Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients

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Abstract
A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5′-untranslated region (5′-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5′-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5′-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5′-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.