A novel method to precisely quantify hepatitis B virus covalently closed circular (ccc)DNA formation and maintenance

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.