The Preliminary Research On The Anti-virus Effect Of Interferon Gamma Against Foot And Mouth Disease

Abstract:FMDV is a single-stranded RNA virus that can lead to foot and mouth disease of cloven-hoofed species, causing the appearance of blisters in hoof, tongue. It includes seven FMD virus serotypes, at least65subtypes, with high variability rusulting in challenging tasks on its prevention. Interferon gamma belongs to type II interferon family, which was confirmed to resist the virus replication in the host. To study the antiviral mechanism of interferon, the PK15cells were treated with interferon gamma in presence or absence of FMDV.To study of the mechanism of resistance interferon against FMDV comprehensively and accurately, high-throughput sequencing was used to check comprehensive gene expression profiles in different treatments. We established four experimental groups:a group without interferon gamma and was not infected with FMDV(IFN-0), a group without interferon gamma was infected with FMDV(FIFN-0), a group treated with interferon gamma was infected with FMDV(FIFN-5), a group treated with interferon gamma was not infected with FMDV(IFN-5).14670genes were found to be co-expressed in IFN-0, FIFN-0and FIFN-5groups, counting for57.9%of the total genes. There were no signifcant differences for most genes expressions between different groups. Based on the filtering by FDR<0.05and fold change, differently expressed genes were sorted. Comparing the treatment IFN-0to treatment FIFN-0,191genes were up-regulated and124genes down-regulated.33genes were up-regulated and62genes were down-regulated in the treatment FIFN-0compared to the treatment FIFN-5. In the IFN-0vs FIFN-0comparison group, the differently expressed genes were enriched by GO significance analyses in biological processes terms, such as a negative regulator of biological processes, a positive regulator of cellular processes. However, for FIFN-0vs FIFN-5comparison group, the differently expressed genes were classified into the responses to the virus, representing antigen processing、immune response、defense response, etc, which provide us more evidences for anti-virus effects of interferon-gamma against FMDV. Pathway significant enrichment analysis showed that differentially expressed genes were significantly enriched in p53pathways in IFN-0vs FIFN-0comparison group. While in FIFN-0vs FIFN-5group, differentially expressed genes were majored in antigen processing presenting pathway, phagosome pathway, complement and coagulation cascades pathway. Increased expressions of TAP1and OAS1were also confirmed by Western blotting in IFN-gamma treatments compared to non-IFN-gamma treatments. There was less spliceosome of PARP protein both in IFN-0and FIFN-5groups compared to FIFN-0, which means that cell apoptosis is induced by FMDV infection with absence of IFN-gamma and inhibited by the treatment together with IFN-gamma.We validated the results of the sequencing experiments by real-time quantitative PCR focusing on9genes depending on the GO and Pathway enrichment results, such as OAS1, OAS2, MX1, IFIT1, RIG-Ⅰ, CTGF, F3, RCAN1and PLA U. The expression patterns of these9genes between different groups were consistent in qPCR and RNA-seq assays, indicating that the sequencing results are accurate and reliable. OAS1, OAS2, MX1, IFIT1, RIG-Ⅰ expressions are increased significantly in FIFN-5groupcompared to FIFN-0group, which belong to interferon-induced gene famliy. While, CTGF, F3, RCAN1and PLA U expressions are much higher in FIFN-0group compared to FIFN-5group. These4genes are involved in cell apoptosis. It has been demonstrated that FMDV infection will induce cell apoptosis. Generally, after adding interferon, the expression of antigen-presenting pathway is enriched significantly, following the enhancement of the immune capacity of host which can strengthen its anti-virus capabilities. We also amplified the full length fragment of TAP1gene and constructed pcDNA3.1, pEGFP recombinant vector, to make preparations for its antiviral mechanism in PK15cells.These results demonstrate that the treatment of interferon gamma will induce innate and adaptive immune responses, and inhibit apoptosis in PK15cell, resulting in the resistance effect of interferon gamma against foot and mouth disease virus.