HBV Heterogeneity Determined By Ultra-deep Pyrosequencing And Its Correlation With Antiviral Efcacy Of Lamivudine

Background/Aims：We previously reported that Hepatitis B virus (HBV) heterogeneitywithin reverse transcriptase (RT) was a predictor of antiviral efficacy based onclone-based sequencing (CBS). Next-generation sequencing (NGS) technologies allow formassive parallel picoliter-scale amplifcation and detection of individual DNA molecules,providing the potential to reduce the time and complexity for DNA sequencing without theneed for cloning. Here, by comparing ultra-deep pyrosequencing (UDPS) with CBS incharacterizing the genetic heterogeneity of HBV quasispecies within the RT region, weevaluated the performance of UDPS in the analysis of HBV viral populations. In part two,the aim was to investigate dynamic changes of hepatitis B virus (HBV) quasispecieswithin the reverse transcriptase (RT) region based on UDPS method during the early stageof lamivudine treatment and its correlation with antiviral efcacy.Patients and methods：HBV genomic DNA was extracted from serum samples of thirtyone antiviral treatment na ve chronic hepatitis B (CHB) patients. The RT region’squasispecies were parallel analyzed using CBS and UDPS. Quasispecies heterogeneitycharacterization was conducted using bioinformatics analysis. Quasispecies complexitywas calculated based on formula (Sn=i(pi ln pi)/lnN). In part two, thirty-fvechronic hepatitis B patients received lamivudine treatment for at least48weeks. Sixteenpatients responded to lamivudine, while nineteen patients were partial responders. HBVDNA was extracted from serum samples at baseline and week4. Three sequentialoverlapping400-bp segments covering the RT coding region were amplifed andpyrosequencing. Quasispecies heterogeneity characterization was conducted using bioinformatics analysis. Quasispecies complexity and diversity within the RT region wereanalyzed at baseline and week4, and evolutionary patterns of quasispecies in respondersand partial responders were studied.Results：The number of qualified strains obtained by UDPS was much larger than that byCBS (P<0.001). Pearson analysis showed that there was a positive correlation ofquasispecies complexity at nucleotide level between the two methods (P<0.05), whilecomplexity derived from UDPS was higher than that from CBS (P<0.001). Prevalencestudy of variations within RT region showed that CBS detected an average of9.7±1.1amino acid substitutions/sample, and UDPS detected an average of16.2±1.4amino acidsubstitutions/sample. The phylogenetic analysis based on UDPS data showed more geneticentities than that on CBS data. In part two, the quasispecies complexity value ofresponders were signifcantly higher than that of partial responders for the frst and secondRT regions at baseline (P <0.05), and the quasispecies complexity value of responderswere signifcantly higher than that of partial responders for the third RT region at week4(P <0.05). The main parameters of quasispecies diversity (including the mean geneticdistance and the number of non-synonymous substitutions per non-synonymous site) ofresponders were signifcantly higher than that of partial responders for all three RT regionsat baseline (P<0.05), and the main parameters of quasispecies diversity of responders weresignifcantly higher than those of partial responders for the first and third RT region atweek4(P <0.05). Furthermore, average changes and net changes in the mean geneticdistance at amino acid level and the number of non-synonymous substitutions pernon-synonymous site were signifcantly higher than those of partial responders for the firstRT region (P <0.05).Conclusions：Viral heterogeneity determination by UDPS technique is more sensitive and efficient in terms of low abundant variations detection and quasispecies simulation thanthat by CBS method although imperfectly, thus sheds light on the future clinicalapplication of NGS in HBV quasispecies studies. The baseline heterogeneity and dynamicchanges of HBV quasispecies within the RT region showed distinct patterns betweenresponders and non-responders during early stage of lamivudine treatment based on UDPSmethod. High complexity and diversity of quasispecies at baseline and the dynamicchanges of those during the frst4weeks were correlated with lamivudine antiviralefficacy.