The Study Of SFTSV Infection For The Regulation Of Key Molecular Expression On Host Innate Immune And Inhibition Of IFITM3 For SFTSV

Recently,severe fever with thrombocytopenia syndrome virus(SFTSV)is first discovered of a new type of virus in our country t,belonging to the members of bunyaviridae of phlebovirus.It can cause people fever with thrombocytopenia syndrome,mainly fever,thrombocytopenia,leukopenia and gastrointestinal diseases and severe cases died of multiple organ failure,the fatality rate is as high as 10 ~ 30%.There are currently no effective treatments for the disease that poses a great threat to people life and health.Therefore,it laid the theoretical basis to explore the SFTSV pathogenesis and host antiviral mechanism for the development of an effective treatment method.The host innate immune response is the first line of defense against a variety of virus infection,which is mainly started by the type I interferon induced and NK cell activated.Toll-like receptors(TLRs)and RIG-I receptors play an important role in the interferon antiviral effect.Type I interferon induces tyrosine kinase signal converter and activation of transcription factor(Jak/STAT)signaling pathway by strengthening STAT1 phosphorylation level and interferon stimulated gene activity components(ISRE),resulting in hundreds of interferon stimulated gene(ISGs),thus host cells showe antiviral effect,limit virus replication and spread.Interferon induced transmembrane protein is a a small molecule transmembrane protein stimulated by type I interferon,which can block the virus into cells as host limit factor.Interferon induced transmembrane proteins,including IFITM1,IFITM2 and IFITM3,have broad spectrum antiviral effect.The studies prove IFITMs have obvious inhibitory effect for RVFV and so on several kinds of buniyavirus.Now,no reports have been related about its influence on SFTSV infection.Objective: To initially explore the regulation of SFTSV infection inherent antiviral role of the host cell signal transduction pathway and antiviral factor IFITMs.At present,it is not reported that IFITM3 has inhibition on SFTSV,so this research is studied by analyzing the expression regulation of antiviral pathway of key molecular including IFITM protein expression regulation function and the influence of IFITM3 on SFTSV infection to explore the relationship between IFITM3 and SFTSV and its antiviral mechanism.There is still no effective drug or vaccine for SFTSV,so the study will be completed to provide new ideas and perspectives for the development of specific drug molecules or treatment.Contents: 1.SFTSV regulation for key molecules of innate immune pathways.2.Establishment of HEK293 cell line stably induced IFITM3.3.Inhibition of IFITM3 on fever with thrombocytopenia syndrome virus infection.Methods: 1.SFTSV regulation for key molecules of innate immune pathways According to the key related molecules on the natural immune signaling pathways,we choose TLR3~8,RIG-I,MDA5,My D88,TBK-1,IRF7,IFN-?,STAT1 ~6,IFITM1,IFITM2,IFITM3,PKR,Mx1,OAS1,ISG15 and ISG56.Expression changes of key molecular in the inherent immune is detected by fluorescence quantitative PCR in different time points of SFTSV infection,evaluating SFTSV effects on innate immune signaling pathways.2.Establishment of HEK293 cell line stably induced IFITM3 Using the Tet-On3 G induced eukaryotic expression system,p TRE3G-IFITM3 built and p LVX-Tet3 G will be to transfected into 293 cells under G418 and puro pressure to filter induced 293 cell lines stably expressing IFITM3,which is identified by Western blot,MTS and flow cytometry analysis.3.Inhibition of IFITM3 on fever with thrombocytopenia syndrome virus infection The experiment of SFTSV infection with 293-Tet3G-IFITM3 and MDCK Tet3G-IFITM3 cell line built analyze and evaluate IFITM3 inhibition of SFTSV infection or entry by indirect immunofluorescence,fluorescence quantitative PCR and flow cytometry.Results: 1.The key molecular in the inherent immune are deteced by fluorescence quantitative PCR in THP-1 cells infected by SFTSV,such as toll-like receptors(TLRs)and its downstream molecular TBK-1,RIG-I receptors,STATs and its molecular PKR,to evaluate expression change of key downstream molecular in natural immune signaling pathways.The results showed that in the infection of THP-1 cells by SFTSV after different time points,the expression of TLR7 and TLR8 gene in TLR3~TLR8 toll-like receptors are mainly enhanced,RIG-I and MDA5 gene expression greatly raised,and its downstream My D88 expression increased slightly and IRF7 and IFN-? significantly enhanced.In JAK/STAT pathway,STAT1 and STAT2 expression significantly increased,and interferon stimulated gene of its downstream,including IFITM1,IFITM3,PKR and so on increases obviously.The above results suggest the host cell starts the antiviral natural immune response pathways after SFTSV infection of THP-1 cells.After IFN-? processes HEK293 and A549 cells infected by SFTSV,STAT1,STAT2,IRF9,IFITM1,IFITM2 and IFITM3 gene expression levels were lower than that of normal cells processed by IFN-?,suggesting SFTSV can inhibit the signaling pathway induced by exogenous IFN-?.2.Reacting carrier carrying IFITM3 and regulatory carrier will be cotransfected into 293 cells by the use of Tet-On3 G induced expression system,under the pressure of G418 and puro to build 293 cell lines stably expressing IFITM3,namely 293-Tet3G-IFITM3,which is analyzed and appraised right by western blot,MTS and flow cytometry.Results showed that stably-expressing IFITM3 293 cell lines is built successfully,The percentage of the expression IFITM3 of 293 cell that has no apparent influence on cell viability induced by Dox is nearly 100%.3.By SFTSV infection experiments,IFA results show that 293 and MDCK cells expressing IFITM3 induced by Dox are resistant to SFTSV infection.RT-q PCR results show that the induced IFITM3 expression of Dox can inhibit SFTSV early infection.FCM detection results show that IFITM3 can inhibit SFTSV infection;Low p H can enhance SFTSV infection,but does not affect IFITM3 antiviral effect on SFTSV.