The Complex Patterns Of Quasispecies In The Reverse Transcriptase Region Of Hepatitis B Virus In Telbivudine Treatment

Background and Aim:Telbivudine(Ld T)is an anti-hepatitis B virus(HBV)agent for its inhibitory effect on virus replication.There is a frequent emergence of drug resistance mutations in the virus population in the course of treatment,which are predominantly located in the reverse transcriptase(RT)region.Long-term anti-viral therapy increases the risk of development of drug resistant strains with the RT region in the viral polymerase gene.The evolutionary dynamics of HBV quasispecies with LdT mono-therapy has yet to be fully assessed.Using the technology of next generation sequencing,we characterized the components and dynamic changes of quasispecies during the course of LdT treatment,which may underlie drug resistance in chronic hepatitis B patients.Methods:1.We collected and extracted the HBV genomic DNAs from the 21 chronic HBV-infected patients' serum of five time points(i.e.,baseline,weeks 1,3,12 and 24 after LdT treatment).Nested PCR was performed to amplify the HBV-RT regions.PCR products were performed with Sanger and Ion Torrent PGM sequencing.2.We used a pipeline to process the empirical PGM data,including pre-processing of raw sequencing data,error correction,alignment,quasispecies reconstruction,and calculating complexity and diversity of quasispecies.3.We compared the discrepancy of the complexity and diversity of quasispecies with different treatment group.Results: We constructed a bioinformatics pipeline for analyzing Ion Torrent PGM sequencing data for HBV RT region,including error-correction and quasispecies construction.During follow-up,we noted that the changes of HBV DNA levels were significantly associated with virologic response(the complete response,HBV DNA level ?60 IU/ml at 24 weeks of treatment;the incomplete(partial)response,60< HBV DNA level ?2000 IU/ml;and inadequate response,>2000 IU/ml)and HBV genotype(ANOVA,P=9.9e-07 and 1.9e-05,respectively).The complexity of viral quasispecies(the Shannon entropy,Sn)did not differ with time points,genotype and response(ANOVA,P=0.50,0.16,and 0.69,respectively).The changes of mean genetic distance(?)were not significantly associated with genotype(P=0.45 and 0.13 for the nucleotide and amino acid level,respectively)and response(P=0.95 and 0.35).In addition,the changes of dN and dS were not significantly associated with genotype(P=0.33 and 0.19)and response(P=0.17 and 0.06).Conclusion: There is no significant change of the complexity and diversity of quasispecies in the HBV-RT region associated with LdT mono-therapy for 24 weeks between the groups of complete and incomplete/inadequate response.