Molecular Mechanism Of The Interferon Stimulating Gene Viperin On Restricting Neurotropic Infection Of Tembusu Virus

Tembusu virus（TMUV）belongs to the genus Flavivirus,family Flaviviridae.In recent years,TMUV variants infection mainly cause severe neurological symptoms and encephalitis in ducks and geese.So,the neurotropic infection of TMUV is increasing,which brings new challenges to the prevention and control of the disease.Interferon-induced protein Viperin is an antiviral effector induced by dependent/independent type I interferon（I-IFN）.It has broad-spectrum antiviral function and is widely involved in virus infection process and host innate immune response.Previous studies have shown that Viperin plays an essential role in the innate immune response against flavivirus infection of the central nervous system（CNS）,and is a key factor in the study of the immune mechanism of viral neurotropic infection.However,whether Viperin exerts antiviral effects in TMUV-infected nerve cells and nerve tissues and the mechanism of action still need to be further explored.In this study,mouse neuroblastoma cells（Neuro-2a）were used as research objects to establish a TMUV-infected nerve cell model to explore the mechanism of Viperin on restricting neurotropic infection of TMUV.A mouse infection model was constructed by lentiviral therapy to reveal the role and molecular mechanism of CNS innate immunity in antiviral infection,providing a new target for screening against TMUV infection,and providing a theoretical basis for innate immune response to other neurotropic virus infections.1.The effect of Viperin on TMUV replication in Neuro-2a cellsIn order to explore the role of Viperin in the immune response between TMUV infection and host cell virus,the monoclonal antibody against Viperin was prepared and identified.Secondly,Neuro-2a cells were used to establish a cell model of TMUV infection to study the innate immune induced by TMUV infection and its relationship with Viperin.By overexpression and knockdown of Viperin,the replication level of TMUV-infected Neuro-2a cells was determined by RT-q PCR and Western blot.Furthermore,according to Viperin structure and function domain,Viperin truncated genes was constructed to determine the antiviral function domain of Viperin.The results showed that three hybridoma cell lines,named E8F1B12,E8F1E1 and E6E7G1,were obtained,which could secret anti-Viperin antibody.TMUV AQ-19 strain（MOI=1）infection with Neuro-2a cells after 48 h can cause cellular pathologies,as infection time increases,the level of TMUV E protein expression continues to increase,and Viperin expression also increases with it.Then,Viperin can limit TMUV replication after overexpression,while knockdown Viperin is able to promote the replication of TMUV.Finally,to identify the functional domain of Viperin,the N-terminal domain of Viperin was found to significantly inhibit the replication of TMUV.This study proves that Viperin is a positive regulator in the Neuro-2a cells of antiviral immune response.2.The the molecular mechanism of Viperin on restricting TMUV replicationIn order to explore the molecular mechanism of Viperin on restricting TMUV replication,we first analyzed the effects of Viperin on the TMUV replication cycle by the TCID₅₀ and q PCR methods.Then,Viperin’s effects on the cellular lipid structure of cholesterol and potassium phosphorus by ELISA.We selected the two molecules MβCD and Myriocin that can damage cellular Lipid structure to explore the effect of lipid cellular replication on TMUV.Finally,the virus target proteins for the action of Viperin were clarified by building a carrier of vertical nucleus expression with the HA/Flag label,using immune co-deposition and cell co-positioning.The results showed that on the TMUV absorption and invasion phase,the viral copies were no difference between Viperin overexpression group and the empty vector control group（P>0.05）,while in the release phase the TCID₅₀ results in the upper clearance of the vacuum carrier cell was higher than the Viperin overexpression group,so that Viperin restrict TMUV replication by affecting the budding release of the TMUV.In addition,after overexpression,Viperin consumed the lipid components of the cell membrane cholesterol and potassium phosphorus（P<0.05）,thereby destroying the liposuction structure,MβCD and Myriocin had dose-dependent inhibitory effects on TMUV replication on Neuro-2a cells,and the two drugs had no differences in the TMUV absorption and entry phases,while in the budding release phase,significantly inhibited the level of expression of TMUV E protein and virus titer.Therefore,Viperin restricts the budding of TMUV by affecting the structure of the lipid in Neuro-2a cells by consuming the main component of lipid.Finally,it was identified that Viperin can interact with TMUV NS1.Overall,Viperin is able to inhibit TMUV replication in ways such as interaction with virus and affecting cellular structure.3.Effect of Viperin on the pathogenicity of TMUV in mouse brainIn order to further explore the effect of Viperin on the pathogenicity of TMUV infection in vivo,the intracranial infection model of mice susceptible to Viperin lentivirus was established based on the intracranial infection model of ICR mice constructed in our laboratory,and the effect of Viperin on neurotropic infection of TMUV was analyzed.The lentivirus triplasmid system（packaging plasmid,envelope plasmid and transfer plasmid）was used to construct the transfer plasmid carrying Viperin,and the three plasmids were co-transfected into 293T cells,to obtain the lentivirus carrying Viperin and purify and concentrate.The ICR mice were infected with lentivirus by intracranial injection,and the mental state,survival and expression of Viperin in the brain of the mice were observed.Finally,24 h after Viperin lentivirus infection,TMUV infection was carried out.The mental state and survival rate of mice were observed.The pathogenicity of Viperin on TMUV CNS infection was determined by HE staining of pathological sections,immunohistochemistry,TUNEL and q PCR.The results showed that the titer of the prepared lentivirus could reach3.7×10⁷TU/m L after concentration and purification,which could meet the requirements of animal experiments.After lentivirus infection,the mental state of mice was not affected except on the day of injection,the body weight of mice gradually increased with the increase of age,and there was no death,and the safety was good.In addition,the expression of Viperin in the brain of mice was obvious and could last for 6 days.After TMUV infection,the survival rate of mice in the lentivirus intervention group was higher than that in the empty vector group.On the 5th day after TMUV infection,all mice in the empty carrier group showed mental depression,hind limb paralysis and other neurological symptoms,while on the 7th day after infection in the Viperin lentivirus intervention group,3 mice showed neurological symptoms,and the rest were normal.In addition,the expression levels of inflammatory factors（IL-1β,IL-6 and TNF-α）in the Viperin lentivirus treatment group were significantly lower than those in the empty vector group.These results indicate that Viperin can reduce the nerve damage which caused with TMUV infection in the brain of mice and play an antiviral role in the CNS of mice,thus these results provide a target for the prevention and control of TMUV.In summary,this study confirmed the mechanism of Viperin on restricting neurotropic infection of TMUV from animal models and cell models.The results of this study provide new ideas for further clarifying the interaction between host and TMUV,and screening antiviral targets.