Experimental And Clinical Study On Dynamic Detection Of Hepadnaviral Covalently Closed Circular DNA In Serum

An important development in the research on hepatitis B virus (HBV) was the discovery that human HBV virus is the prototype for a family of viruses with common biological characteristics, which is referred to as hepadnaviridae. Covalently closed circular DNA(cccDNA), serving as the original template of hepadnavirus replication, is the first replicative intermediate emerging during the life cycle of hepadnavirus, which indicates establishment of viral infection and origination of the replication cycle. A stable pool of cccDNA, varying from 5 to 50 copies per cell, has been found in each nucleus of persistently-infected hepatocyte, and is thought to be one of the main factors responsible for reoccurrence of chronic hepatitis B after withdrawal of antiviral agents. Therefore, determination of HBV cccDNA does not only play an vital role in the development and evaluation of antiviral agents, but also may provide a useful guide to the efficacy of antiviral treatment and the likelihood of long-term response in patients with hepatitis B, and may aid in the decision of when to cease therapy. However, quantification of HBV cccDNA seems to be too far from clinical application until now, presumably because cccDNA only exists in the nuclei of hepatocytes and detection of cccDNA is exclusively dependent on specimens from liver biopsy, which makes it not so convenient and acceptable as to monitor dynamics of serum HBV load.Fortunately, it seems promising to bridge the gap between laboratory research and clinical application of monitoring the HBV cccDNA dynamics based on a few reports published in recent years, which suggested that HBV cccDNA could occur in the peripheral blood and was significantly correlated to the serum ALT level, viral load as well as to intrahepatic total HBV DNA, and could be dynamically depressed by antiviral agents such as lamivudine. Yet doubt on the insufficiently demonstrated specificity of their assays to detect HBV cccDNA posted an unavoidable challenge to the reliability of their conclusions. In this research, we aim to confirm whether cccDNA is detectable or not in the serum of Shanghai brown ducks with DHBV infection and of patients with hepatitis B, based on a specific and sensitive strategy to determine HBV cccDNA developed by our laboratory.This research was composed of three parts:Ⅰ. Comparative study on the clearance dynamics and stability of hepatitis B virion and cccDNA in the blood;Ⅱ. Dynamic detection of duck hepatitis B virus cccDNA in the serum of ducks with liver injury;ⅢDynamic detection of hepatitis B virus cccDNA in the serum of patients with hepatitis B.PartⅠComparative study on clearance dynamics and stability of hepatitis B virion and cccDNA in the bloodObjectives To confirm whether it is feasible to detect HBV cccDNA based on analysis of its clearance dynamics and stability in the blood of ducks. Materials and Methods①twelve 1-week-old ducklings were averaged into group A and group B, and each duckling was intravenously injected with 1 milliliter of serum containing 1.2×109 copies of HBV virion (group A) or plasmid pBS HBV 3.6Ⅱ(group B). At 0.25h, 2h, 4h, 6h, 8h, 12h, 24h and 32h after injection, serum HBV DNA level in each duckling was quantified by fluorescent PCR.②fifty microliter of plasma containing 1.2×108 copies/ml of HBV virion or pBS HBV 3.6Ⅱ(cccDNA) were treated by 5 units of RQ1 DNase at 37℃for 1h, then the difference of serum HBV DNA before and after treatment by DNase was documented.③one and half milliliter of plasma containing 1.2×108 copies/ml of HBV virion or pBS HBV 3.6Ⅱ(cccDNA) were incubated at 37℃for 4h, 8h, 12h, 16h, 24h, 2d, 3d, 4d, 5d, 6d and 7d, and stability of HBV virion or plasmid(cccDNA) in the plasma was dynamically analyzed by fluorescent PCR. Results①No significant difference is observed in the haemodynamics between HBV virion and cccDNA, both of which are characteristic of one-phase exponential decay, with a mean half-life 4.536±0.769 h and 4.511±0.791 h, respectively. Both of them could be detected from 0.25h to 24h after intravenous administration, and became undetectable at 32h.②In the plasma, cccDNA could be degraded by two log10 when treated with 5 units of RQ1 DNase, while HBV virion was not obviously affected. But no significant reduction of HBV virion or cccDNA in the plasma were observed when they are incubated at 37℃for 4h to 7d. Conclusions HBV cccDNA is similar to HBV virion both in characteristics of clearance dynamics and in stability, which makes it technically feasible to detect serum HBV cccDNA.PartⅡDynamic detection of duck hepatitis B virus cccDNA in serum of ducks with liver injuryObjectives To confirm whether DHBV cccDNA could be detected in the serum of ducks with DHBV infection and liver injury. Materials and Methods①A selective fluorescent PCR system based on a primer set spanning the two gaps(DR regions) in DHBV genome was established to preferentially amplify DHBV cccDNA and not (or less) the replicative intermediates, and the efficiency of this system to discriminate cccDNA and rcDNA was verified with purified DHBV rcDNA molecules and plasmid containing whole DHBV DNA sequence (cccDNA). Selective extraction of HBV cccDNA based on the co-precipitation of potassium dodecyl sulfate (PDS) and protein, and further purification with plasmid-safe ATP-dependent DNase (PSAD), were integrated with selective fluorescent PCR system to ensure the specific amplification of DHBV cccDNA under high DHBV rcDNA background.