Serum Amyloid A Regulates Toll-like Receptor 2/4-Mediated IFN-β Signaling Pathway Inhibiting Marek’s Disease Virus Replication

Marek’s disease virus(MDV)is a highly avian oncogenic herpes virus,which can cause immune inhibitory tumor diseases in broiler chickens and laying chickens.MDV has been widely popular in our country and causes huge economic losses to the breeding industry.MDV activates the host’s strong immune response,causing rapid up-regulation of acute response phase proteins(APPs)including serum amyloid A(SAA)in a short time.SAA is an APP widely found in mammals and birds,it not only inhibits the infective ability of multiple viruses through directly binding to viral proteins,but also plays an immunomodulatory function through activating innate immune signaling pathways.In recent years,more and more studies have demonstrated that SAA can regulate innate immunity and play an antiviral function.However,the role of SAA in MDV infection remains unclear.To determine the role of SAA in MDV infection,we explored the induction effect of MDV infection on SAA in vivo and in vitro.Through in vivo and in vitro detection,we found that MDV infection significantly increases SAA expression in chicken and chicken embryo fibroblast(CEF)cells.SAA,as an important APP,can regulate host innate immunity,this suggests that SAA may be involved in host resistance to MDV infection.To clarify the relationship between SAA expression and MDV infection,we constructed the eukaryotic expression plasmid of SAA and the interference RNA(si RNA)targeting SAA.Through overexpression and interference experiments,we found that the viral titer of MDV significantly decreased and increased,which proved that SAA could inhibit MDV replication.In order to confirm whether SAA has immunoregulatory effect and exerts antiviral function through immune regulation,chicken interferon Beta(IFN-β),interferon regulatory factor 7(IRF7)and nuclear transcription factor κB(NF-κB)luciferase reporter genes were constructed.Dual luciferase assay demonstrated that SAA regulates IFN-β signaling pathway through regulating IFN-β and IRF7 promoter activation in CEF cells.Subsequently,in order to explore whether the regulatory effect of SAA on IFN-β can be achieved through the mediation of Toll-like receptor 2/4(TLR2/4)receptor,the activator of TLR2/4 receptor was used in this study.We found that SAA can regulate innate immunity through regulating TLR2/4-mediated IFN-βsignaling pathway when TLR2/4 signaling pathway was activated.To further confirm the regulatory effect of SAA on TLR2/4-mediated IFN-β signaling pathway during MDV infection,we explored the regulatory of SAA on TLR2/4 signaling pathway during MDV infection.The results showed that SAA regulates the expression of downstream factors related to IFN-βsignaling pathway which mediated by TLR2/4,type I interferons(IFNs)and interferon stimulator genes(ISGs)during MDV infection and promote IRF7 phosphorylation.Finally,in order to determine whether SAA inhibits MDV replication through TLR2/4-mediated IFN-βsignaling pathway,we used inhibitors of TLR2/4 pathway,and found that after inhibition of TLR2/4 signaling pathway,the expression and production of IFN-β mediated by SAA and the inhibition of SAA on MDV replication decreased significantly.These results suggest that SAA can increase rapidly after MDV infected,regulating TLR2/4-mediated IFN-β signaling pathway,exerting anti-MDV function.In conclusion,this study demonstrates for the first time that SAA can inhibit MDV replication and revealed the molecular mechanism of SAA against MDV through immune regulation.This study provides a new perspective for understanding the role of avian SAA in avian herpesvirus infection and provides a new strategy for the control of oncogenic herpes virus infection through APPs.