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Abstract
Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever (http://imed.med.ucm.es/PVS/) and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA.
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Figure 1.
Multiple sequence alignment of NS1 proteins from avian influenza virus reported in Korea using Multalin. The NS1 protein sequences were obtained from GenBank. Multiple sequence alignment analysis of NS1 proteins using Multalin displayed 84% homology.
Figure 2.
Detection of anti-NS1 antibodies by synthetic peptide-based ELISA in chicken sera. (A) NS1-1, (B) NS1-2, (C) NS1-3, and (D) NS1-4. Dashed horizontal line represents the cut-off value calculated by adding an OD 450 nm mean value and two standard deviations (SD). The cut-off values for the test are (A) 1.122, (B) 1.127, (C) 0.601, and (D) 1.787. Serum sample is positive when OD value is greater than the line.
Figure 3.
Detection of anti-NS1 antibodies by synthetic NS1-5 peptide-based ELISA in chicken sera. Dashed horizontal line represents the cut-off value calculated by adding an OD 450 nm mean value and two standard deviations (SD). The cut-off value for the test is 1.257. Serum sample is positive when OD value is above line.
Table 1.
GenBank accession number of NS1 proteins in Korea avian influenza
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