
- A distinct 22-nt deletion mutant in the X gene region was discovered in the cccDNA population.

## Abstract

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.

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.

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.

Hepatitis B virus (HBV) double-stranded linear DNA-derived covalently closed circular DNA (dsl-cccDNA) is generated during viral replication through the nonhomologous end-joining (NHEJ) self-ligation pathway. This process often introduces insertions and deletions (INDELs) near the joint site, distinguishing dsl-cccDNA from authentic cccDNA. While most dsl-cccDNA species are replication-defective, some remain functional and may contribute to viral antigen expression, immune evasion, and HBV integration, complicating HBV pathogenesis. In this study, we confirmed the presence and abundance of dsl-cccDNA in the livers of 32 treatment-naïve chronic HBV (CHB) patients [22 HBeAg(+) and 10 HBeAg(-)] and characterized INDEL patterns by positions and lengths. We observed high INDEL heterogeneity near DR1, a defining feature of dsl-cccDNA, with greater dsl-cccDNA abundance and diversity in HBeAg(+) patients compared to HBeAg(-) patients. Additionally, we discovered a distinct 22-nt deletion mutant in the X gene region in cccDNA population. This is the first study to characterize dsl-cccDNA in liver samples from both HBeAg(+) and HBeAg(-) CHB patients. Our discoveries warrant further investigation into the potential impact of dsl-cccDNA on HBV pathogenesis and therapeutic outcomes.