Study On The Effect Of Natural Immunity Factors On SFTS Virus Infection

Innate immunity factors, which include cytokines, complements and antimicrobial proteins, play important roles in the antiviral effects. They initiate and participatelin the innate immune response to virus infection. Cytokines are a kind of low molecular weight soluble proteins which are produced from cells stimulating by immunogen, mitogen or other factors. They can regulate innate and adaptive immunity, promote hematopoiesis, stimulate cell activation and proliferation, etc. As the first discovered cytokine, interferon (IFN) is a kind of protein with broad-spectrum of antiviral activity, and could regulate immunity and cell growth. According to the different sources and physicochemical properties, they can be divided into Ⅰ, Ⅱ and Ⅲ three types. Type Ⅰinterferon includes IFN-α, IFN-β,etc. While a sole member makes up the type Ⅱ IFNs that is called IFN-γ.A novel phlebovirus in the Bunyaviridae family named severe fever with thrombocytopenia syndrome (SFTS) virus was isolated in China in recent years and it was identified to be the cause of SFTS. Patients clinically presented with acute fever, thrombocytopenia and leucopenia, some of whom rapidly developed multiorgan dysfunction and the case fatality rate was 10-15%. SFTSV poses a constant threat to public health in China. However, neither vaccines nor specific treatments are available.Understanding the molecular pathogenesis of SFTSV will provide new strategies to the prevention and control of SFTS.For the invading virus, interferon does not play a direct role in antiviral process in human body, but instead recognize viral nucleic acids by Toll-like receptors and RIG-like receptors, and then stimulate a series of downstream signal transducer to produce effector proteins, like interferon-stimulated genes (ISGs), which play the antiviral effects directly. This process was regulated by multiple mechanisms, and a lot of innate immunity factors are involved in this process. Thus, to study the effect of innate immunity factors on SFTS virus infection, we selected some representative factors related to type Ⅰ interferon (IFN-α) and type Ⅱ interferon (IFN-y), determine their mRNA expression levels and SFTS virus copies under the conditions of interferon stimulation and (or) SFTS virus infection by quantitative real-time RT-PCR assay.The results showed that mRNA expression levels of these innate immunity factors increased significantly when stimulated separately with IFN-a or SFTS virus. However, when stimulated with IFN-a and SFTS at meantime, the induction of IFN-a on these factors were significantly inhibited. The inhibitory effect was not obvious in the innate immunity factors associated with IFN-y. Results in THP-1 cells were the same as those in 293T cells. Although the SFTS virus infection could be inhibited significantly by IFN-α, the high viral copies of virus’ mRNA could still be detected in THP-1 cells stimulated by IFN-al. The inhibitory effect of IFN-γ on SFTS virus was not obvious.We also chose CCL5/IL-6/IL-8, which are specific target genes of transcription factor NFKB, and determined their mRNA expression levels under the conditions of lipopolysaccharides (LPS) stimulation and (or) SFTS virus infection by quantitative real-time RT-PCR assay, to study their effect on SFTS virus infection.The results showed high does LPS can induce the expression of CCL-5, IL-6 and IL-8 significantly, and the infection of SFTS virus has little effect on the induction of LPS on CCL-5 and IL-8.The results above showed IFN-a can effectively inhibit the infection and replication of SFTS virus, and SFTS virus can also inhibit the production of IFN-a. So in the following experiments, we selected a group of interferon stimulated genes IFITM1, IFITM2 and IFITM3, which are induced by IFN-a, to study the inhibitory effects of these factors on SFTS virus infection at the cellular level.Interferon induced transmembrane proteins is a set of antiviral protein encoded by the interferon stimulated gene IFITMs. They can be effectively induced by IFN-a. In the IFITM family, IFITM 1, IFITM2 and IFITM3 have highly homologous sequences, and showed a broad spectrum of antiviral activity. All of they can inhibit some bunyavirus infection significantly, such as Rift Valley fever virus, Dengue virus and Hantaan virus. The effect on the same virus is not same because of selective antiviral function of IFITM1/2/3. We don’t know whether IFITMs can inhibit the infection of SFTS virus, and there has not been reported at home or abroad.We used IFN-a to stimulate 293T cells, amplificate IFITM 1/2/3 genes by reverse transcription PCR. Cloning it into eukaryotic expression vector pCAGEN, and transfected into Vero cells and THP-1 cells. To detect the overexpression of IFITM1/2/3 in Vero cells and THP-1 cells by indirect immunoinfluscent assay and Western Blot. To detect the infection of SFTS virus by real time fluorescence quantitative PCR at different time points.Results of IFA and Western Blot showed IFITM 1/2/3 have good antibody binding activity in Vero cells and THP-1 cells. Results of real time fluorescence quantitative PCR showed IFITM1/2/3 have a significant inhibitory effect on SFTS virus infection in the cells, and there is no obvious difference between each group. At the same time, over expression of IFITM 1/2/3 have a more significant inhibitory effect on low doses of SFTS virus infection.So, we have successfully constructed the eukaryotic expression vector of IFITM1/2/3, and confirmed that IFITM1/2/3 have a significant inhibitory effect on SFTS virus infection at the cellular level.