| Infectious bursal disease is an acute, highly contagious and immunosuppressive avian disease caused by Infectious Bursal Disease Virus (IBDV), whose genome consists of two segments of double-stranded RNA (dsRNA). The segment A contains two partially overlapping open reading frames (ORFs). The larger ORF encodes a precursor polyprotein in the form of NH2-pVP2-VP4-VP3-COOH. The polyprotein processes the proteolytic activity of viral protease VP4 to generate VP2, VP4 and VP3. VP4 is the viral protease, which plays an important role in the maturation of viral proteins. In the preceding, much shorter and partially overlapping open reading frame. Segment A also encodes the nonstructural protein VP5, which is involved in virus release and apoptosis.The type I interferon (IFN) has a crucial role in the first line of defense against virus infectious. The generation and working of type I IFN depend on IFN production and IFN working signal pathway. Specifically, type I IFN is generated by the cells being attacked by virus, which works on the membrane receptor of neighbouring non-infected cells to maintain an antiviral state. Studies have indicated that congenital immunity can be suppressed through inhibiting the transcription of IFN genes by IBDV. And it is also verified that IBDV can down-regulate type I IFN. However, which viral protein does is indistinct.In the present study, all the structural and nonstructural protein genes of IBDV were cloned and inserted to the mammalian expression vector pCI-neo, with co-transfected Vero cells by the luciferase reporter gene, to investigate the interplay between VP4, VP5 protein and RIG-I induced signaling pathway, IRF3 and NF-κB production or working signal pathway, especially Jak-Stat signaling pathway. This study presents a better understanding of mechanism underlying IBDV infection and pathogeny, and may lead to successful strategies for targeting VP4 and VP5 in prevention and control of IBDV. Experiment I. Construction and expression of recombinant expression vectorsThe VP1, VP2, VP3, VP4, VP5 genes of CV03 and VP4, VP5 genes of B87 were cloned using specific primers with Flag-tagged. The target genes were retrieved and cloned into pMD19-T vector, and confirmed by sequencing. Then the positive genes were inserted into pCI-neo Mammalian Expression Vector. After identification by restriction analysis, the recombinant vectors were transfected into Vero cells, and the expression products were analysised by western-blotting. IFA further characterized the expression of recombinant vectors.Experiment II. The influence of VP4, VP5 on the production and working signal pathway of type I IFNIn order to point out which protein of the IBDV is responsible for the antagonist effect on type I IFN, a transient co-transfection with constructs of individual viral proteins together with the IFN-β-promoter-luciferase reporter was applied. The result showed a significant reduction in expression from the IFN-β promoter in Vero cells transfected with VP4 and VP5. The similar experiment was performed by the comparation of CV03 and B87. Two different strains of IBDV both decreased the activity of INF-β promoter induced by RIG-I. To further investigate the mechanism of down-regulation of IFN-β by VP4 and VP5, the VP4 and VP5 gene recombinant expression vectors of two strains were co-transfected with p4xIRF3-Luc and pNF-κB-Luc. The expressions of IRF3 promoter were apparently reduced both by CV03 and B87, and activated the expression of NF-κB. Therefore, VP4 and VP5 could down-regulate the expression level of IFN-β via RIG-I and IRF3 by inducing IFN production pathway, rather than inducing the NF-κB. Jak-Stat transduction signaling pathway acts a significant role in type I IFN exerting antiviral activity. To investigate the effect of VP4 and VP5 on working signal pathway of type I IFN, especially the Jak-Stat signaling, the reporter gene pISRE-Luc was created and co-transfected with VP4 and VP5 recombinant expression vectors of CV03 and B87. The result indicated that the VP4 and VP5 greatly reduced the expression of ISRE promoter to weaken the effect of IFN through the Jak-Stat signaling pathway. |
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