title: Evaluation of intra-host variants of the entire hepatitis B virus genome.
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 evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of 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 epidemiological settings.
authors.affiliation: Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America. dcq6@cdc.gov
authors.forename: Sumathi
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Xiangjun
authors.initials: X
authors.lastname: Zhai
authors.forename: Hong
authors.initials: H
authors.lastname: Thai
authors.forename: Davis S
authors.initials: DS
authors.lastname: Campo
authors.forename: Guoliang
authors.initials: G
authors.lastname: Xia
authors.forename: Lilia M
authors.initials: LM
authors.lastname: Ganova-Raeva
authors.forename: Jan
authors.initials: J
authors.lastname: Drobeniuc
authors.forename: Yury E
authors.initials: YE
authors.lastname: Khudyakov
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D016679:Genome, Viral; D005838:Genotype; D006509:Hepatitis B / Q000235:genetics* / Q000635:transmission* / Q000821:virology; D006515:Hepatitis B virus / Q000145:classification* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral