The Analysis Of The Character Of Quasispecies Of The Reverse Transcirptase Region Of HBV Covalently Closed Circular DNA And The Observation Of Mutations Associated With Drug Resistance Of HBV Covalently Closed Circular DNA

The study on the virus quasispecies and dynamics distribution of popoluation ofmutations of drug resistance,is the key to clarify the the drug resistance of virus and theoccurance of the virus breakthrough.This research was investigated from two aspects: The first part was mainly concernedabout the quasispecies of reverse transcriptase(RT) region of HBV covalently closedcircular DNA(cccDNA) in liver and of HBV DNA in serum in the urtreated chronichepatitis B patiens and the correlation between them.The second part was mainly about theobservation of the changes of the mutations associated with drug resistance of HBVcccDNA.Ⅰ.To explore the quasispecies of RT region of HBV cccDNA in liver and of HBVDNA in serum in the urtreated chronic hepatitis B patients and the correlation betweenthem.Objective To investigate the quasispecies of RT region of HBV cccDNA in liverand of HBV DNA in serum in the urtreated chronic hepatitis B patients and the correlationbetween them. Methods We choosed26cases in the untreated chronic hepatitis Bpatients and their liver tissues and serum were reserved,and then we apply the QIAampDNA Mini Kit to extract HBV related genes in the liver tissues and the serum；andplasmid-safe ATP-dependent DNase (PSAD) was used to purify the extracted products ofliver tissues；Then we conduct nested PCR by primers set spanning the two gaps(DRregions) in HBV genome to amplify RT regions of HBV cccDNA. A selective Polymerasechain reaction (PCR) system based on primers set spanning the two gaps(DR regions) inHBV genome was used to preferentially amplify the region encoding lamivudine, adefovirand enticavir resistant substitutions of HBV cccDNA；By applying T-A cloning,weamplified the RT region of HBV cccDNA in liver and of HBV DNA in serum；Then weconducted the cloning,including establishing reorganization-plasmid,makingreorganization-plasmid and enzyme digestion.According to the sequencing results, weanalysed the quasispecies of RT region of HBV cccDNA in liver and of HBV DNA inserum and the correlation between them. Results The amount of qusispecies of RTregion of HBV cccDNA was four to twenty-two, the amount of qusispecies of RT region of HBV DNA in serum was three to twenty,there was no statistical significance betweenthem(p>0.05)；The quasispecies complexity and diversity were not significantly differentbetween the RT region of HBV cccDNA in liver and HBV DNA in serum in nucleotidelevel and in amino acid level(p>0.05). Conclusions We proved that the quasispecies ofcccDNA exists; The amount of quasispecies of RT region was not significantly differentbetween the RT region of HBV cccDNA in liver and HBV DNA in serum.In addition, Thequasispecies complexity and diversity were not significantly different between the RTregion of HBV cccDNA in liver and HBV DNA in serum. Ⅱ.The observation of the variant strains associated with drug resistance of cccDNAin the treated chronic hepatitis B patientsObjective To investigate the natural mutations associated with drug resistance ofHBV cccDNA and explore the proportions between wild and variant strains in the chronicHBV infected patients existed mutations associated with drug resistance of cccDNA.Methods We choosed13cases who received liver puncture twice between12months,wereserved their liver tissues and serum,and extract HBV related genes in the liver tissues andthe serum respectively,the extracted products of liver tissues were further purified, HBVDNA and cccDNA were quantified by using Real-time fluorescent quantitative PolymeraseChain Reaction (Real Time PCR). By PCR, nested PCR, direct sequencing,we detected themutations associated with drug resistance of HBV cccDNA before and after treatment.Results We found that three patients (three out of thirteen)exists the natural mutationsassociated with drug resistance of HBV cccDNA, the mutations associated with drugresistance of HBV cccDNA happend to one patient (one out of thirteen) who took NAs.The proportions between wild and variant strains in these four patients existed mutationsassociated with drug resistance of cccDNA change after taking NAs. Conclusions1.Ithas been proved that the natural mutations associated with drug resistance of cccDNAexists.2. The proportions between wild and variant strains of the mutations associated withdrug resistance of cccDNA in patients who took NAs after taking NAs may change.