The Quasispecies Analysis Of The Relapsed Patients With Chronic Hepatitis B After Adefovir Dipivoxil Standard Treatment

Objects: Nucleotide analogs are mainstream therapy forpatients with chronic hepatitis B. The relapse of nucleotide analogs treatedpatients is wildly concerned. As HBV existing and transmitting in theform of quasispecies, quasispecies composition of HBV is the key point ofchoosing anti-HBV therapeutic regimen for nucleotide analogs treatedpatients, and there are few research about HBV quasispecies compositionof nucleotide analogs treated then relapsed patients both in China andabroad. These researches about relapse are focus on Lamivudine only, lackof researches about other nucleotide analogs such as adefovir dipivoxil. Indomestic China, some clinical researches reveal that it is effective to reuseadefovir dipivoxil in retreatment of relapsed chronic hepatitis patientsafter cessation of standard adefovir dipivoxil treatment. And there are fewresearches about the quasispecies composition of these relapsed patients.Therefore, our research focus on the exact quasispecies composition ofadefovir dipivoxil standard treated then relapsed patients after drugcessation with chronic hepatitis B and the quasispecies diffidence betweenthe treated then relapsed patients and treatment naive patients. So that wecould provide some therapy advices for adefovir dipivoxil treated thenrelapsed patients after drug cessation with chronic hepatitis B inaccordance with virology insight of the exact HBV quasispecies. Methods: Standard treated then relapsed patients after adefovir dipivoxil cessationwith chronic hepatitis B and treatment na ve patients with chronichepatitis B were recruited and their serum were drew for further blood test.First, HBV-DNA was extracted from their serum then HBV RT gene wasamplified through nested-PCR. Half of the PCR products were directlysequenced, the other half of PCR products were used for TA-cloning inwhich they were connected to T-Vectors after purified through agarose gelelectrophoresis. Then, the T-Vectors were transfected to competent cell ofTOP10coli bacillus. Thirty positive bacterial colonies screened byX-gal-IPTG-AMP-LB agar medium of each serum simple were sent tothird-party sequencing company named Life technology to forward andreversely sequence the target HBV fragments connected to T-vector insidethe positive colonies coli bacillus. The forward and reverse sequencingresults were assembled by DNASTAR SeqMan. These sequences wereused for removing vector sequences, multiple alignment, drug resistancemutations analyses, calculating quasispecies complexity and diversity andevolutionary tree analyses by bioinformatics softwares. Statistic data wasanalyzed by SPSS19.0software package. Results:1. More than93.3%strains of the HBV quasispecies of the relapsed patients and treatmentna ve patients are wild strains. Wild stain is the dominant strain of theirHBV quasispecies. There are10drug resistant clones and740wild clonesin the relapsed group. There are8drug resistant clones and472wild clones in the treatment na ve group. There are no statistic differencebetween the two group of patients (Chi-square test, P>0.05).2. Thequasispecies complexity of the relapsed patients and the treatment na vepatients are0.974(0.044) and0.922(0.081) respectively, P>0.05, there areno statistic difference between the two group of patients. The comparisonbetween quasispecies diversity (d/dS/dN) of relapsed patients andtreatment na ve patients shows P>0.05and there are no statistic differencebetween the two group of patients.3. The Spearman rank correlation testof quasispecies complexity and diversity with HBV-DNA level showsthere are no correlation between them.4. Many drug resistance mutationswere detected in HBV quasispecies of recruited patients, includingentecavir resistance mutations rtI169T/rtS202I/rtM250V, lamivudinetelbivudine resistance mutations rtV173L/rtL180M/rtM204I/rtM204V andadefovir dipivoxil resistance mutations rtA181V, however, mutationrtT184G and rtN236T were not detected. We did detect drug resistancemutations in treatment na ve patients, including rtI169V，rtA181V，rtS202I，rtL180M and rtM204I/V. Only one sequence possessing adefovirdipivoxil resistance mutation A181V was found in a treatment na vepatient.5. No known drug resistance mutations was detected in sequencesof direct nested-PCR products. However, many drug resistance mutationsmentioned before were detected in sequences of TA-cloned nested-PCRproducts.6. Phylogenetic tree showed that the sequences (direct sequences of nested-PCR products) of relapsed patients and treatment na ve patientsare not clustered and their genetic distances are very close (blow0.04).Conclusions:1.Wild strain is the dominant strain of the standard treatedthen relapsed patients after adefovir dipivoxil cessation. The comparisonbetween the quasispecies complexity and diversity of the relapsed patientsand the treatment na ve patients shows no statistic difference. It is efficientfor them to reuse adefovir dipivoxil after relapse.2. There are many kindsof known drug resistant mutations in the quasispecies of treatment na vepatients although wild strain is dominant. Sequencing by TA-clonednested-PCR products is more accurate and better than direct sequencing ofnested-PCR products in detecting drug resistance mutations.