Establishment of a high sensitive indirect ELISA for detecting specific antibodies against H9 subtype avian influenza virus / 生物工程学报

H9 subtype avian influenza virus causes worldwide epidemic, resulting in enormous economic losses of poultry production. In the present study, an indirect ELISA method was established for more accurate and specific detection. The recombinant protein of the globular head domain of HA of H9 subtype avian influenza virus was used as antigen. Specific blocking buffers and dilution buffers were determined to increase the sensitivity and specificity. The sensitivity of ELISA was higher than that of hemagglutination inhibition (HI) test. The coating antigen is very specific and no cross-reactivity with positive serum against H3N2, H5N2 and H7N9 subtype influenza viruses, Newcastle disease virus, avian infectious bronchitis virus, avian infectious disease virus, and egg drop syndrome virus. Two hundred of clinical sera samples were examined. The results indicate the coincidence rate between ELISA and HI test reached 97%. In addition, there was a positive correlation between OD450 values and the logarithm of HI titer to the base 2 of an individual serum sample (R2=0.981 1).

Proteomic Analysis of the Serum from Chicken Infected by Avian Influenza Virus

Avian influenza (AI) is an infectious, low pathogenic virus that is endemic all over the world and poses a potential threat to the poultry industry. Vaccination is a widely used effective method to prevent avian influenza virus. Here we employed a comparative proteomics approach [two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)] to characterize proteome in the sera from the specific pathogen free (SPF) chickens, the vaccinated chickens, and the naturally infected chickens. We identified total 58 proteins that were differentially expressed in the sera of three groups. Among them ovotransferrin and vitamin D-binding protein were more expressed in the sera of naturally infected chickens compare with other groups. Our results suggested that the level of these two proteins in the serum may help to discriminate the naturally infected chicken from the vaccinated chicken.

Detection of the Avian Influenza Viruses Nonstructural Protein 1 for Distinction between Vaccinated and Infected Chickens Using Synthetic Peptide-Based ELISA

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.