| Objectives:Previous studies have focused on the effect of early quasispecies on the efficacy of short-term antiviral therapy(typically 3 years or less).To observe the long-term antiviral efficacy of nucleos(t)ide analogues(NAs)in prospective cohort of patients with chronic hepatitis B virus(HBV)infection(in patients with chronic hepatitis B and hepatitis B virus-related cirrhosis)and its effect on clinical outcomes.To study the quasispecies heterogeneity and evolutionary mechanism of hepatitis B virus during long-term antiviral therapy.Methods:This study was a prospective,open,random,real world of observational study.107 cases of chronic HBV infection,including chronic hepatitis B patients and patients with HBV related liver cirrhosis were enrolled in the study.All patients accepted the NAs monotherapy and were divided into two groups according to initial NAs therapies,entecavir treatment group(treated with entecavir)and non-entecavir group(treated with lamivudine,adefovir dipivoxil or telbivudine).Patients were followed prospectively every 24-48 weeks and the longest follow-up was about 10 years.Clinical data was collected.The virological,biochemical and serological response and hepatocellular carcinoma(HCC)and other clinical outcomes were analyzed.Due to the limitations of PCR amplification and cloning sequencing technology,24 patients from the first stuy were divided into three groups:untreatment group(UTG),treatment one year group(TOG),and treatment ten years group(TTG).For TOG,we divided them into group A(1 year<HBV response time<3 years)and group B(3 years<HBV response time<6 years)according to the time of viral response after one year of treatment.Serum from patients was conducted HBV quasispecies research.HBV RT quasispecies were researched using polymerase chain reaction(PCR),cloning and sequencing,to explore the relationship between virological response and the characteristics of quasispecies of HBV during 48 weeks treatment and HBV quasispecies evolutionary mechanism.Full-length genome amplification was performed,and the HBV strain was cloned to conduct a comprehensive analysis of mutations and to analyze quasispecies variation in relation to the coexistence of HBsAg and anti-HBs.Results:1.After long-term NAs antiviral treatment,Patients HBV DNA log10 IU/mL after treatment reduced to 1.28(1.28,1.40);lower than 6.92(5.06,7.89)before treatment(P<0.05).Entecavir group has lower HBV DNA level than non-entecavir group,especially on year 1,2,6,7 and 9.7 cases of patients were got HBsAg loss during treatment.All these 7 cases got early virological response at week 24 or 48.HBsAg was lower than 1500 IU/ml in 67.9%patients.HBeAg loss rate was 71.21%and HBeAg seroconversion rate was 40.91%.A total of 6 cases of malignant tumor patients had been found during the follow-up period(5.61%,6/107),including HCC and non-HCC malignant tumor.There are three patients(2.80%,3/107)with HCC,and all were in entecavir treatment group,and died during follow-up.There is no significant difference between two treatment groups for cumulative incidence of HCC,malignant tumor and death(P>0.05).APRI after treatment was 0.44(0.31,0.56),lower than 1.05(0.53,1.75)before treatment(P<0.05).The liver stiffness value was 4.9(4.0,6.2)KPa at the end of follow-up.At the end of follow-up,optimized therapy or therapy alteration rates were 4.17%for entecavir group and 6.78%for non-entecavir group.There is no significant difference between two treatment groups(P>0.05).The overall rates of virologic breakthrough is 26.17%,and 18.75%for entecavir group and 32.20%for non-entecavir group.There is no significant difference between two treatment groups(P>0.05).Undetectable HBV DNA at 1 year of treatment were protective factors for virological breakthroughs(HR0.235(0.103,0.538))(P<0.05).2.Two patients of group A were detected resistant mutations,accounted for 40%;6 patients of group B were detected resistant mutation,accounted for 75%;3 patients of TTG were detected resistant mutations,accounted for 50%.De novo mutations at resistance loci almost were in series with the known drug resistance loci,a few exist alone with low frequency.Patients without TDF treatment were detected TDF resistant mutation(A194T),suggested the presence of pre-existing mutations.Insertions,deletions,or termination codon mutations were detected in 50%of patients in both group B and TTG.3.For nucleotide and amino acid levels of RT,the complexity of TOG was significantly higher than that of UTG(P<0.05),and the average genetic distance of TOG or TTG was significantly higher than that of UTG(P<0.05).At nucleotide level of RT,the complexity of group A(Sn=0.9734)were significantly higher than that of group B(Sn=0.8875,P=0.0393)and TTG(Sn=0.8524,P=0.0141).The average genetic distance,the average number of synonymous substitution and the average number of nonsynonymous substitutions of group A and group B,were lower than TTG,but no statistical significance(P>0.05).At amino acid level of RT gene,group A had a significantly higher complexity(Sn=0.8874)than TTG(Sn=0.7098,P=0.0473),but the average genetic distance among three groups had no significant difference(P>0.05).4.Phylogenetic tree analysis showed average branch length of group A(0.0028±0.0005)and group B(0.003±0.0004)were significantly greater than that of TTG(0.002±0.0004)(P<0.001),but difference between group A and group B was not significant(P=0.6368).The results suggested virological response group of HBV evolved faster and accumulated more variations than that of TTG.Compared with TOG(group A and group B),UTG and TTG had a simpler topological structure.The mean branch length(0.0029±0.00029)of TOG(group A and group B)was significantly higher than that of UTG(0.0012±0.0003,P=0.0137).5.The selection pressure of three groups of patients with HBV RT(?=dN/dS)were analyzed.The results showed that three groups of HBV had undergone positive selection.TTG has accumulated more positive selection signals than that of other groups.For the genotype B,C,B/C,three groups of patients had similar co value distribution trends.In the B/C genotype,the selection pressures between group A(?=0.296±0.0332)and group B(?=0.291±0.0469)had no difference,were lower than that of TTG(?=00.5679±0.1722).6.The complexity(nucleotide level)of RT quasispecies was negatively correlated with the corresponding HBV DNA load(P=0.0163 and R=0.6493)at one year.The complexity(nucleotide and amino acid levels)of RT quasispecies was negatively correlated with the corresponding ALT(P=0.0437,R=0.5661;And P=0.0117,R=0.673).The average genetic distance,average number of synonymous substitution and the average number of synonymous substitutions showed no correlation with corresponding HB V DNA loads and ALT levels.7.Phylogenetic analyses and genotype identification suggested that A02 was infected with a putative genotype I strain.Unusual deletions of PreS(11 of 16 clones(69%)in PreS1)and amino acid variants of HBsAg(11 of 16clones;69%)were observed in the A02 quasi species.Conclusions:Long-term antiviral treatment with NAs can benefit patients to varying degrees,which is closely related to HBV quasispecies heterogeneity.Under the pressure of antiviral drugs,HBV RT quasispecies heterogeneity is associated with the time of nucleoside analogue antiviral treatment.The high complexity of HBV quasispecies may prompt that response is good.HBV quasispecies with high complexity are in favour of HBV DNA or ALT levels drop,but low levels ALT go against HBV DNA clearance.Group B and TTG have accumulated more resistant mutations,missing,or termination codon mutations than group A,which prompts these mutations against virological response,may be related to immune escape.TTG has more simple evolution model and more positive selection signals,which may be associated with virological breakthrough.The propensity for PreS1 deletions and the complexity of HBsAg variants in HBV isolates from a single patient infected with HBV genotype I could explain the coexistence of HBsAg and anti-HBs and the resistance to current antiviral therapies. |
PDF Full Text Request