Research On The Existing Situation Of HBV In PBMC Of Post-liver Transplant Chronic Virus Hepatitis B Patients

Part oneA new and specific PCR method for the detection of HBV cccDNAHepatitis B virus (HBV) covalently closed circular DNA (cccDNA), is a continuous double chain ring, while other DNA forms contain a gap on each chain at the position of direct repeat 1 sequence (DR1) and direct repeat 2 sequence (DR2), respectively. At present it is still difficult to detect with high sensitivity and specificity and quantify the HBV cccDNA .We applied a highly sensitive PCR approach to discriminate between the HBV relaxed circular DNA (rcDNA) genome present in virions and the cccDNA generated by the DNA repair machinery of the host cell nucleus. We have devised a differential PCR protocol, designated paired comparative PCR (pcPCR) as Stoll-Becker et al, and add a couple of primer in the first round of nested PCR to amplify the segment of HBV S region .The PCR assay for detection of cccDNA was determined with a serial dilution of plasmid DNA (PBHB4) containing a full-length HBV genome, the sensitivity of nested PCR for detection of cccDNA is about 7.9×10~5 copies/ml,meanwhile, the PCR assay for detection of rcDNA was determined with a serial dilution of DNA prepared from DANE viral particles, the sensitivity is over 1.1 xlO11 copes/ml.In order to confirm the clinical practicality of the detection system, we took two extracted liver tissue DNA and two serum-extracted DNA with high HBV DNA copy from HBV infected patient as templates." Results showed that the presence of HBV cccDNA were detected in two liver tissue DNA sample , and no positive result in two clinical serum sample .In Conclusion, This assay proved to be effective for rapid and sensitive detection of HBV cccDNA with high specificity and efficacy. Part twoDetection of HBV DNA and HBV cccDNA in peripheral mononuclear blood cells from post-transplantation patients using the polymerase chain reactionObjective: By investigate Research on the exist situation of HBV in PBMC of post-liver transplant chronic virus hepatitis B patients, we explore the relationship between HBV infection within PBMC and HBV reinfection. Material and Methods.- Blood specimens taken from 30 following recipients intermittently after liver transplantation, all the recipients received lamivudine monoprophylaxis or combined with HBIG. HBV DNA in serum and PBMC were detected by Fluorescence quantitative polymerase chain reaction (FQ-PCR)respectively. And partial PBMC samples with high HBV DNA loading were subsequently assayed for HBV cccDNA by a newestablished selected PCR system with high specificity and efficacy to discriminate HBV cccDNA The experimental results were collected and analyzed with clinical dataResults: (1) the overall 1-year, 2-year and 3-year rates of HBV reinfection in this group were 11.3%> 34.85% and 58.36%, respectively.(2)All 8 patients with HBV reinfection were detected the presence of HBV DNA in their PBMC, and HBV DNA were found in 96.8%(21/22) patients without HBV reinfection. In addition, of 6.7%(5/30) patients, HBV DNA negative result were presented at least one time. (3) No HBV cccDNA positive result were gained in 32 PBMC samples with high HBV DNA burden (>106 copies/ml cell disruption).Conclusion: Those findings indicated the HBV DNA in PBMC could persist for a long-term in general recipients despite protective measures being taken for prevention HB recurrence, but no evidence for HBV replication in PBMC, and PBMC maybe play an important role in transmission HBV partical from other extrahepatic HBV infection tissues to allograft.