Evaluation of a reverse hybridization assay for genotyping of hepatitis C virus

• Published in: Journal of hepatology Vol. 23; no. 6; pp. 654 - 661
• Main Authors: Zeuzem, Stefan, Rüster, Brigitte, Lee, Jung-Hun, Stripf, Tobias, Roth, W.Kurt
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier B.V 01.12.1995; Elsevier
• Subjects: Base Sequence; Biological and medical sciences; Genome, Viral; Genotype; Hepacivirus - classification; Hepacivirus - genetics; Hepatitis C virus genotype/subtype; Human viral diseases; Humans; Infectious diseases; Medical sciences; Molecular Sequence Data; Nucleic Acid Hybridization; Phylogenetic analysis; Polymerase Chain Reaction; Reverse transcription; Sequence analysis; Viral diseases; Viral hepatitis; Phylogenetic analysis; Polymerase chain reaction; Reverse transcription; Hepatitis C virus genotype/subtype; Sequence analysis; Human; Evaluation; RNA-directed DNA polymerase; Enzyme; Transferases; Hepatic disease; Hybridization; Infection; Virus; Nucleotidyltransferases; Microbiological investigation; Viral disease; Digestive diseases; Flaviviridae; Genome; Hepatitis C virus; Viral hepatitis C
• ISSN: 0168-8278; 1600-0641
• DOI: 10.1016/0168-8278(95)80030-1

Background/Aims: Several strains of the hepatitis C virus exist; distinct genotypes and subtypes can be identified by sequence comparison of the viral genomes. Recent evidence that the genotype/subtype of hepatitis C virus may infuence the clinical course of chronic hepatitis C and the response to interferon- a therapy for this disease suggests that methods to identify the genotype may become clinically useful. In the present study we evaluated a recently introduced reverse hybridization assay. Methods: HCV-RNA was isolated from serum samples from 61 consecutive patients attending our out-patient clinic and subsequently sequenced in the 5′-noncoding and the nonstructural-5 region by the dideoxynucleotide chain termination method. HCV-genotyping was performed by phylogenetic analysis of nonstructural-5 sequences. The amplification product for the reverse hybridization assay was obtained by “nested” polymerase chain reaction using biotinylated primers corresponding to the 5′-noncoding region. The assay is based on hybridization of the resulting polymerase chain reaction product with oligonucleotide probes immobilized as paralleled lines on membrane strips. Results: According to the phylogenetic analysis of the nonstructural-5 region the prevalence of hepatitis C virus subtypes was as follows: 1a 18%. 1b 51%, 2a 3%, 2b 3%, 2c 7% and 3a 18%. The reverse hybridization assay correctly identified each hepatitis C virus genotype (1, 2, and 3). However, differentiation of hepatitis C virus subtypes was insufficient. 1 11 HCV-1a isolates was incorrectly classified by the reverse hybridization assay as HCV-1b and vice versa 3 31 HCV-1b isolates as HCV-1a. Classification of hepatitis C virus subtypes 2a, 2b and 3a was correct, but 4 4 HCV-2c isolates were misinterpreted by the assay a HCV-2a. Conclusions: The reverse hybridization assay can differentiate between hepatitis C virus genotypes 1, 2, and 3, but is not completely reliable for hepatitis C virus subtyping.