Evolution Of Hepatitis B Virus Quasispecies And It's Correlation With Drug Resistant During Antiviral Therapy Of HBV Chronically Infected Patients

Nearly 400 million people are chronically infected with Hepatitis B virus worldwide,prolonged infections may result in severe liver diseases with eventual progression to hepatitis,cirrhosis and hepatocellular carcinoma. Clinical application of Nucleotide analogs(NAs) is the milestone of antiviral therapy because of its excellent effect of inhibition on virus replication and relatively low in cost and side effect.But NAs also have distinct defects.On the one hand,HBV could happen to replicate once without the drugs,on the other hand,long-term nucleoside therapy may select drug-resistant mutants on HBV polymerase gene from serum of patients and the consequent drug-resistant phenomenum will result in failure of clinical therapy. In the present study,serum sample were taken at several different time points from 6 HBV chronically infected patients before and during lamivudine(LAM) monotherapy and entecavir(ETV) rescue therapy.The HBV DNA polymerase gene was amplified,subcloned and sequenced, HBV wild strains and total virus were quantitative detected by amplification refractory mutation system real-time PCR(ARMS-PCR). Dynamics of virus quasispecies in the samples are monitored and analyzed. Following were the results:Wild type(WT) YMDD variants were the dominant population in baseline from patients without drug therapy.The LAM-resistance variants(rtM204V/I±rtL180M) emerged and gradually increased under LAM administration.The constitute of HBV population was more and more complex until completely replacing with the LAM-resistant variants. The dynamics of the RT quasispecies during entecavir rescue therapy showed that in the three patients,the LAM-resistant variants(rtM204V/I±rtL180M) always represented 83% in the HBV population.In the other patients,WT YMDD variants reverted to be the dominant population under ETV rescue therapy.No variants relative to ETV resistante were found in all these patients. Meanwhile,the in vitro drug sensitivity assay was ultilized on preliminary study of population durg resistance of one patient and expected to find out the relationship between the evolution of HBV quasispecies and drug resistance. Phenotypic assay results showed that different kinds of artificial populations were still sensitive to ETV,and increasing the ratio of WT in the mixed plasmids,the viral replication of transfected cells seemed rather sensitive to ETV. We draw a conclusion that the distribution of virus quasispecies were always dynamic variation in patient treated with nucleotide analogs. Two different patterns of dynamic HBV quasispecies in the six patients under ETV rescue treatment may result in different possibility to ETV resistance with long-term ETV administration. The sensibility of population to drug may be determined by the predominance variants.