Avian Influenza Virus Regulateed Chicken Mavs Mediated Antiviral Signal Pathway

Avian influenza viruses(AIVs)could cause the respiratory and systemic infection of poultry.Highly pathogenic avian influenza virus(HPAIV)could be replicated systematically and produces neurologic dysfunction and death in poultry,it could also occasionallyinfect humans causing clinical symptoms and death.As the natural reservoirs of influenza viruses,waterfowloftenshow little or no clinical signs.However,AIVs from waterfowl are often highly pathogenic to terrestrial birds,especially chickens.The pathogenicity of AIVs was dependent on multiple factors,and the hostimmune response was a very important factors.The current study focused onthe host innateimmune response of mammals,but a few studies focused on the host innate immune response ofpoultry.To understand the innate immune system in host defense against AIVsinfections,we used iTRAQ quantitative proteomic techniques to analyze chicken embryo into fibroblasts(CEF)and duck embryo into fibroblasts(DEF)infectedby AIVs.Bioinformatics analysis showed that most of the identified proteins related to cytoskeleton,stress response,antioxidant,energy metabolism,macromolecular biosynthesis,ubiquitin-proteasome pathway regulation,immune response and antigen processing presenting,signal transduction pathway.We identified more proteins in infected CEF than in infected DEF,but the number of differential protien in infected CEF was less than in infected DEF.Moreover,the number of total protein and differential protein variedin different infection times.Patheway analysis showed that the number of up-regulator proteins varied in different infection times and different host.Both TLRs and MDA5/MAVS signal pathway were activatedin infected CEF.In order to identify the chicken MAVS gene and prediction structure,the chicken MAVS gene was cloned from CEF cells by RT-PCR.The results showed that the CDS of chicken MAVS gene was 1926 bp,encoding 641 amino acids.Sequence analysis showed that chicken MAVS had low homology with the MAVS in mammals.Structural prediction showedchicken MAVS containedA caspase activation and recruitment domain(CARD),a proline-rich region domain(PRR)and a transmembrane domain(TM),which was similar to mammalian MAVS.Tissue distribution analysis indicated chicken MAVS expressed in the most organs of the chicken,and were higher in the trachea,liver and spleen.We investigated the structure and function of chicken MAVS.Overexpression of the chicken MAVS in DF1 cellsup-regulatedthe expression of TRAF3,IFN-? and IL6.Therefore,RLRs signal pathway could be effectively activated by chicken MAVS.Overexpression ofCARD domain,1-191 aa domain,1-525 aa domain and TM domain showed that 1-525 aa was the key domain of chicken MAVS-mediaed antiviral immune response.In order to understand the mechanism of interaction between avian influenza virus and host innate immune signal pathway,NS1 and PB2 genes of DK212 virus were co-expressed with chicken MAVS gene in DF1 cells.Our resultsshowed that NS1 and PB2 proteins inhibited the chicken MAVS-induced IFN-?production.Co-IP analysis further indicated thatNS1 protein could not directly interact with chicken MAVS,but PB2 protein could directly interact with chicken MAVS protein.Further analysis showed thatthe cap-binging domain and the unclear import domain of PB2 were the key domainto the interaction of PB2 with chicken MAVS.In summary,chicken MAVS played an important role in chicken antiviral natural immune response,and the 1-525 aa was the key domain of MAVS-mediaed antiviral immune response.The NS1 and PB2 proteins of avian influenza virus could inhibit the MAVS-mediated antiviral innate immune signal pathway.The PB2 protein could directly interact with the chicken MAVS protein,and the cap-binging domain and the unclear import domainwere the key of the PB2 protein interact with the chicken MAVS.