The Detection And Analysis Of Natural Mutations Associated With Drug Resistance Of HBV Covalently Closed Circular DNA

This research was composed of two parts:Ⅰ.Detection of natural mutations associated with drug resistance of HBV covalently closed circular DNA.Ⅱ. Cloning analysis of variant strains associated with drug resistance of HBV ccc DNA.Ⅰ. Detection of natural mutations associated with drug resistance of HBV covalently closed circular DNA.Objective To determine the existence of mutations associated with drug resistance of HBV covalently closed circular DNA in the untreated chronic HBV infected patients, and find out the distinction of variant strains between in the liver tissues and in the serum. Methods①Selected 85 cases in the untreated chronic HBV infected patients and reserved their liver tissues and serum.②The QIAamp? DNA Mini Kit (tissue) and directly boiling were used to extract HBV related genes in the liver tissues and the serum respectively.③The extracted products of liver tissues were further purified with plasmid-safe ATP-dependent DNase (PSAD). HBV DNA and ccc DNA were quantified using Real-time fluorescent quantitative Polymerase Chain Reaction (Real Time PCR).④Measured serum immunological markers associated with HBV and serum biochemical index (ALT), and analysed the relationship among them and HBV load statistically.⑤All the samples were amplified by nested PCR with the corresponding primers.A selective Polymerase chain reaction (PCR) system based on primers set spanning the two gaps(DR regions) in HBV genome was used to preferentially amplify the region encoding lamivudine, adefovir and enticavir resistant substitutions of HBV cccDNA.⑥The positive PCR products were directly sequenced commercially, and observed the results.⑦Analysed comparatively intrahepatic HBV ccc DNA, HBV rc DNA and serum HBV DNA genetic sequence. Results The level of Intrahepatic HBV cccDNA, HBV rcDNA and the serum level of HBV DNA were positively correlated with each other(P < 0.05). There was no statistical significance between the level of ALT and the load of HBV DNA(P>0.05). DNA sequences of the whole 85 cases were successfully tested. Mutations were detected in intracellular HBV cccDNA, rcDNA and serum HBV DNA of 2 patients (2.4%):one is rtM204I(No.44),the other is rtV214A(No.49).There was also another case of sequencing map with miscellaneous peaks(No.85). Conclusions There were mutations associated with drug resistance of HBV ccc DNA in the naive patients, so it proved that the existence of natural mutations of HBV ccc DNA.Ⅱ. Cloning analysis of variant strains associated with drug resistance of cccDNAObjective To determine the proportions between wild and variant strains in the chronic HBV infected patients existed natural mutations associated with drug resistance of cccDNA. Methods①Selected the specimens of chronic HBV infected patients existed natural mutations associated with drug resistance of cccDNA as cloning templates, and the methods of nested PCR was the same as the part one.②Complete the operations according to the specific steps of clone mini kit(purification, connection, transformation and etc). Detect the cloning results, and pick out 50 monoclonal colonies randomly from each specimen, then sequenced commercially.③Analysed comparatively the sequencing results, determined the proportions of wild and variant strains further. Results①Cloning analysis of 204 site: Intrahepatic HBV ccc DNA:ATG/ATT/ATC=10/38/2; Intrahepatic HBV rc DNA: ATG/ATT/ATC=3/45/2; HBV DNA of the serum: ATG/ATT/ATC=0/50/0.②Cloning analysis of 214 site: Intrahepatic HBV ccc DNA: GTA/GCT/GTT=20/29/1,Intrahepatic HBV rc DNA: GTA/GCT =5/45,HBV DNA of the serum: GTA/GCT=10/40.③The case of No.85 which appeared miscellaneous peaks showed most sites related with drug resistance were nonsense mutations, but rt A181T was found in one case of fifty clones in the intrahepatic ccc DNA, while rtM250V was found in one case in HBV DNA of the serum. Conclusions Variant strains mixed with wild plants existed natural mutations associated with drug resistance of cccDNA in the chronic HBV infected patients. The proportions are not the same in intrahepatic ccc DNA,intrahepatic rc DNA and HBV DNA of serum. The nonsense mutations did not affect the expression of HBV genes.