Recombinant HCV type 2b Nonstructural Protein 5 (a.a. 2212-2313) [GST]

A set of 150 synthetic peptides spanning the proteins NS3-NS4-NS5 of the hepatitis C virus (HCV) was synthesized and tested with a panel of 20 sera obtained from HCV-infected patients. Of 62 peptides prepared from the NS3 region, none exhibited strong antigenic reactivity. Rather, five peptides from this region demonstrated specific reactivity with only 5-10% of anti-HCV-positive sera. Nonetheless, it is well known that the NS3 region contains strong antigenic epitopes. These epitopes appear to be modeled in a functionally active manner with recombinant proteins and cannot be mimicked properly with short synthetic peptides. This finding suggests that the major NS3 antigenic epitopes are conformationally dependent. Seven of 20 peptides prepared from the NS4 region were immunoreactive. Five peptides from this region demonstrated very strong HCV-specific antigenic reactivity, Four of the five peptides belong to the recognized immunoreactive 5-1-1 region located inside the C100-3 antigen. One peptide demonstrating immunoreactivity with approximately 90% of anti-HCV-positive sera was found outside the C100-3 region at the C-terminal part of the NS4 protein. Of 68 peptides synthesized from the NS5 protein, 30 were immunoreactive. Six of the 30 demonstrated immunoreactivity with 35-50% of anti-HCV-positive sera. Thus, the NS4 and NS5 regions of the HCV polyprotein contain a large number of specific, broadly reactive, linear antigenic epitopes. The highly antigenic reactivity of the NS5 region suggests that this protein may have significant diagnostic potential. (C) 1995 Academic Press, Inc.

The use of phylogenetic analyses of partial NS5 and core regions for hepatitis C virus (HCV) genotyping was evaluated by analysing seven Honduran and 24 European HCV strains. Core primers were designed with which HCV genotypes 1, 2, and 3 were readily amplified. The reliability of phylogenetic analysis of a Ill-bp core sequence was verified by comparing the typing results with those obtained using the whole core gene of 52 reference strains. Accordant genotypes (la, Ib, 2b, and 3a) were obtained when phylogenetic analyses were undertaken on both the partial core and a 222-bp NS5 sequence in ail of the European HCV strains. Genotypes la, Ib, and 3a were identified among the Honduran strains by phylogenetic analysis of the partial NS5 sequence. Interestingly, two of three Honduran type 3a strains, as determined by the NS5 sequence analysis, turned out to be type la by core sequence analysis. These two strains were also classified as type la, but not 3a, by a core type-specific PCR. Furthermore, the E2/NS1 regions were similar to HCV-PT, a representative strain of genotype la. The results indicate that chimeral HCV strains exist, although in most cases a good concordance is found when phylogenetic analysis of partial core and NS5 sequences are used for genotyping. This finding should be taken into account when HCV is genotyped by phylogenetic analysis of a partial HCV sequence from a single genomic region. (C) 1996 Wiley-Liss, Inc.