Method For HBV CccDNA Quantitation In Liver Tissue Of Patients With Chronic Hepatitis B And Its Clinical Application

Object: Hepatitis B virus covalently closed circular DNA(HBV cccDNA)is the original template for the replication of pre-genomic RNA(pgRNA)of HBV.It is of great significance for the replication of hepatitis B virus and the establishment of infection status.Previous literatures have reported some methods for quantitative detection of HBV cccDNA,each with its own advantages and disadvantages.This study intends to use the method of Fluorescence quantitative polymerase chain reaction(FQ-PCR)to detect the HBV cccDNA quantification.To evaluate the effect of antiviral treatment on the HBV cccDNA level,we used the FQ-PCR method in HBV related ESLD and CHB patients..Methods: using pHBV1.3-B6 plasmid containing the HBV gene as a positive control,we designed specific PCR primers and probes for HBV cccDNA,HBV Total DNA,and HBV pre-genome pgRNA quantification.We established a standard curve for FQ-PCR detection according to different copy number of positive control plasmid.We also established the standard curve for Jukart cells number and DNA quantification of the housekeeping gene ?-actin.The number of hepatocytes was then obtained by PCR method of ?-actin gene.Finally,HBV cccDNA,HBV Total DNA and HBV pgRNA quantification in each hepatocyte could be obtained through FQ-PCR method.The method was verified in HePGAD38 cells and THP-1 cells.The liver tissue samples of 5 patients with chronic hepatitis B(CHB)treated with nucleotides(NAs)were collected at baseline and at 48 weeks after enrollment.We also collected the liver tissues from 11 patients with end-stage liver disease(including cirrhosis and liver cancer).The relevant virological biomarkers including hepatocyte HBV cccDNA,HBV Total DNA and HBV pgRNA quantification and its significance were studied.Results:1.The level of HBV cccDNA,HBV Total DNA and HBV pgRNA were successfully detected in HePGAD38 cells [with the mean 3.89 copies/cell(IQR 3.44-4.49)? 1289.43 copies/cell(IQR 859.96-6038.50)and 6.42 copies/cell(IQR 5.06-7.85)respectively],while the negative control THP-1 cells failed to detect the relevant indicators.2.Among 11 patients with end-stage liver disease associated with HBV infection,the level of HBV cccDNA and HBV Total DNA in liver tuiss of patients with HBeAg-negative chronic hepatitis B treated by NAs were significantly lower than that in CHB patients without the treatment of NAs(respectively,P <0.0001;P=0.0004).The hepatocellar HBV cccDNA quantification in HBeAg-negative patients treated by NAs with serum HBV DNA-positive were higher than those NAs treated patients with serum HBV DNA-negative(P=0.0238).However,there was not significantly difference in the level of HBV Total DNA in ESLD between HBV DNA positive and HBV DNA negative patients(P>0.05).3.The median of HBV cccDNA,HBV Total DNA and HBV pgRNA in the liver tissues of 3 HBeAg-positive patients were respectively 0.93 copies/cell?1.62 copies/cell and 0.05 copies/cell.The liver tissues of 2 HBeAg-negative patients were detected.The median of HBV cccDNA,HBV Total DNA and HBV pgRNA were respectively 0.05 copies/cell?0.38 copies/cell and 0.01 copies/cell.The HBV cccDNA,HBV Total DNA and HBV pgRNA content in HBeAg-positive patients were higher than those in HBeAg-negative patients,but the difference was not statistically significant(P>0.05).After 48 weeks of continuing NAs antiviral therapy,the HBV cccDNA,HBV Total DNA and HBV pgRNA median in the liver tissues of 3 HBeAg positive patients were respectively 0.3 copies/cell?0.22 copies/cell and 0.06 copies/cell.The median of HBV cccDNA,HBV Total DNA and HBV pgRNA in liver tissue of 2 HBeAg negative patients were respectively 0.05 copies/cell ? 0.22 copies/celland 0.00 copies/cell.After 48 weeks of treatment,the HBV cccDNA and HBV total DNA levels in HBeAg-positive patients were lower than those at baseline,but the difference was not statistically significant(P>0.05).After 48 weeks of treatment,the HBV total DNA and pgRNA levels in HBeAg-negative patients were lower than those at baseline,but the difference was not statistically significant(P>0.05).Conclusion: The method for detecting liver tissue HBV cccDNA quantification was successfully established.Antiviral theatment could decrease the level of hepatic HBV cccDNA and HBV Total DNA quantification in HBeAg-negative ESLD patients.Hepatocellar HBV cccDNA quantification is an accurate and reliable indicators for evaluating the antiviral efficacy of patients with clinical chronic hepatitis B.The shortcoming of this study is that the sample size is small and it needs to be further expanded.