Accurate representation of the hepatitis C virus quasispecies in 5.2-kilobase amplicons

Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 10⁵ IU/ml, 15 of 16 (94%) at 10⁴ to 10⁵ IU/ml, 6 of 7 (86%) at 10³ to 10⁴ IU/ml, and 2 of 3 (67%) at less than 10³ IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated daring amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.