Hepatitis B virus (HBV) cccDNA replicative but not transcriptional activity is significantly impaired in the liver of HBeAg-negative patients

Background/Aim: Knowledge of factors regulating covalently circular closed DNA (cccDNA) pool size and hepatitis B virus (HBV) production may help understanding mechanisms of viral clearance. To evaluate activity of cccDNA in chronically infected (CH-B) patients, we analysed serological parameters (HBV-DNA titres and HBsAg), as well as intrahepatic HBV DNA and RNA species in HBeAg-positive and HBeAg-negative patients. Methods: Sera and liver needle biopsy tissues from 80 CH-B patients. HBV transcript-specific primers were used to reverse transcribe pregenomic and sub-genomic HBV RNAs, as well as to quantify by real-time PCR and FRET-hybridisation probes, levels of total intrahepatic HBV-DNA, -RNA and cccDNA copy number, from each liver biopsy. HBsAg was quantified using the Laurell test. Results: We observed profound differences for all serological and intrahepatic parameters, when HBeAg-positive (n=33) and HBeAg-negative (n=47) patients were compared: median serum HBV-DNA (4E+07 vs. 2,5E+02; p<0,0001), HBsAg levels (35,5µg/ml vs. 6,9µg/ml; p<0,0001), intrahepatic HBV-DNA (137,2 copies/cell vs. 1,02 copies/cell p<0,0001) and cccDNA (3,1 vs. 0,1 copies/cell p<0,0001) in HBeAg-positive vs. HBeAg-negative patients were detected, respectively. Although HBeAg-negative patients had smaller amounts of cccDNA and total intrahepatic HBV DNA, we found a significantly higher proportion of cccDNA in the liver of these patients (9,9% vs. 1,7%; p=0,002), indicating reduced cccDNA replicative activity in HBeAg negative patients. Accordingly, the ratios between HBV-DNA viral titres and cccDNA copy number were also significantly lower in HBeAg-negative patients (3-log; p<0,0001). Interestingly, the ratio of HBsAg levels and cccDNA in HBeAg positive and negative patients were comparable (22 vs. 19), indicating that the transcriptional activity was equal in both groups. These findings could both be confirmed by the analysis of viral RNA transcripts. Our RNA data revealed that the cccDNA transcriptional activity was not different in both groups, whereas the replicative activity was significantly impaired in the liver of HBeAg-negative patients. Conclusions: We observed profound differences in term of viral production between HBeAg positive and negative patients, which appeared to be due not only to the lower amount of cccDNA, but also to an impaired replicative activity of the cccDNA in HBeAg-negative patients, despite a similar cccDNA transcriptional activity. HBV cccDNA