Selection and application of serotypical synthetic peptides derived from hepatitis C virus NS5A region.
BACKGROUND
Numerous studies have reported a relationship between hepatitis C virus (HCV) genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay (ELISA) method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a (NS5A) region was developed.


METHODS
Based on 45 sequences, representing HCV genotypes 1 - 6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182 - 2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1 - 6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis.


RESULTS
The correspondence of amino acids in HCV NS5A region with amino acids in positions 2182 - 2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182 - 2211 (R1), 2272 - 2301 (R7) and 2302 - 2331 (R9): the highly variable 2212 - 2241 (R3) and 2257 - 2286 (R6). Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera. Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis.


CONCLUSIONS
The major antigenic regions in HCV NS5A region were at 2212 - 2241 (R3), 2272 - 2301 (R7) and 2302 - 2331 (R9). Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.
Dou
Xiao-guang
XG
Department of Infectious Disease, Shengjing Hospital Affiliated to China Medical University, Shenyang 110004, China. DOUXG@CMU2H.COM
Zhang
Lin
L
Li
Zhi-wei
ZW
Feng
Guo-he
GH
Chang
Joy
J
Fildes
Howard
H
Khudyakov
Yuri
Y
D000595:Amino Acid Sequence; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D012703:Serotyping; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000276:immunology*; D012324:RNA-Dependent RNA Polymerase
D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase