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Updated: Mar 11, 2026
Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection
Published on: December 15, 2023
Simple and Reliable Method to Quantify the Hepatitis B Viral Load and Replicative Capacity in Liver Tissue and Blood
Claudia Minosse1, Sabrina Coen1, Ubaldo Visco Comandini2
1Laboratory of Virology, National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
Insights
A new method accurately quantifies hepatitis B virus (HBV) DNA in patients, revealing treatment impacts viral replication but not long-term colonization. This aids in understanding chronic hepatitis B (CHB) and personalizing treatment strategies.
Area of Science:
- Hepatology and Virology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Chronic hepatitis B (CHB) requires understanding intrahepatic viral dynamics for functional cure.
- Intrahepatic covalently closed circular DNA (cccDNA) is the key marker of HBV infection and parenchymal colonization.
- Elucidating relationships between cccDNA and peripheral markers can avoid invasive liver biopsies.
Purpose of the Study:
- To develop and validate a reliable method for quantifying HBV cccDNA and total DNA in clinical samples.
- To establish a straightforward assay for intracellular HBV molecular markers.
- To enable better assessment of viral load and treatment response in CHB patients.
Main Methods:
- Development of a plasmid construct as an isomolar multi-standard for HBV quantitation and cellular normalization.
- Validation of a real-time assay for specific cccDNA quantification.
- Analysis of liver biopsies and peripheral blood cells (PBMCs, granulocytes) from CHB patients.
Main Results:
- A novel assay accurately quantifies HBV cccDNA and total DNA in various clinical samples.
- Treatment significantly reduced HBV replicative capacity (total DNA/cccDNA) but had minimal effect on parenchymal colonization.
- Analysis of peripheral blood cells provided further insights into viral dynamics.
Conclusions:
- A validated, straightforward method for quantifying intracellular HBV molecular parameters has been developed.
- This assay can improve CHB prognosis assessment and guide tailored, time-limited treatment strategies.
- Widespread use of this assay facilitates a more rational approach to CHB management.
Background:
A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients.
Objectives:
The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples.
Patients And Methods:
Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays.
Results:
A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed.
Conclusions:
A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies.
