Evaluation of Intra-Host Variants of the Entire Hepatitis B Virus Genome 英文参考文献.docVIP

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Evaluation of Intra-Host Variants of the Entire Hepatitis B Virus Genome 英文参考文献.doc

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Evaluation of Intra-Host Variants of the Entire Hepatitis B Virus Genome 英文参考文献

EvaluationofIntra-HostVariantsoftheEntireHepatitisB VirusGenome SumathiRamachandran1*,XiangjunZhai2,HongThai1,DavisS.Campo1,GuoliangXia1,LiliaM.Ganova- Raeva1,JanDrobeniuc1,YuryE.Khudyakov1 1Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America,2JiangsuCentersforDiseaseControl,Jiangsu,China Abstract Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluationofgeneticrelatednessamongHBVstrains.AlthoughanalysisofHBVcompletegenomesusinggeneticcloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approachtowholegenome(WG)HBVquasispeciesanalysisbasedonend-point,limiting-dilutionreal-timePCR(EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispeciesfromserumsamplesofpatients(n=38)infectedwithHBVgenotypesA,B,C,D,EandG.Phylogeneticanalysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub- populationsofthevirus.AcriticalobservationwasthatHBV-WGconsensussequencesobtainedbydirectsequencingof PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiologicalsettings. Citation:RamachandranS,ZhaiX,ThaiH,CampoDS,XiaG,etal.(2011)EvaluationofIntra-HostVariantsoftheEntireHepatitisBVirusGenome.PLoSONE6(9): e25232.doi:10.1371/journal.pone.0025232 Editor:Jean-PierreVartanian,InstitutPaste