ABSTRACT
Nonstructural 3 (NS3) protein of hepatitis C virus (HCV) is one of the antigens commonly used in diagnostic assays for antibody to hepatitis C virus. However, immune response to the NS3 protein from one genotype may not cross-react with that from other genotypes. In the development of an anti-HCV assay, the NS3 genes from genotypes 1 and 3 commonly found in Thailand were amplified and cloned into a bacterial expression system. These recombinant NS3 proteins were immunogenic and reacted with plasma samples of Thai patients infected with various HCV genotypes. Interestingly, the NS3 proteins from the Thai genotypes could react with 3 plasma samples from HCV infected Thai blood donors, which could not bind to the NS3.1 protein in the commercial HCV immunoblot kit using antigen from HCV genotype 1. This finding supports our prior observation that the appropriate HCV antigens used in a diagnostic assay should be derived from the virus genotypes commonly found in that geographical region.
Subject(s)
Amino Acid Sequence , Base Sequence , Biomarkers/blood , DNA Primers/genetics , DNA, Viral/genetics , Electrophoresis, Polyacrylamide Gel , Genetic Vectors/genetics , Genotype , Hepacivirus/chemistry , Hepatitis C/diagnosis , Humans , Immunoblotting , Molecular Sequence Data , Nucleic Acid Amplification Techniques , Protein Isoforms/genetics , Recombinant Proteins/diagnosis , Sensitivity and Specificity , Thailand , Viral Core Proteins/genetics , Viral Nonstructural Proteins/diagnosisABSTRACT
New sets of primers for amplification of hepatitis C virus (HCV) RNA were designed from the conserved regions of American and Japanese isolates of HCV. Primers set A amplified parts of the 5’-untranslated and core gene regions, whereas set B amplified parts of the core and envelope gene regions. PCR amplification were carried out from HCV RNA isolated from sera of 13 Thai patients with antibody to HCV, using these newly developed primers. HCV RNA was detectable in 8 patients (61.5%). Of those PCR positive samples, only 4 patients (50%) were positive with primer set A, whereas 7 patients (87.5%) were positive with primer set B. Interestingly, 4 patients were tested positive with only set B, 3 patients with both sets A and B, and just one patient with set A only. This result suggests that PCR assay as a diagnostic tool for HCV may need to be carried out with more than one primer set. The relatively low percentage of PCR positivity of the HCV from Thai patients using primer set A, which was previously identified as the most conserved region of the HCV genome, also indicates that the sequence of the Thai isolates of the virus may be different from those of the American and Japanese strains.