Replicative but not transcriptional activity of HBV'cccDNA is impaired in HBeAg-negative patients

Background/Aim: Recent studies indicated that intrahepatic levels of both total hepatitis B virus (HBV) DNA and nuclear covalently closed circular DNA (cccDNA), the template of HBV transcription, differed significantly between HBeAg-positive and HBeAg-negative patients. To investigate whether viral replicative and transcriptional activity directly correlate with cccDNA levels and phase of infection, we analyzed HBV DNA and RNA levels in liver biopsies from 42 HBeAg-positive and 77 HBeAg-negative patients and correlated those values with serological parameters.

Methods: Intrahepatic DNA and RNA species were extracted simultaneously from 119 liver biopsies. HBV-specific primers were used to detect and quantify, by real-time PCR and FRET-hybridisation probes, levels of pregenomic and sub-genomic HBV RNAs, as well as total intrahepatic HBV DNA and cccDNA copy number, from the same liver biopsy specimen. HBsAg was quantified using the Laurell test.

Results: Intrahepatic median levels of pregenomic RNA were significantly higher (p<0,001) in HBeAg-positive patients (524 pgRNA copies/cell) compared to levels in HBeAg-negative patients (3 pgRNA copies/cell). Replicative activity varied significantly between the two groups, since HBeAg-positive patients produced considerably higher amounts of pgRNA per cccDNA molecule (214 vs. 40 pgRNA/cccDNA; p=0,005). Total HBV RNA contents (pgRNA + subgenomic RNA) directly correlated with cccDNA copy number (rSP=0,7) and therefore, where higher among HBeAg-positive patients (524 vs. 39 total HBV RNA copies/cell; p<0,001). Although HBeAg-negative patients had a lower proportion of pgRNA, levels of subgenomic RNAs per cccDNA molecule were similar in both groups (357 vs. 746 RNA copies/cccDNA), indicating that the production of envelope proteins depended on cccDNA levels. In fact, there was a good correlation between levels of serum HBsAg, total HBV RNA and cccDNA.

Conclusions: HBV replicative activity (pgRNA/cccDNA) was significantly lower in the liver of HBeAg-negative patients, while the transcriptional activity (subgenomic RNAs/cccDNA) was similar and correlated with cccDNA pool size, validating the notion that measurement of serum HBsAg levels may serve as non-invasive surrogate parameters to monitor intrahepatic cccDNA loads.