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Updated: Jun 24, 2025
Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection
Published on: November 7, 2018
Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy
Zerui Yang1, Jingyan Zeng2, Yueyue Chen1
1Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, 400016, China.
Insights
Hepatitis B virus (HBV) DNA integration in plasma cell-free DNA (cfDNA) is detectable in patients with chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). An improved detection strategy reveals integration characteristics across disease stages.
Area of Science:
- Virology
- Genetics
- Oncology
Background:
- Hepatitis B virus (HBV) integration into host DNA is a key event in liver disease progression.
- Detecting HBV DNA integration in cell-free DNA (cfDNA) across different liver disease stages (CHB, LC, HCC) remains challenging.
Purpose of the Study:
- To develop and optimize a strategy for detecting HBV DNA integration in plasma cfDNA.
- To characterize HBV integration events in HBV-infected individuals with varying liver disease severity.
Main Methods:
- Developed an improved cfDNA detection strategy using DNA probe capture and next-generation sequencing.
- Validated the strategy in artificial DNA samples and HBV-infected HepG2-NTCP cells.
- Analyzed plasma cfDNA from 25 HBV-infected patients (CHB, LC, HCC).
Main Results:
- Successfully detected HBV integration with high sensitivity and accuracy.
- Identified 333 integration breakpoints in 88% of clinical samples.
- Observed significantly higher integration in HCC compared to CHB and LC patients.
- Integration breakpoints were randomly distributed on human chromosomes and enriched in the HBV genome around nt 1800, predominantly in gene-coding regions.
- Both NHEJ and MMEJ mechanisms were involved in HBV integration.
Conclusions:
- The optimized strategy enables sensitive detection of HBV DNA integration in plasma cfDNA.
- HBV DNA integration is detectable across CHB, LC, and HCC stages.
- Integration patterns and mechanisms provide insights into HBV-associated liver disease pathogenesis.
Abstract:
The landscape of hepatitis B virus (HBV) integration in the plasma cell-free DNA (cfDNA) of HBV-infected patients with different stages of liver diseases [chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC)] remains unclear. In this study, we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA, based on DNA probe capture and next-generation sequencing. Using this optimized strategy, we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection, with high sensitivity and accuracy. The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals (CHB, LC, and HCC) were further investigated. A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25 (88%) clinical HBV-infected samples, respectively. In vivo analysis showed that the normalized number of support unique sequences (nnsus) in HCC was significantly higher than in CHB or LC patients (P values < 0.05). All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800. The majority of integration breakpoints (61.86%) are located in the gene-coding region. Both non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) interactions occurred during HBV integration across the three different stages of liver diseases. Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients, including those with CHB, LC, or HCC, using this optimized strategy.
