Assessment of hepatitis C virus sequence complexity by electrophoretic mobilities of both single-and double-stranded DNAs

Abstract

To assess genetic variation in hepatitis C virus (HCV) sequences accurately, we optimized a method for identifying distinct viral clones without determining the nucleotide sequence of each clone. Twelve serum samples were obtained from seven individuals soon after they acquired HCV during a prospective study, and a 452-bp fragment from the E2 region was amplified by reverse transcriptase PCR and cloned. Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined heteroduplex analysis (HDA) and a single-stranded conformational polymorphism (SSCP) method to determine the number of clonotypes (electrophoretically indistinguishable cloned cDNAs) as a measure of genetic complexity (this combined method is referred to herein as the HDA+SSCP method). We calculated Shannon entropy, incorporating the number and distribution of clonotypes into a single quantifier of complexity. These measures were evaluated for their correlation with nucleotide sequence diversity. Blinded analysis revealed that the sensitivity (ability to detect variants) and specificity (avoidance of false detection) of the HDA+SSCP method were very high. The genetic distance (mean +/- standard deviation) between indistinguishable cloned cDNAs (intraclonotype diversity) was 0.6% +/- 0.9%, and 98.7% of cDNAs differed by <2%, while the mean distance between cloned cDNAs with different patterns was 4.0% +/- 3.2%. The sensitivity of the HDA+SSCP method compared favorably with either HDA or the SSCP method alone, which resulted in intraclonotype diversities of 1.6% +/- 1.8% and 3.5% +/- 3.4%, respectively. The number of clonotypes correlated strongly with genetic diversity (R2, 0.93), but this correlation fell off sharply when fewer clones were assessed. This HDA+SSCP method accurately reflected nucleotide sequence diversity among a large number of viral cDNA clones, which should enhance analyses to determine the effects of viral diversity on HCV-associated disease. If sequence diversity becomes recognized as an important parameter for staging or monitoring of HCV infection, this method should be practical enough for use in laboratories that perform nucleic acid testing.

FIG. 1

Example of combined HDA and SSCP analysis in one gel, illustrating the importance of driver selection. The E2 region of HCV was amplified from serum by RT-PCR and cloned, and the combined HDA+SSCP procedure was performed. Representative results are shown. (A) Lanes 1 to 4 contain DNA from four cloned cDNAs, with a driver sequence selected according to details given in Materials and Methods. (B) Lanes 1 to 4 contain DNAs from the same four clones, with a different driver sequence from the same sample. S, SSCP; H_n, heteroduplex bands; H₀, homoduplex bands.

FIG. 2

Intraclonotype versus interclonotype diversity, by specimen. For each specimen, intraclonotype (A) and interclonotype (B) nucleotide sequence comparisons were made and expressed as the mean pairwise genetic distance. Error bars indicate ±1 SD.

FIG. 3

Frequency distributions of intraclonotype and interclonotype diversity for all samples. All possible pairwise sequence comparisons were performed and classified as interclonotype or intraclonotype based on the results of HDA, the SSCP method, or the HDA+SSCP method. A histogram of the resulting percent diversity is displayed.

FIG. 4

Measures of complexity. The clonotype distributions for samples D and G were plotted, and the normalized Shannon entropy values (H′) are indicated in the inset.