Inhibitory Effect Of Porcine Hemagglutinating Encephalomyelitis Virus Infected Macrophages On Type Ⅰ Interferon

Porcine hemagglutinating encephalomyelitis(PHE)is one of the swine’s infectious disease that are caused by porcine hemagglutinating encephalomyelitis virus(PHEV).According to the main characteristics of the disease,it divided into two types: vomiting consumption and encephalomyelitis,The main characteristics respectively showed vomiting and neurological symptoms.At present,the disease has ocuured in many countries including China and led to huge economic losses to the pig industry.In the early stage of infection,geneogenous immunizing cells secrete type I-interferon(I-IFN)to protect the host from the virus infection.However,some pathogens can escape the recognition of IFN through their own characteristics.In the early stage of PHEV infection,whether the body exert antiviral effects by secreting this cytokine is unclear.I-IFN is an important cytokine which are involved in the innate immune response.In this experiment,to better understand what I-IFN reaction after PHEV infection,extracted brain and spinal cord’s total protein after infected BALB/c mice five days with PHEV replication reaching peak,and detected IFN-β protein by ELISA method.The result showed that the brain tissue IFN-β secretion is about 80 ng/L,in spinal cord,there is no significant difference between experiment group and blank control group.Thus,PHEV can replicate in the brain and spinal cord,but has no ability to induce high expression of IFN-β,which may be an important reason for neurological symptoms.These studies indicate that PHEV can evade the immune recognition of IFN-β in vivo.PHEV can break through the peripheral immune after infect body,the study of our group found that DC can not play the role of anti-PHEV by secreting IFN.As another important peripheral immune cell,macrophage’s ability to deal with virus and its innate immune response is unclear.First we detected PHEV replication in peritoneal macrophages,RAW and J774 A.1 cell after infected 24 h and showed viral positive signals,the results indicate that PHEV can infect and reply in macrophages.Macrophage is the main source of I-IFN,in order to understand the IFN’s response to PHEV,IFN-β m RNA level in RAW and J774 A.1 cells were detected by RT-PCR,VSV infection was a positive control,the result showed that PHEV infected J774 A.1 cell 24 h and VSV infected RAW 9 h can strongly induce IFN-β,however,RAW cell didn’t detect IFN-βsecretion until infected 36 h,indicating that IFN was inhibited during the early phase of infection in RAW cells.About IFN-β protein level in macrophages,both peritoneal macrophages and two cell lines’ supernatant were detected by ELISA,the results showed that PHEV infected RAW and peritoneal macrophages 24 h did not detecte IFN-β,but the amount of IFN-β in J774.1 up to 1 200 ng/L,the results showed,on early infective stage,PHEV could not induce peritoneal macrophages and RAW cells secreting IFN.IRF-3 is an upstream transcription factor that regulates the production of I-IFN,which is usually found in the cytoplasm.Once activated,IRF-3 will transfer from cytoplasm to nucleus.In order to study the IFN inhibition mechanism in macrophage,we detected the nuclear localization of IRF-3 by immunofluorescence after PHEV infected RAW and J774 A.1 cells.The results showed that PHEV infected 24 h,IRF-3 still located in the cytoplasm.Suggested that PHEV infection did not activate the IRF-3 pathway in macrophages,and PHEV may inhibit the production of IFN-β by inhibiting IRF-3 or its upstream regulatory factors.To understand the role of pattern recognition receptors in macrophages after PHEV infetion,we used live and inactivated virus infected J774 A.1 cells and detected by RT-PCR.the result showed that inactivated virus have no ability to induce IFN-β,suggested that cytoplasmic pattern recognition receptors are able to participate with PHEV recognition.There are three kinds of cytoplasmic pattern recognition receptors,RIG-I,MDA5,LGP2,in order to screen the main receptor,using RNA interference technology to transient silence RIG-I,MDA5,LGP2.The results showed that PHEV infected cells 24 h,the IFN-β protein in MDA5 transiently silenced cells was significantly reduced,suggested that MDA5 is involved in the process of IFN procuction during PHEV infection.In addition,si MDA5 can significantly enhance the replication of PHEV,characterized by the up-regulated expression of viral protein expression,all results suggested that MDA5 is involved in the recognition of PHEV infection,the pathway could play a role of anti-PHEV.In summary,PHEV can escape the antiviral innate immune of IFN-β and be recognized by MDA5 receptor,PHEV escape the peripheral immune defense and enter central nervous system to play pathogenic role,eventually cause nerve damage.The results of this study revealed that the key pattern recognition receptors in the process of PHEV infection and its antiviral innate immune function,it can provide a reference for further research on the mechanism to escape from the host innate immune recognition.