The immunoreactivity profile of different HCV genotypes on immunoblot assay and its implications in the development of diagnostic assays.

The immunoreactivity profiles of plasma samples obtained from patients infected with different hepatitis C virus (HCV) genotypes were studied using immunoblot assay containing multiple HCV antigens. The immunoblot assay was found to be positive in 81.5% of 195 blood donors who had anti-HCV antibodies as detected by second generation enzyme immunoassays. The samples reacted preferentially with the viral core, NS3-1 and NS5 antigens, and these reactivities were not influenced by HCV genotype. However, the reactivities with NS3-2 and NS4 antigens varied depending on HCV genotypes. The samples from patients infected with HCV genotype 1 reacted well with NS3-2 and NS4 antigens whereas those with other genotypes did not. In addition, samples with the unclassified HCV genotype reacted poorly with all antigens, except NS3-1. This study demonstrates the importance of the core, NS3-1 and NS5 antigens in the detection of antibodies against HCV, especially in areas where more than one genotypes of HCV are present. It also demonstrates that there is a need for further improvement of the currently used assays as new HCV genotypes are recently discovered.

Improved amplification system for detection of hepatitis C virus genome that simultaneously differentiates viral genotypes.

An improved system for amplification of hepatitis C virus genome (HCV) was developed based on a multiplex nested polymerase chain reaction format. Two sets of oligonucleotide primers were used simultaneously. One was derived from the conserved sequences in the 5' non-coding region of the viral genome which can bind to the viral genome of all genotypes. The other set of primers was designed from a sequence in the nonstructural-5 region of HCV. HCV genotypes 1 and 3 can be differentiated by the banding patterns of amplified DNA products. All of 39 samples containing the HCV genotype 1 could be amplified with primers in the 5' non-coding region only, whereas 92% of those with genotype 3 could be amplified by both primer sets. In addition, HCV RNA can be detected in 81% of 84 anti-HCV-positive blood donors and in 0% of 34 anti-HCV-negative cases. Of the HCV RNA-positive specimens, 69% showed genotype 1-like patterns while 31% showed genotype 3-like patterns. The detection rate of HCV RNA in this study was much higher than that in our previous report due to the improvement of new primers which can detect all genotypes of the virus. In conclusion, this improved amplification system is a sensitive method for rapid identification of HCV RNA in clinical specimens that can simultaneously differentiate the two most common genotypes of HCV found in Thailand.