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

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.

Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous, but Not Heterologous, Challenge

“…1A). 5=-UTR (33) and core-envelope 1 (core-E1) (34) analysis of week 2 viruses, amplified by reverse transcription (RT)-nested PCR detecting the four strains with equal sensitivity (29,33,34), detected only SA13(5a). The animal developed acute hepatitis with elevated serum alanine aminotransferase (ALT) levels from weeks 12 to 18 (Fig.…”

Viral hepatitis in Denmark

“…The most common HCV genotypes are genotypes 1a (43%), 3a (39%), 1b (11%), and 2b (6%) in Denmark (27). However, genotype 4 has an increased prevalence of approximately 2% among patients with hepatitis C referred to hospital departments in Denmark (28).…”

Clinicopathologic correlates of hepatitis C virus in brain: A pilot study

“…The first round of the nested PCR amplification of the HCV E1 region was performed using external E1 primers previously described (Corbet et al, 2003) in a 20-μl final reaction mixture containing 2 μl of template cDNA (from the reverse transcription reaction), 1× Phusion HF buffer, 200 μM of each dNTP, 0.5 μM primers, 3% DMSO, and 0.4 U Phusion DNA polymerase (Finnzymes). Cycle conditions were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s and 72°C for 20 s, and a final elongation at 72°C for 10 min.…”

“…Reverse transcription of the 5′ UTR and E1 regions was performed using the external reverse E1 primer of (Corbet et al, 2003), (see table 1). The 20-μl final reaction mixture contained 0.5 μg of total tissue or plasma RNA, 2.5 μM primer, 1× Transcriptor RT reaction buffer, 20 U Protector RNase Inhibitor, 1 mM of each dNTP, and 10 U Transcriptor Reverse Transcriptase (Roche).…”

“…For the nested, second round of the process, 2 μl of the first round reaction product were added to 18 μl of new reaction mixture containing 1× Phusion HF buffer, 200 μM of each dNTP, 0.5 uM primers, 3% DMSO, and 0.4 U Phusion DNA polymerase (Finnzymes) for a final volume of 20 μl. The internal E1 primers (Corbet et al, 2003) were used. The cycling conditions were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 69°C for 2 s, and 72°C for 18 s, and a final elongation at 72°C for 10 min.…”