Typing of hepatitis C virus genomes by restriction fragment length polymorphism

Abstract

Recently, we reported that hepatitis C virus (HCV) can be classified genetically into two types, HCV-K1 and HCV-K2, which show 67% and 71% identity at the nucleotide and amino acid sequence levels in a 340 bp region which encodes the NS5 gene Gly-Asp-Asp motif. To develop a rapid method to classify the genomes of HCV isolates, we identified restriction fragment length polymorphisms (RFLPs) in reverse transcriptase-polymerase chain reaction products encoding a portion of the NS5 gene. AluI and AccII enabled HCV to be classified into the K1 and K2 types, and Sau96I enabled classification into the Kl type, and the K2a and K2b subtypes. These RFLPs also generally allow Japanese isolates to be distinguished from the prototype (PT, an isolate from the U.S.A.), which is a K1 type. Sequence analysis of the 5'-untranslated regions of Japanese isolates revealed near identity between the K1 type and PT, and 93 to 94% identity between the K1 and K2 types, indicating that there are type K1-and K2-specific RFLPs in this region. Our results suggest that the nucleotide sequences of the K1 and K2 types are different throughout the HCV genome. The incidence of HCV types K1, K2a and K2b, and PT in 50 samples was 74%, 16%, 8% and 2%, respectively.

Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5' non-coding region

“…Previous work has demonstrated that all six currently defined major genotypes of HCV (Simmonds et al, 1993a;Bukh et al, 1993) can be distinguished on the basis of RFLP analysis of RT-PCR products derived from the 5'NCR (Nakao et al, 1991;McOmish et al, 1993McOmish et al, , 1994Simmonds et al, 1994b). In one previous study, amplified DNA is cleaved with HaeIII-RsaI in one reaction and with MvaI-HinfI in a second, and the virus genotype is deduced by the combination of electropherotypes produced .…”

Use and interpretation of virological tests for hepatitis C

“…6 In clinical practice, HCV can be genotyped by direct sequence analysis, by reverse hybridization to genotype-specific oligonucleotide probes, or by restriction fragment length polymorphism analysis. [29][30][31] Two standardized kits based on PCR amplification of the 5' noncoding region are commercially available. The Trugene HCV 5'NC Genotyping kit (Visible Genetics, Toronto, Ontario, Canada) 32,33 is based on direct sequencing of PCR amplicons and sequence comparison with a reference sequence database.…”

Diversity of Hepatitis C virus in Southern India Based on 5′UTR Sequence

“…RFLP analysis of 5 0 UTR was employed by Nakao et al [13] making use of the conserved type-specific point mutations in the 5 0 UTR. Okamoto et al [14] designed type-specific primers from the core region of HCV to develop a nested PCR method for genotyping.…”