The Effect Of PA-X Protein Of H9N2 Subtype Avian Influenza Virus On Innate Immunity Of Mucosal Dendritic Cells

H9N2 subtype low pathogenicity avian influenza virus(AIV)not only has pathogenicity to poultry,but also can directly infect humans across species and provide internal genes for other subtypes of influenza viruses,resulting in an increaseed viral susceptibility to humans.Therefore,H9N2 subtype AIV poses a potential threat to the global poultry industry and human health.As a new protein encoded by the PA gene,PA-X protein is currrently considered to be a virulence factor of H9N2 subtype AIV,and is also related to the regulation of host's innate immune,especially induces an immunosuppression.Dendritic Cells(DCs),as important antigen presenting cells,are the key to regulate the host's innate immune response.It is still unclear whether the PA-X protein of H9N2 subtype AIV can regulate the innate immune function of DCs,thereby affecting viral pathogenicity to poultry and mammals.In this study,we firstly genereated the H9N2 subtype PA-X deficient strain and the H1N1 subtype PA-X deficient strain to compare the replication levels in vitro and pathogenicity to mammals in vivo between parental strains and PA-X deficient strains.Secondly,we evaluated the effect of the parental strain and PA-X deficient strain on the innate immune response of murine primary DCs in vitro and nasal mucosal DCs in vivo.Finally,we established an induction culture model of chicken bone marrow-derived DCs in vitro,and initially explored the effect of H9N2 subtype parent strain and PA-X deficient strain on the innate immune response of chicken DCs.This study is the first to analyze the effect of PA-X protein on innate immunity of mammalian and avian hosts at the DCs level,which provides a basis for revealing the pathogenic mechanism of H9N2 subtype AIV.1.Generation of H9N2 PA-X deficient virus strains and their pathogenicity in mammalsH9N2 subtype avian influenza virus(rTX),H1N1 subtype influenza virus(rPR8)and their PA-X deficient viruses(rTX-APAX and rPR8-APAX)by site directed mutagenesis and reverse genetics were sucseccfully generated.The knockdown or deletion of PA-X protein was verified by western blot,and the pathogenicity of the above-mentioned strains to mammals was further evaluated in vivo and in vitro.The results showed that the deletion of PA-X attenuated the early growth and replication levels of H9N2 subtype AIV and H1N1 subtype influenza viruse on MDCK mammalian cell lines.Murine pathogenicity assay in vivo showed that,the lung pathological changes were weakened,the lung viral load and the inflammatory cytokine IL-6,TNF-?,and IP-10 expression levels were significantly reduced when infection of PA-X deficient virus strain compared with the parent strain.Therefore,the PA-X protein of H9N2 subtype AIV significantly increased the pathogenicity of H9N2 subtype influenza viruses to mammals.2.Effect of H9N2 PA-X deficient virus strain on the level of innate immunity in murine DCsFirstly,we cultured murine bone marrow-derived DCs which had a typical dendritic structure by light microscope and the purity was>91.7%identifited by flow cytometry.DCs can be transformed into a mature state and expressed CD86 highly under the lipopolysaccharide(LPS)stimulation.Therefore,the cultured DCs can be used for subsequent experiments.Subsequently,the parental strains(rTX,rPR8)and PA-X deficient viruses(rTX-APAX,rPR8-APAX)were used to infect murine DCs.The result of flow cytometry showed that the deletion of PA-X significantly enhanced the ability of H9N2 and H1N1 to infect DCs.Morever,the morphological index of DCs were significantly promoted,the expression level of phenotypic markers(CD80,CD86,CD40,and MHCII),early activation markers(CD69)and migration markers(CCR7)on DCs was significantly increased.At the same time,the secretion levels of the pro-inflammatory cytokine IL-1?,IL-6,IL-12,and TNF-? were increased,and the secretion level of the anti-inflammatory cytokine IL-10 was decreased.In the mixed lymphoid reactions assay between infected DCs(MOI=0.5)and CD4+T cells(DCs:T=1:1/1:5),DCs infected by PA-X deficient virus significantly stimulated the proliferation of CD4+ T cells.Finally,after the intranasal infection of each recombinant viruse into mice,the deletion of PA-X of H9N2 subtype virus significantly promoted DCs recruitment under the nasal mucosa and draining lymph nodes.DCs involved in this process were mainly CD11b+ subtype.The above results indicated that the PA-X protein of H9N2 subtype AIV significantly suppressed the innate immunity of murine DCs.3.Effect of H9N2 PA-X deficient virus strain on the level of innate immunity in chicken DCsFirstly,the recombinant chicken GM-CSF and IL-4 were combined to induce and culture chicken bone marrow-derived DCs.The results showed that the cultured cells grew in a typical colony pattern and had obvious dendritic structure observed by light microscope.The expression level of phenotypic markers CD40 and CD86 were increased significantly under the stimulation of LPS identifited by flow cytometry.The above results indicated that the cultured DCs were in accord with the morphological and functional requirements,which can be used in subsequent experiments.Secondly,chicken DCs were infected by the parental strain(rTX)and PA-X deficient virus(rTX-?PAX).It showed that the ability of H9N2 PA-X deficient virus to infect chicken DCs and stimulate the maturation of chicken DCs was significantly enhanced,which was manifested by significant increases in the expression of NP protein and phenotypic markers of chicken DCs(CD86,CD40,and MHCII)identifited by flow cytometry;meanwhile,the inflammatory cytokine IL-6,IL-12 and IL-1? expression levels were significantly increased when IFN-? and IL-10 were significantly decreased.The above results preliminarily indicated that the PA-X protein of H9N2 subtype AIV suppressed the innate immunity levels of chicken DCs.