It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.
Animals ; Cattle ; Enzyme-Linked Immunosorbent Assay/veterinary ; Foot-and-Mouth Disease/*diagnosis ; Foot-and-Mouth Disease Virus/*isolation&purification ; Goats ; Sensitivity and Specificity ; Vaccination ; Viral Nonstructural Proteins/*chemical synthesis ; Viral Vaccines

OBJECTIVETo identify the location of major immunodominant antigenic region and study the relationship between the gene heterogeneity and immunoreactivity via detecting antigenic reactivity of synthetic peptides deriving from immunodominant region in different genotypes of hepatitis C virus (HCV) NS5a gene.

METHODSIn total, 305 non-identical 30-mer long and overlapping by 15 aa peptides derived from HCV NS5a region from codon 2,182 to 2,343 among 45 unique published HCV sequences in GenBank corresponding to different genotype were designed and synthesized. The amino acid sequences of all peptides were compared with DNA Star software. The antigenic reactivity of those peptides was detected with indirect ELISA with both anti-HCV and anti-NS5 positive serum.

RESULTSThe sequences showed highly conserved among HCV genotype in regions 2,272-2,301 and 2,302-2,331 as compared to regions 2,212-2,241 and 2,257-2,286. The peptides basing on amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331 showed stronger immunoreactivity than any other peptides. Eighteen peptides derived from this region showed a broad immunoreactivity, 3 of them could react with 96% of anti-HCV positive sera. Whereas the immunoreactivity of the peptides derived from the region showing highly variable among HCV genotype was found to react more strongly with homologous-genotype sera.

CONCLUSIONThe major linear antigenic region was located at amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331; the short synthetic peptide derived from NS5a region at position 2,212-2,241, 2,272-2,301 and 2,302-2,331 can be used for efficient detection of HCV antibody; some peptides showed genotype specific immuunoreactivity.

Amino Acid Sequence ; Antigenic Variation ; Antigens, Viral ; blood ; immunology ; Epitope Mapping ; Genotype ; Hepacivirus ; classification ; genetics ; immunology ; Hepatitis C ; immunology ; virology ; Hepatitis C Antibodies ; blood ; immunology ; Humans ; Immunodominant Epitopes ; Peptides ; chemical synthesis ; immunology ; Recombinant Proteins ; genetics ; immunology ; Sequence Homology, Amino Acid ; Viral Nonstructural Proteins ; genetics ; immunology