Antagonistic Effect On Interferon And Interaction With Host Of H5N1 Subtype Avian Influenza Virus With Partial Deletion In NA And NS1 Genes

H5 subtype avian influenza virus (AIV) is an important zoonotic pathogen, which becomes the dominant strain with partial deletion in both neuraminidase (NA) and nonstruturall (NS1) genes under the natural selection. However the mechanism of evolution for the virus has not yet been studied and reported.Studies showed that NA and NS1 protein could help avian influenza virus antagonistic interferon. NA protein of 49-48 deleted 20 aa, enhanced the proliferation ability of the virus under a certain concentration of interferon. NS1 protein 80-84 deleted 5 aa can also improved the proliferation of virus, but the specific mechanism was unclear.1. Construction of expression plasmid and preparation of polyclonal antibodies aganist NA and NS1.In order to obtain the antibodies which can be used to detect the expression of NA and NS1 protein, The NA and NS1 genes were cloned into the prokaryotic vector PET-32a orderly. After the recombinant plasmid was transduced into E.coli BL21 cells, the expression of NA and NS1 were induced by IPTG. The antisera against NA and NS1 protein were geneated by immunizing BABL/c mice with purified NA and NS1 protein.2. Antagonistic effect on interferon in H5N1 subtype Avian Influenze Virus with partial deletion in NA.For the study of antagonism interferon of NA protein of recombinant, the experiment adopted the eukaryotic expression plasmid pcDNA3.1-NA transfect into Vero cells, Western-Blot detected the protein expression level. The test determined the proliferation abilitystrain of AIV on the Vero cells. Results showed that under the effect of different concentration of interferon, antagonism interferon levels of the four strains from low to high in turn order were:A+A+<S+S-< A-S+< A-S-. Under the same condition, we transfected pcDNA3.1-NA into Vero cells, antagonism interferon ability of virus on Vero cell was improved, it showed that AIV NA protein can influence the abilitytype of antagonism IFN I.3. Proteomic analysis of lung tissues of duck infected with H5N1 AIVIn this study, the differentially expressed proteins were investigated in H5N1 avian influenza virus (AIV) infected duck by two dimensional gel electrophoresis (2-DE). The lung tissues were lysised from the AIV infected or control groups were extracted at 1,3,5 day post-inoculation and separated by 2-DE. Five protein spots were found to have notably differential expression between the 2 groups (ratio>2, p<0.05), up-regulated in the AIV infected duck five of them were identified by MALDI-TOF/TOF, including:CD81, ATPase, Gammaglobulin, myosin and aryotic initiation factor. The transcriptional levels of CD81 was verified by real-time PCR, which was completely consistent with the proteomic analysis result.This study proved that in the H5N1 AIV, the deletion of NA gene enhanced the ability of antagonism interferon than the deletion of NS1 gene. CD81,eukaryotic transcription initiation factor were found in A+S+ and A-S-strain infected ducks. The results preliminary explored the interaction between the H5N1 subtype of avian influenza and host.