Effect Of IRF3/7-mediated Innate Immunity On HY12 Enterovirus Replication

Bovine enterovirus(BEV)and Coxsackievirus,Enterovirus 71 and Poliovirus belong to the family of Picornaviridae within a member of Enterovirus.BEV infection mainly causes digestive and respiratory symptoms of cattle,which seriously threatens the healthy development of cattle industry.In 2012,we isolated a strain of enterovirus from cattle with cough and diarrhea in Jilin Province,named HY12.HY12 virus is the first domestic E enterovirus.Since BEV infection is a new infectious disease,the immune mechanisms involved in the pathogenesis of BEV and host antiviral responses are still unclear.In this study,we employed HY12 virus to infect a murine macrophage RAW264.7 cell line and an ICR mouse model to explore the expression of IFNs,IRF3,IRF7,major TLRs by immunology,molecular biology,histopathology and immunohistochemistry.In vivo and in vitro results showed that HY12 virus infection triggered the type IFN-I expression,and the expression was in a time-dependent manner.Further exploring the mechanism of IFN-I production,it was found that TLR3 and TLR7 levels were positively correlated with the expression of IFN-α and IFN-β after HY12 infection.Since IRF3 and IRF7 are two important downstream transcription factors of TLR3 and TLR7,we have also detected the activation of IRF3 and IRF7 in HY12-stimulated RAW264.7 cell.The results showed that the expression of IRF3 and IRF7 increased significantly at 4 h,reached its peak at 8 h,and then decreased gradually,which is consistent with in vivo result.Immunohistochemistry assay showed that HY12 virus in different tissues(heart,liver,spleen,lung,kidney,intestine and brain)gradually decreased,with the increase of IRF3 and IRF7 expression.These results suggested that IRF3 and IRF7 may be involved in the process of anti-HY12 infection in the body.To further investigate the role of IRF3 and IRF7 in HY12 viral replication,we overexpressed IRF3 and IRF7 in RAW264.7,respectively,and tested the titer of viral and the expression of structural protein VP2 after HY12 infection.The results showed that the viral titer was significantly reduced after HY12-stimulated RAW264.7 cell with overexpression of IRF3 and IRF7 compared with RAW264.7 infected with only HY12 virus.It was further confirmed by IPMA and Western blot that overexpression IRF3 or IRF7 inhibited the expression of HY12-VP2 protein.Taken together,these findings indicated that IRF3 and IRF7 are involved in the innate immune response of host anti-HY12 virus,and the activation of IRF3 and IRF7 plays a significant role in inhibiting HY12 virus infection.The above research results not only provide guidance for further revealing the pathogenesis of BEV infection,but also provide theoretical basis and experimental basis for clinical prevention and treatment of the disease.