Establishment Of Indirect Immunofluorescence Assay (IFA) With NA Protein From H5N1 Subtype AIV

Avian Influenza (AI) is one of the fatal infectious diseases caused by influenza A virus in avian. It first reported of AI was in Italy in 1878. Subsequently, different strains were found and caused enormous economic losses to the avian industry throughout the world. At Present, Highly pathogenic avian influenza(HRAI) is listed in A infectious diseases by Office International Des Epizooties(OIE).Recently, HPAIV broke out among poultry throughout Asia,leading to not only tremendous economic losses in poultry industry,but also human infections and deaths. Vaccine inoculation is the key method to against from AI infection. But there was no way to distinguish between vaccinated and naturally infected birds through serological test and it is impossible to detect or control AI early and undertake permissive trade. However, the usage of a heterologous virus possessing a different NA subtype was suggested as a possible solution to the problem of detecting vaccinated birds infected with field virus. Therefore the homologus differential diagnostic method through serological test was created.A pair of primer was designed by the analysis of the reported H5N1 subtype strains NA gene sequences available in GenBank (DQ201830.1).A 1.4 kb gene fragment was amplified by RT-PCR, using H5N1 AIV nucleinic acid as template. Results of homology analysis showed the homology between this amplified DNA fragment and a published NA gene (A/Goose/Huadong/ 1/2000(H5N1), in Genbank reached 100%. NA gene was inserted in EcoRⅠand PstⅠmultiple cloning sites of plasmid pFastBacTM HTA and named positive recombinant transfer vector as pF-NA.The pF-NA was transformed into DH10Bac. Recombinant bacmid rAcBacmid-NA with the insertion of NA gene was obtained by specific transposition, between pF-NA and AcMNPV bacmid. Recombinant virus vAc-NA was obtained by transfecting purified rAcBacmid-NA into Sf9 insect cells. Recombinant baculovirus was harvested 6 d later. Results of PCR indentification showed hybrid NA gene has been recombinant correctly with baculovirus DNA namely pF-NA was infected tautologically on monolayer Sf9 cell to obtain high titer P3.To express recombinant NA interest protein, P3 was infected on Sf9 monolayer cell for 4 dand the CPE cell was harvested. The analysis results of SDS-PAGE and Western-blot showed that AIV NA gene was expressed in baculovirus, a 52 kD protein was detected according with the theoretic molecule weight and the recombinant protein could be recognized by 6×His monoclonal antibody. The result of indirect immunofluorescence Assay (IFA) indicated that the expressed protein possessed the antigenic specificity which could be recognized by AIV H5N1 subtype positive serum and was displayed on the surface of Sf9 cell.Initial establishment indirect immunofluorescence detected sera antibody with NA protein. Subsequently, Our experiments researched the conditions about MOI value, the best time of protein expression and cell fixed agent.Virus titer of recombinant baculovirus was 0.5×108 pfu/ml by plaque counting. MOI value was 7, the time of protein expression was 120 h and cell fixed agent was 4% polyoxymethylene by square matrix titration. Sera from chickens inoculated H5N2 subtype inactivated vaccine, no-inoculated, infected N1 subtype field virus were to detect sera. The sera dilution were determined to be 1:20 and the incubation time was 1 h at 37℃; Fluorescein isothiocyanated (FITC) conjugate goat anti-chicken IgG was the second antibody and the dilution was 1:60. The incubation time was 0.5 h at 37℃.NA-IFA, differential diagnosis method was developed through above searching experiment scenarios. To evaluate the specificity and sensitivity of the NA-IFA through aforesaid method, 120 serum samples were collected from experimentally manipulated chickens. Using HI as the reference methods, the specificity of the NA-IFA was 100% and its sensitivity was 98.3%. To test the reproducibility of NA-IFA, 8 serum samples with different antibody levels were tested 3 times with ELISA plates coated with the same batch of NA protein. Each serum sample was assayed 3 times with each batch of coated plates in different time. The result showed there was no significant difference within sample groups. To further evaluate the specificity of the test, the NA-IFA was used to test a panel of sera, including sera from chickens with infectious bursal disease (IBD), Newcastle disease (ND), infectious bronchitis (IB), and sera from H5N2 subtype vaccinated chickens, N1 subtype AIV infected chickens and SPF chickens. Only sera from N1 subtype AIV infected chickens was found positive by NA-IFA, which indicated the NA-IFA was highly specific.Morever,we developed diagnosis kit based on above NA-IFA.The kit was stable well when storing at 4℃for 7d, 37℃for 2d and -20℃for 7 months up to now.The kit was convenient, economic and simple. Results of above experiment indicated that the NA-IFA can be used to differentiate N1 subtype AIV infected animals from H5N2 subtype vaccinated ones. Potentially, it is a significant diagnostic tool for AI eradication campaigns and control programs.