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Updated: May 16, 2026

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Published on: November 7, 2018

Generation of HBV cccDNA using single-stranded M13 phage DNA for authentic minichromosome functionality.

Yumeng Li¹, Ting Hua¹, Menghan Hao¹

¹MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai, China.

Journal of Virology

|May 14, 2026

Summary

Researchers developed M13 phage-derived DNA to create authentic Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) models. This breakthrough enables better study of HBV cccDNA minichromosomes and antiviral drug development.

Keywords:

antivirals covalently closed circular DNA hepatitis B virus minichromosome ssDNA

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Published on: December 29, 2015

Area of Science:

Virology
Molecular Biology
Hepatology

Background:

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as nuclear minichromosomes, sustaining viral RNA transcription and persistent infection.
The low copy number and lack of authentic in vitro models for HBV cccDNA impede research and therapeutic development.

Purpose of the Study:

To develop a novel method for generating sequence-authentic HBV cccDNA in vitro.
To establish an authentic model for studying HBV cccDNA minichromosome functionality and host-virus interactions.

Main Methods:

A novel M13 phage-based system and deoxyribozyme were used to produce full-length single-stranded DNA (ssDNA) corresponding to HBV cccDNA strands.
ssDNA was cyclized and ligated to produce HBV cccDNA (McccDNA) in vitro.
McccDNA was transfected into hepatic cells to form minichromosomes and assess biological activity.

Main Results:

McccDNA successfully formed minichromosomes in transfected hepatic cells, recapitulating key HBV life cycle stages.
McccDNA accurately transcribed viral RNAs, mimicking infection-derived transcripts more faithfully than recombinant cccDNA.
The McccDNA model demonstrated sequence-specific nucleosome organization and responded to antiviral drugs, allowing for targeted mutagenesis studies.

Conclusions:

McccDNA generated from M13 phage DNA serves as an authentic in vitro model for HBV cccDNA.
This model recapitulates natural HBV cccDNA properties, facilitating the study of minichromosome functionality and antiviral strategies.