Hepatitis C virus heteroduplex tracking assay for genotype determination reveals diverging genotype 2 isolates in Italian hemodialysis patients

Abstract

A heteroduplex tracking assay (HTA) was developed for genetic analyses of the hepatitis C virus (HCV) using single-stranded probes from the core (C)/E1 region. Nucleotide sequencing of reverse transcriptase (RT)-PCR products from 15 Italian dialysis patients confirmed the specificity and accuracy of the HTA genotyping method, which identified 5 of 15 (33.3%) 1b, 7 of 15 (46.7%) 3a, and 3 of 15 (20%) type 2 infections. The genotypes of an additional 12 HCV antibody-positive blood donors from different geographical locations were also in agreement with the genotypes determined by the Inno-LiPA HCV II kit (Innogenetics) and/or restriction fragment length polymorphism (RFLP). Isolates which had between 35 to 40% nucleotide divergence from control subtype 1a, 1b, 2a, 2b, or 3a standards could be typed. Surprisingly, HTA detected one 1b-2 coinfection which was missed by DNA sequencing. Three samples that were designated non-2a or 2b type 2 by HTA were found to be type 2a by both RFLP and direct nucleotide sequencing of the 5' untranslated region. The genetic distance between patient type 2 and control 2a, 2b, and 2c isolates indicated that a new subtype was present in the population being studied. Serotyping (RIBA serotyping strip immunoblot assay kit) of 23 dialysis patients showed that the genotype could be determined in 6 of 8 (75%) C/E1 RT-PCR-negative and 15 of 23 (65.2%) RT-PCR-positive samples, indicating that the two tests complement each other.

FIG. 1

Single-stranded probes (lanes 1a, 1b, 2a, 2b, and 3a in panels A to E, respectively) were hybridized to PCR products from either the same control serum samples from which they were derived, forming homoduplexes (h), or from dialysis patient sera, forming heteroduplexes (numbered lanes), and were electrophoresed on MDE gels.

FIG. 2

Phylogenetic analysis of partial C/E1, E1, or 5′ UTR nucleotide sequences from dialysis patients (HTA 1 to 4, 7, 18, 20 to 26, 28, 30, 33, and 35) and published subtypes 1a (HCV-1 [10]), 1b (HCV-J [22]), 2a (HC-J6 [29]), 2b (HC-J8 [29]), 2c (S83 [4]), and 3a (NZL-1 [31]). Nucleotide coordinates are according to reference .

FIG. 3

Single-stranded probes (1a, 1b, 2a, 2b, and 3a) were hybridized to either PCR products from which each single-stranded probe was derived (lanes 1a, 1b, 2a, 2b, 3a in panels A to E) or to HCV antibody-positive sera from the United States (lanes b to j), Egypt (lane a), and Holland (lanes k and l). The single-stranded probe alone is designated by the letter p.