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Updated: Dec 13, 2025
Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection
Published on: December 15, 2023
A novel method to precisely quantify hepatitis B virus covalently closed circular (ccc)DNA formation and maintenance
Thomas Tu1, Benno Zehnder2, Bingqian Qu2
1Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany; Storr Liver Centre, Westmead Institute for Medical Research, Westmead Hospital and University of Sydney, Westmead, NSW, Australia.
Insights
A new assay, cinqPCR, accurately quantifies Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). This method enables precise measurement of cccDNA formation, crucial for developing new HBV therapies.
Area of Science:
- Virology
- Molecular Biology
- Hepatology
Background:
- Hepatitis B virus (HBV) infection persists due to its episomal genome, covalently closed circular DNA (cccDNA).
- Accurate quantification of cccDNA is challenging due to viral replicative intermediates.
- Understanding cccDNA formation is key to developing effective HBV treatments.
Purpose of the Study:
- To develop and validate a novel, precise assay for quantifying HBV cccDNA.
- To investigate the kinetics of cccDNA formation during HBV infection.
- To establish a screening method for anti-cccDNA therapeutics.
Main Methods:
- Development of a novel cccDNA quantification assay (cinqPCR) using restriction enzymes.
- Validation of cinqPCR against classical methods in vitro and in vivo (humanized mice).
- Application of cinqPCR to determine cccDNA formation timing and efficiency.
Main Results:
- cinqPCR provides accurate, sensitive, and specific absolute quantification of cccDNA without a standard curve.
- cccDNA formation was detected within 12 hours post-inoculation.
- cccDNA formation efficiency decreased after 28 hours post-inoculation despite ongoing viral DNA synthesis.
Conclusions:
- cinqPCR is a highly precise and sensitive method for HBV cccDNA quantification.
- The assay facilitates detailed studies on cccDNA formation kinetics.
- cinqPCR serves as an ideal platform for screening anti-cccDNA therapeutic candidates.
Abstract:
Hepatitis B virus (HBV) is the major cause of virus-associated liver disease. Persistent HBV infection is maintained by its episomal genome (covalently closed circular DNA, cccDNA), which acts as a template for viral transcripts. The formation of cccDNA is poorly characterised due to limited ability to quantify it accurately in the presence of replicative intermediates. Here, we describe a novel cccDNA quantification assay (cccDNA inversion quantitative PCR, cinqPCR), which uses restriction enzymes to invert a DNA sequence close to the gap region of Genotype D HBV strains, including the isolate widely used in experimental studies. Importantly, cinqPCR allows simultaneous normalisation to cellular DNA in a single reaction, provides absolute copy numbers without requiring a standard curve, and has high precision, sensitivity, and specificity for cccDNA compared to previous assays. We first established that cinqPCR gives values consistent with classical approaches in both in vitro and in vivo (humanised mice) HBV infections. We then used cinqPCR to find that cccDNA is formed within 12 h post-inoculation (hpi). cccDNA formation slowed by 28 hpi despite de novo synthesis of HBV DNA, indicating inefficient conversion of new viral genomes to cccDNA within infected cells. Finally, we show that cinqPCR can be used as a 96-well screening assay. Thus, we have developed an ideal method for testing current and future anti-cccDNA therapeutics with high precision and sensitivity.