②Forty 1-day-old ducklings were inoculated with DHBV-positive serum first intravenously and then intraperitoneally 3 days later. Twenty four ducks with persistent DHBV infection were picked out and averaged into A, B, C and normal control (NC) group. Ducks in A group were intravenously administrated with D-galactoamine (D-GalN) at a dose of 2.2 g/kg, and were further given lipopolysaccharide (LPS) intraperitoneally at a dose of 100μg/kg 3 hours later. For group B, ducks were pretreated with D-GalN and LPS as those in group A. Twenty-four hours after D-GalN injection, they were intragastrically administrated with carbon tetrachloride (CCl4) at a dose of 10 ml/kg every 12 hours for two times. Ducks in group C were treated similarly as those in group B in spite of a different dose of CCl4 (15 ml/kg). Contrarily, ducks in the NC group were only administrated with normal saline. At 0 h, 24 h, 36 h and 48 h after treatment, sera were collected from each duck for determination of serum DHBV load, DHBV cccDNA and alanine aminotransferase (ALT), and ducks were eventually sacrificed to obtain liver specimens for pathological assessment of the liver lesions and determination of intrahepatic total DHBV DNA and DHBV cccDNA. Results①Templates containing 103 to 108 copies/ml of plasmids(DHBV cccDNA) were all efficiently amplified by the selective fluorescent PCR system for detecting DHBV cccDNA. In contrast, when 103 to 108 copies/ml of purified DHBV rcDNA were amplified with the same system, only minimal fluorescence signal was observed in 107 and 108 copies/ml of DHBV rcDNA templates. Such unspecific signal was undetectable when templates were pre-treated with PDS-protein co-precipitation and PSAD purification, which indicated that DHBV cccDNA would be specifically detected even in high rcDNA background.②No obvious pathological changes were observed in the liver of ducks from NC group, while indexes of liver injury were significant different between group A, B and C. One duck from group B and 4 ducks from group C died at the end of experiment. DHBV cccDNA was undetectable in the sera of ducks from NC and A group at all time points. On the contrary, DHBV cccDNA, varying from 3.17×103 copies/ml to 1.72×104 copies/ml, were detected in the sera of 2 ducks from group B and 3 ducks from group C at 36 h after treatment, whose livers were seriously damaged. The occurrence of DHBV cccDNA in the serum was significantly affected by the degree of liver injury, while no significant association with serum ALT level and DHBV load as well as with the level of intrahepatic total DHBV DNA and DHBV cccDNA was observed. Conclusions①The strategy integrating PDS-protein co-precipitation , PSAD purification and selective fluorescent PCR amplification could detect DHBV cccDNA specifically and sensitively .②DHBV cccDNA could be detected in the serum when the liver of duck was seriously damaged. The incidence of DHBV cccDNA in the serum was significantly associated with the severity of liver injury, while was insignificantly associated with serum ALT level and DHBV load as well as with the level of intrahepatic total DHBV DNA and DHBV cccDNA.PartⅢDynamic detection of Hepatitis B virus cccDNA in serum of patients with hepatitis BObjectives To confirm whether HBV cccDNA could be detected in the serum of patients with hepatitis B. Materials and Methods Fifty-seven patients, including 26 patients with mild chronic hepatitis B (MCHB) and 31 patients with severe hepatitis B (SHB), were investigated. Twenty-six serum specimens were collected from patients with MCHB (1 specimen for each patient), and 60 serum specimens were obtained from patients with SHB (1 to 4 specimens for each patient at different time during the course of disease). The prothrombin time (PT), serum HBV load and biochemical indexes for liver function were documented at each time of collecting specimens. Results Serum HBV cccDNA, varying from 1.25×103 copies/ml to 4.88×104 copies/ml, was detectable in 1 patient with MCHB and in 13 serum specimens from 13 patients with SHB, which was significantly different between two groups of patients.The sensitivity to diagnose of SHB by detection of HBV cccDNA was only 41.94%, and the accuracy was 66.67%.By logistic regression analysis,incidence of HBV cccDNA in the serum was found to be significantly associated with the level of PT, while no significant association with sex, age, serum HBV load and ALT level was observed. Conclusions HBV cccDNA could be detected in the serum of patients with hepatitis B and was significantly associated with the severity of hepatitis B instead of serum HBV load or ALT level, but serum HBV cccDNA is not an appropriate index for evaluating the severity and the prognosis of hepatitis B, and detection of intrahepatic cccDNA could not be replaced by detection of serum cccDNA.