Experiment And Clinical Study Of HBV Integration And Quatitative Detection Of Intrahepatic CccDNA In Hepatitis B Patients

Hepatitis B virus (HBV) infection is a predominant global health concern with>350million individuals chronically infected worldwide. Approximately one million mortalities occur annually due to the long-term complications associated with HBV infection. The most important reason for virological breakthrough, relapse and HCC occurring in many HBV infected patients lies in the fact that covalently closed circular DNA (cccDNA) pool and HBV integration may persistently exist in the liver of these patients. In this study we established a simplified, specific SYBR Green I Real-Time PCR (RT-PCR) method to detect intrahepatic (IH) cccDNA levels in60patients, including acute hepatitis B (AHB) and chronic hepatitis B (CHB), in order to confirm a threshold of IH cccDNA levels which can predict the cure of CHB. Otherwise, we detected the general condition of HBV integration in part of the patients by a "short reads" whole genome paired-end deep sequencing.Part I The establishment of SYBR Green I RT-PCR method for cccDNA detectionObjectives: A simplified, specific and sensitive SYBR Green I RT-PCR for cccDNA detection was established.Methods: The SYBR Green I RT-PCR for cccDNA detection was improved in several ways such as PSAD degrading, the temperature of fluorescence measuring and the extracting of liver DNA.Results: A simplified, specific and sensitive SYBR Green I RT-PCR for cccDNA detection was established with the lower limit of24copies/μlConclusions: The establishment of simplified, specific and sensitive RT-PCR for cccDNA detection makes it possible to clinically detect IH cccDNA levels in a large number of hepatitis B patients.Part II Predictive value of IH HBV cccDNA in patients with acute hepatitis B and patients with chronic hepatitis B receiving anti-viral treatmentObjectives: This study aimed to investigate the predictive value of IH HBV cccDNA in acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients receiving anti-viral treatment. Methods:IH cccDNA and tDNA levels, serum HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and alanine aminotransferase (ALT) levels were detected in11AHB patients,46CHB patients receiving anti-viral treatment among which21patients remaining primary treatment failure,11patients achieving virological response (VR), and15patients achieving both VR and HBsAg seroclearance.Results:The median IH cccDNA and tDNA levels of the AHB patients (0.002copies/cell and0.04copies/cell) were significantly lower than those in the CHB patients. In the CHB patients, the median IH cccDNA level in those achieving both VR and HBsAg seroclearance (0.012copies/cell) was significantly lower than those remaining primary treatment failure (4.18copies/cell, P﹤0.0001) but not than those achieving VR (0.039copies/cell, P=0.169). The median IH tDNA level in those achieving both VR and HBsAg seroclearance (0.096copies/cell) was significantly lower than those remaining primary treatment failure (371copies/cell, P﹤0.0001) and those achieving VR (1.62copies/cell, P=0.001). No significant difference was found between the area under receiver operating characteristic curve (ROC) of IH tDNA and IH cccDNA levels to predict the likelihood of achieving both VR and HBsAg seroclearance (0.96and0.88, P>0.10). IH cccDNA levels was positively correlated with serum ALT (P=0.024), HBeAg (P=0.001) and IH tDNA levels (P﹤0.0001) but not with serum HBV DNA (P=0.12) and HBsAg levels either in HBeAg positive patients (P=0.84) or in HBeAg negative patients (P=0.146).Conclusions:IH cccDNA could persist in AHB patients and CHB patients achieving both VR and HBsAg seroclearance after anti-viral treatment. The detection of IH cccDNA levels had potential value to predict successful therapeutic response in CHB patients receiving anti-viral treatment.Part Ⅲ Detection of HBV integration in hepatitis B patients by whole genome sequencing and Sanger sequencingObjectives:To investigate the occurrence of HBV integration in HBV carriers, AHB patiensts and CHB patient receiving anti-viral treatment.Methods:HBV integration was detected in2HBV carriers,3AHB patients and13CHB patients, among which5patients remaining primary treatment failure,6patients achieving VR and2patients achieving both VR and HBsAg seroclearance by "short reads" whole genome paired-end deep sequencing. Then routine PCR and Sanger sequencing were used to verify the HBV integration breakpoints with reads N﹥=2and to detect the unknown sequences in the HBV integration breakpoints.Results: HBV integration occurred in all hepatitis B patients and the total number of integration sites was2083. While the average number of all patients was138.2±379.9, the higher numbers were248.5±57.3in patients remaining primary treatment failure and18.6±13.7in patients achieving VR respectively. Correlation analysis indicated that the number of integration sites was positively correlated with IH cccDNA levels, average coverage and IH HBsAg score (P<0.0001respectively).14HBV integration breakpoints with reads N＞=2were successfully validated by routine PCR and Sanger sequencing, among which integration breakpoint Chrl6:51320015was found in all18patients.Conclusions:HBV integration can occurred in all hepatitis B patients, and the number of integration sites was positively correlated with IH cccDNA levels, average coverage and IH HBsAg score. Integration breakpoint Chr16:51320015was a major integration site (MIS), which may occur in many hepatitis B patients.Part IV Study of HBV-human chimeric transcription and quantitative detection of integration breakpoint Chr16:51320015Objectives:To study HBV-human chimeric transcription of14HBV integration sites been proved, and to quantitatively detect the copies of integration breakpoint Chr16:51320015in18hepatitis B patients.Methods: Reverse transcription PCR was used to detect HBV-human chimeric transcription of14integration breakpoints, and molecule clone and RT-PCR was used to detect the copies of integration breakpoint Chr16:51320015in18patients.Results: HBV-human chimeric transcription was found in integration breakpoints1,3,4,5,6,12and14, among which except integration breakpoint4was S gene and integration breakpoint6was pre-C gene respectively, all others were X gene in viral sequence. The average level of integration breakpoint Chrl6:51320015in18patients was1.46x10-2±4.94×10-2copies/cell (3.48×10-5～0.212copies/cell).Conclusion:X gene integration may be one of the most important conditions for HBV-human chimeric transcription, and HBsAg may be produced by integrated S gene. Clonal expansion with high copies of integration breakpoint Chr16:51320015persisted in all18patients.