Study On The Interaction Of H7 Avian Influenza Virus HA Protein With Immune Cells And Its Mechanism

Influenza virus,a family of Orthomyxoviridae,is a pathogens of causing influenza.In recent years,the avian influenza virus H5N1 and H7N9 have caused hundreds cases of infection world-widely each year[1].Compared with seasonal influenza,the mortality rate for avian influenza virus patients are significantly higher(60%and 36%in H5N1 and H7N9,respectively)[2.3.4].The high pathogenicity of avian influenza virus is related to over-reaction immune responses such as "cytokine storm",but the mechanism is unclear and the specific pathogene factor in avian influenza virus which cause "cytokine storm" is un-located.Influenza virus HA protein is a membrane protein which locates on the surface of the virus and infected cells,so it is possible for that HA protein directly binds and regulates immune cells.So we iniated the study to elucidate the interaction between influenza virus HA protein and immune cells in order to further dissect relation between HA protein and avian influenza virus Pathogenesis.In order to study the interaction between influenza HA protein and immune cells on mammalian cells,we firstly constructed 293A cell lines that could inducible express HA protein by PiggyBac transposon system.The results from flow cytometry showed that approximately 80?90%of the harvested cells expressed HA protein after adding inducer "Cumate",and it suggested that PiggyBac transposon could efficiently and accurately insert influenza HA genes into the cell genome.Westerning Blot showed that the HA protein of all subtypes of influenza viruses was successfully expressed,the amount of HA protein expression is inducer "Cumate" dose dependent.In order to investigate the interaction between influenza virus HA protein and immune cells,we measured the ability of cells expressing various influenza stains HA protein binding PBMC and its various subsets.The results showed that different influenza virus strains HA protein have different affinity with distinct Immune cells:(1)Compared with the cell lines expressing H3N2 HA protein,the cell lines expressing H7N7 and H7N9 HA protein could significantly binding CD4+ T cells and CD8+T cells.The H7N9 HA expressing cell lines majorly bind central memory and the naive CD4+ T cells,while the H7N7 HA expressing cell line had significant affinity for all T cells.(2)Cell lines expressing H7N9 HA protein had a high affinity on B cells,and this effect was not observed in other HA proteins expressing cell lines.To elucidate the preliminary mechanism of binding between H7 HA protein and immune cells,we measure the effect of neuraminidase(NA)on the binding ability of H7 HA expressing cell line.It was found that the removal of sialic acid completely blocked the affinity of H7N7 HA for T cells,and decrease the affinity of H7N9 HA binding B cells.The results suggested that the presence of sialic acid has a significant effect on H7 HA binding immune cells.In order to locate the specific site of H7 HA protein binding to immune cells,we used the H3N2 HA protein as the backbone,and replace its three functional regions with the corresponding segment from H7 HA1 but the cell lines expressing these mutations did not show any significant of the affinity with the immune cells.It indicated the affinity of H7 HA to immune cell need multiple regions coorperation.To located the key amine acid for H7N9 HA binding B cells,we mutated 15 amine acids in H7N9 HA which were different with H7N7 HA protein.The result showed that the mutl1 could significantly inhibit the ability of H7N9 HA binding B cell,but its effect on H7N9 binding CD4+ and CD8+ T cell is not significant.It indicated mutl1 amine acide is the key amine acid for H7N9 HA binding B cells.In conclusion,HA proteins from various influenza virus strain have different affinity to distinct immune cells.Compared with HA protein of H3N2,H7N7 and H7N9 HA protein can significantly bind T cells,but only H7N9 HA protein can strongly bindingB cells.The results of this study provide a new vision for the further study focusing on the pathogenesis of H7N9 avian influenza virus.