Characterization of simple and complex hepatitis C virus quasispecies by heteroduplex gel shift analysis: correlation with nucleotide sequencing.
In infected humans, hepatitis C virus (HCV) exists as a quasispecies typically characterized by multiple nucleotide substitutions within the second envelope gene hypervariable region 1 (HVR1). In the current study, we used heteroduplex gel shift analysis (GSA) of HVR1 sequences amplified directly from patients' sera to define two patterns of HCV quasispecies: (i) simple quasispecies, which gave a mostly homogeneous gel shift profile with a single predominant band and (ii) complex quasispecies, which gave a gel shift profile with multiple bands. Recombinant HVR1 libraries were generated from two patients with complex HCV quasispecies (cases 1 and 2) and two patients with simple HCV quasispecies (cases 3 and 4), and 129 individual clones were analysed by either GSA, nucleotide sequencing or both techniques. In case 1 we identified a highly complex HCV quasispecies with 11 distinct HVR1 variants differing by 1-51 nucleotide changes. We found a general but not absolute correlation between GSA pattern and the number or position of nucleotide changes within HVR1. In case 2, the complex HCV quasispecies consisted of three distinct major variants; GSA of individual HVR1 clones allowed us to reconstruct the complex quasispecies pattern in vitro. In case 3, the simple quasispecies comprised 66% homogeneous clones and 33% unique minor variants differing by 1-3 nucleotides from the consensus sequence. In case 4, the simple quasispecies was 84% homogeneous, but six unique major shift variants were identified among 31 clones by GSA. In summary, HCV quasispecies can be characterized based on GSA profiles following direct PCR amplification of HVR1 sequences from patients' serum; the GSA profiles approximate the clonal population of HCV as determined by clonal analysis. GSA of HVR1 clones showed a strong correlation with nucleotide sequencing.
Wilson
J J
JJ
Viral Hepatitis Laboratory, Department of Laboratory Medicine, University of Washington Medical Center, Seattle 98195, USA.
Polyak
S J
SJ
Day
T D
TD
Gretch
D R
DR
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015752:Genes, env; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D013045:Species Specificity
D009692:Nucleic Acid Heteroduplexes