Hepatitis B virus (HBV) covalently closed
circular DNA (cccDNA) is responsible for viral persistence in the natural course of chronic HBV
infection and during prolonged
antiviral therapy and serves as the template for the production of HBV pregenomic
RNA (
pgRNA), the primary step in HBV replication. In this study, we have developed and applied sensitive and specific quantitative real-time polymerase chain reaction (PCR) assays for the measurement of intrahepatic concentration,
pgRNA production, and replicative activity of cccDNA in liver biopsy samples from 34 non-treated patients with
chronic hepatitis B (CHB); 12
hepatitis B e antigen (
HBeAg)(+) and 22
HBeAg(-). Median copy number for cccDNA was 1.5 per cell and for
pgRNA significantly higher, 6.5 copies per cell, with a good correlation between cccDNA and
pgRNA levels in all samples. In
HBeAg(-) patients, median values of cccDNA and
pgRNA levels were 10-fold and 200-fold lower than in
HBeAg(+), respectively, reflecting the differences in viral activity and clinical characteristics of the two groups. Furthermore, the replicative activity of intrahepatic cccDNA was significantly lower in
HBeAg(-) patients harboring mutant HBV strains than in
HBeAg(+) patients: median 3.5 versus 101
pgRNA copies per cccDNA molecule. In conclusion, the levels of both HBV cccDNA, a marker of HBV persistence, and
pgRNA, an
indicator of viral replication, in the liver of chronically infected patients correlate with viral activity and the phase of HBV
infection. The combined measurement of cccDNA and
pgRNA levels provides valuable information on the presence and replicative activity of intrahepatic HBV cccDNA.