Study Of Relationship Between Hepatitis B Virus Bcp/Pc Region Quasispecies With Relapse On Chronic Hepatitis B Patients With Lamivudine Treatment

Objective:To investigate the relationship between BCP/PC region quasispecies features of hepatitis B virus(HBV) relaxed circular(rc)DNA in serum and covalently closed-circular(cccDNA) in hepatocyte with relapse after meeting the endpoint criteria of2008Asia-Pacific Association guideline(APASL)[1] on chronic hepatitis B (CHB) patients with lamivudine (LAM) treatmentMethods:The liver tissue biopsies of43CHB patients were taken when met the endpoint criteria with LAM treatment, including25patients with LAM-naive,18patients with Nucleos(t)ide analogues (NAs) retreatment who had received LAM-naive. Methods:Two groups of patients were collected serum at the time of baseline of treatment and when they relapsed collected liver tissue biopsy when met the endpoint criteria.Serum rcDNA were extracted by water boiling, hepatocyte full-length HBV cccDNA was amplified with HBV cccDNA special primers by Rolling circle amplification (RCA); HBV genotypes were detected by nest PCR with multiple primers. Serum HBV rcDNA and hepatocyte HBV cccDNA of BCPand PC region were amplified with appropriate primers by nest PCR, the PCR products were recycled after using gel electrophoresis, follow up being cloned into PEASY-Teasy plasmid, the recombinant DNA was transformed into E. coli JM109competent cells Positive clones were identified with inside primers by nest PCR. At last,selected20to32of positive clones for sequencing randomlyResults:(1)There are many mutations in HBV DNA BCP/PC region widespread, whether the patients had received LAM treatment. The top five BCP/PC region mutations points had multiple points were100%at the time of baseline of treatment and when met the endpoint criteria and relapsed,they existed in advantages, sub-advantages and disadvantages cloning.1(7.7%) patients of LAM-naive treatment at baseline existed wild strains,3patients (7.0%) patients (including2patients with LAM-naive treatment,1patient with NAs-retreatment who had received LAM-naive).At the time of baseline of treatment and when met the endpoint criteria and relapsed,the points with high aberration rate were nt1721/1762/1764/1775at BCP, ntl896/1913at PC region,this six points weren’t significantly associated with relapse, P values were greater than0.05. LogisticUnivariate analysis showed that gender, age, HBVfamily history, HBV DNA loads and HBeAg status and ALT at baseline,HBV DNA genotype, the group with NAs treatment, wheather relapse10factors had no significant effect on the detection rate of the6mutation points of serum rc DNA at baseline and cccDNA in hepatocytewhen met the endpoint criteria,P values were greater than0.05.(3) BCP/PC region quasispecies complexity of serum HBV rcDNA at baseline, cccDNA in hepatocyte when met the endpoint criteria, serum HBV rcDNA when relapsed were upward trend in all patients and patients who had relapsed. Quasispecies complexity was higher than quasispecies complexity at baseline,it was statistically significant (0.7753±0.1727VS0.6413±0.2288, P=0.034<0.05). Quasispecies complexity in hepatocyte ccDNA when met the endpoint criteria had not statistically different with quasispecies complexity at baseline and whenrelapsed (P values were0.23,0.245, were greater than0.05).(5)Their quasispecies complexities were not significantly different between LAM-naive treatment group and NAs-retreatment group at the time of baseline of treatment and when met the endpoint criteria and relapsed (P>0.05). The quasispecies complexity over three time samples of patients who had relapsed in LAM-naive treatment group were upward trend; quasispecies complexity were upward trend from baseline to met the endpoint criteria in patients who hadn’t relapsed. In NAs-retreatment group, quasispecies complexity were upward trend from baseline to met the endpoint criteria in patients who relapsed too,follow up appeared downward trend to the time when relapsed,but with a drop of small, quasispecies complexity were downward trend from baseline to met the endpoint criteria in patients who hadn’t relapsed.The differences of quasispecies complexity at the time of baseline and meteting the endpoint criteria between patients with relapsed to patients without relapsed in LAM-naive treatment group and NAs-retreatment group were not statistically significant (P values were greater than0.05).Conclusions:(1) There are many mutations in HBV DNA BCP/PC region widespread, whether the patients had received LAM treatment. At the time of baseline of treatment and when met the endpoint criteria and relapsed,the points with high aberration rate were nt1721/1762/1764/1775at BCP, nt1896/1913at PC region,this six points weren’t significantly associated with relapse. LAM in the treatment of CHB patients at baseline serum and standard of BCP/PC region quasispecies complexity at baseline and met the endpoint criteria and their change trend weren’t associated with relapse.