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Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection
Published on: November 7, 2018
Differential intrahepatic integrated HBV DNA patterns between HBeAg-positive and HBeAg-negative chronic hepatitis B
Daryl T-Y Lau1, Elena S Kim2, Zhili Wang3
1Liver Center, Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA dlau@bidmc.harvard.edu ying-hsiu.su@bblumberg.org guoh4@upmc.edu.
Insights
In chronic hepatitis B (CHB), integrated hepatitis B virus DNA (iDNA) is a key source of hepatitis B surface antigen (HBsAg) in HBeAg(-) patients. This suggests iDNA plays a crucial role in HBsAg production and disease progression.
Area of Science:
- Hepatology
- Virology
- Molecular Biology
Background:
- Hepatitis B surface antigen (HBsAg) production in chronic hepatitis B (CHB) can originate from intrahepatic covalently closed circular DNA (cccDNA) and integrated hepatitis B virus DNA (iDNA).
- Understanding the distinct roles of cccDNA and iDNA is crucial for managing CHB.
Purpose of the Study:
- To investigate and compare the levels and characteristics of cccDNA and iDNA in the liver tissues of treatment-naive CHB patients with different hepatitis B e antigen (HBeAg) statuses.
- To determine the primary source of HBsAg in HBeAg-negative CHB patients.
Main Methods:
- Analysis of liver tissues from 24 HBeAg(+) and 32 HBeAg(-) treatment-naive CHB participants.
- Quantification of cccDNA using real-time PCR after specific enzymatic digestion.
- Detection of iDNA and HBV-host junction sequences via HBV hybridization-targeted next-generation sequencing.
Main Results:
- HBeAg(-) CHB patients exhibited high HBsAg staining despite low cccDNA levels, indicating iDNA as a potential major HBsAg source.
- A significant proportion (78.1%) of HBeAg(-) CHB patients had >50% iDNA of total HBV DNA.
- iDNA integrations were frequently clustered in the DR2-DR1 region in HBeAg(-) CHB (52.4%), suggesting selection advantage.
Conclusions:
- iDNA is likely the primary source of HBsAg in HBeAg(-) CHB, contributing to high HBsAg levels.
- The preferential integration and expansion of iDNA at the DR2-DR1 locus in HBeAg(-) CHB may influence disease natural history.
- These findings highlight the importance of targeting iDNA for effective CHB therapy.
Background:
Hepatitis B surface antigen (HBsAg) can be derived from intrahepatic covalently closed circular DNA (cccDNA) and integrated hepatitis B virus (HBV) DNA (iDNA).
Objective:
We evaluated the cccDNA and iDNA from liver tissues of 24 hepatitis B e antigen (HBeAg)(+) and 32 HBeAg(-) treatment-naïve chronic hepatitis B (CHB) participants in the North American Hepatitis B Research Network.
Design:
For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove relaxed circular DNA and iDNA before real-time polymerase chain reaction. For iDNA detection, total DNA was subjected to HBV hybridisation-targeted next generation sequencing assay for identification of the HBV-host junction sequences. Comparisons of HBV cccDNA and iDNA with other virological biomarkers were assessed.
Results:
Intrahepatic cccDNA, serum HBV DNA, HBV RNA, hepatitis B core related antigen and quantitative hepatitis B surface antigen were higher in HBeAg(+) CHB. Intrahepatic hepatitis B core antigen staining was present in 87% HBeAg(+) but only 13% HBeAg(-) samples (p<0.0001). HBsAg staining was frequent in over 85% in both groups. 23 (95.8%) HBeAg(+) participants had ≤50% iDNA whereas 25 (78.1%) HBeAg(-) participants had >50% iDNA of total HBV DNA in their livers. For HBeAg(+) CHB, the iDNA integration sites were random with only 15.9% localised to the direct repeat 2 (DR2)-DR1 region. For HBeAg(-) CHB, 52.4% of the iDNA integrations were clustered at DR2-DR1. Microhomology-mediated end joining patterns of double-stranded linear DNA HBV integration was more frequent in HBeAg(+) livers.
Conclusion:
HBeAg(-) CHB was associated with high HBsAg staining concentration despite low cccDNA levels suggesting that iDNA was the primary source of HBsAg. The high frequency of DR2-DR1 iDNA distribution in HBeAg(-) CHB suggests the selection advantage and clonal expansion of this integrant in the natural history of CHB.
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