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

Abstract

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 10(5) IU/ml, 15 of 16 (94%) at 10(4) to 10(5) IU/ml, 6 of 7 (86%) at 10(3) to 10(4) IU/ml, and 2 of 3 (67%) at less than 10(3) 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 during 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.

FIG. 1.

Optimization of 5.2-kb nested RT-PCR conditions. (A) Effect of RT primer length and concentration on detection of HCV RNA by 5.2-kb nested RT-PCR and agarose gel electrophoresis of PCR products. Primer lengths and the RT primer concentrations are indicated. The concentration of HCV RNA in a human serum specimen was determined, followed by RNA extraction, dilution in water, and addition to RT reactions in the indicated amounts. (B) Effect of number of PCR cycles on detection of synthetic HCV RNA (transcribed from a 6-kb cDNA clone) using nested RT-PCR and agarose gel electrophoresis. Estimated RNA copies are indicated across the top. RT and PCR were performed as described in Materials and Methods, with the number of PCR cycles in the two nested rounds adjusted as indicated on the left, 20/30 indicating 20 cycles of PCR in the first (outer) round and 30 cycles in the second (inner) round. (−), Negative (water) control; nt, nucleotide.

FIG. 2.

Complexity of cDNA clones obtained by two methods. We amplified 1- or 5.2-kb amplicons from specimens from five study subjects with acute HCV infection. Amplicons were cloned, and clonotypes were identified by using the HDA+SSCP method. Clonotype ratio is the number of clonotypes divided by the number of clones examined. ID, identity.