| Infectious Bursal Disease (IBD), a highly endangering infectious diseases in chicken, is caused by Infectious Bursal Disease Virus (IBDV). Innate immunity is the body's first line of defense against pathogens infection, and plays a very important role during the initiation of the acquired immune response . MicroRNAs (miRNAs),a kind of 21 -25 nt small non-coding RNAs, contribute to the repertoire of host-pathogen interactions during virus infection. In the present study, an in vitro infection pattern was used to investigate the molecular mechanisms of antiviral innate immunity of interferons, p53 and pattern recognition receptors in chicken by using the IBDV as a model virus.1. Differential modulation of interferon and p53 mRNA expression in chickens infected with intermediate and very virulent infectious bursal disease virusInfectious bursal disease virus (IBDV) causes an acute, highly contagious and immunosuppressive disease in chickens. Two serotypes of IBDV, serotype 1(pathogenic) and serotype 2 (non-pathogenic), have been identified.Serotype 1 consists of an intermediate ,virulent (v) or very virulent (vv) strain of IBDV. Both of these strains vary in antigenicity and pathogenesis. The goal of this study was to compare the immunopathogenesis of intermediate virulent strain (B8 7) and very virulent variant strain (QL/12). Four-week-old specific pathogen free chickens were inoculated intraocularly with intermediate virulent strain (B87) and a very virulent variant strain (QL/12). We quantified mRNA transcription in bursal tissue, by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), for the type ? IFN (IFN- ? and IFN-?),Thl cytokines (IFN-?), p53 mRNA and virus load for the first 84 hours after infection of 4-week-old chickens with B87 or QL/12 and compared these with levels in bursal tissue from uninfected age-matched controls.The QL-IBDV produced more pronounced bursal damage as compared to vIBDV. Virus load was significantly higher (P < 0.05) in bursas obtained from QL/12-inoculated chickens than that from B87-inoculated chickens. QL/12 induced significantly higher bursal follicular damage as compared to B87-IBDV.The expression of type I IFN(IFN- ? and IFN-?), Thl cytokines (IFN- y) and p53 mRNA were upregulated by both strains, although to a greater degree by the QL/12 strain.2. gga-miR-2127 is a negative regulator of chicken p53 severing as an antiviral factor against IBDV in chickenInfectious bursal disease (IBD) is a highly risky disease caused by infectious bursal disease virus (IBDV). Although the tumor suppressor protein, p53, can inhibit the replication of a variety of virus, its effect on IBDV replication remains unknown. In the present study, an in vitro infection pattern was used to investigate the effects of p53 on the antiviral effects in chicken by using the IBDV as a model virus. The expression level and activity of p53 were markedly increased in IBDV-infected DF-1 cells. The over-expression of p53 could inhibit IBDV replication and up-regulate the expression level of the antiviral innate immune genes in chicken (IPS-1, IRF3, PKR,OAS, Mx), whereas suppression of p53 could lead to the contrary results. Our results showed that p53 could activate the antiviral innate immune response in chicken to against IBDV infection. Results of bioinformatics and dual-luciferase reporter system showed that gga-miR-2127 could target the 3'UTR of p53. The results of qRT-PCR and immune blotting showed that the over-expression of gga-miR-2127 could down-regulate the expression level of the antiviral innate immune genes of chicken and promote the replication level of IBDV. Our results suggested that gga-miR-2127 targets the 3'UTR of chicken p53 severing as an antiviral factor that enhance antiviral innate immune response to IBDV.3. gga-miR-9* promotes IBDV replication in DF-1 cells by targeting interferon regulatory factor 2MicroRNAs (miRNAs), small non-coding RNAs, contribute to the repertoire of host-pathogen interactions during virus infection. To explore the function of the miRNA of gga-miR-9* in regulating replication of infectious bursal disease virus(IBDV), the gga-miR-9* mimics were synthesized and transfected into DF-1 cells,qRT-PCR and TCID50 analysis indicated gga-miR-9* negatively regulated IBDV-triggered type ? IFN production and promoted IBDV replication in DF-1 cells.Bioinformatics analysis indicated the IRF2 gene had two putative binding sites for gga-miR-9*, which was confirmed by the Luciferase assays. Moreover, western blot analysis and real-time RT-PCR showed that the IRF2 protein level and mRNA level were significantly lower in the IBDV infected gga-miR-9*-transfected-DF-1 cells than that in DF-1 cells. The results indicated that gga-miR-9* down-regulated IRF2 expression at the transcription levels. IRF2 knockdown mediates the enhancing effect of gga-miR-9* on type I IFN-mediated antiviral response. Therefore, we demonstrate that inducible gga-miR-9* feedback negatively regulates host antiviral innate immune response by promoting type I IFN signaling via targeting IRF2.4. Influence of infectious Bursal disease virus infection on the expression of pattern recognition receptors and the initiation of antiviral innate immune responseThe aim of the present study was to determine the influence of infectious bursal disease virus (IBDV) infection on the expression of pattern recognition receptors(PRRs) and the initiation of antiviral innate immune response. The in vitro viral infection model were established by infected DT40 Chicken embryo fibroblast cells with low virulent strain IBDV B87, while the virulent strain IBDV QL/12 infected SPF chicken were used for in vivo assay, the transcriptional expression of PRRs genes (TLR2, TLR3, TLR4, TLR7, MDA5 and LGP2) and the innate antiviral immune relevant genes (IPS-1, IRF3, PKR, OAS and Mx) were assessed by Q-PCR in the different time points following the IBDV infection. Our results shown that in 2 to 24 hours after in vitro IBDV infection, the expression of MDA5, TLR3, chLGP2,IRF3, PKR, OAS and Mx genes were significantly up-regulated, the increased gene expression were around 324-, 22-, 61-, 29-, 16-, 225,000- and 18,700-folds,respectively. After knocking down the chMDA5, chTLR3 and chLGP2 genes by small interfering RNA (siRNA), we found that IBDV infection in DT40 cells failed to initiate the antiviral innate immune responses, as there were no significance change for the relevant gene expressions, including IPS-1,IRF3, PKR, OAS and Mx. In the in vivo IBDV infection experiments, our result showed that, virulent strain IBDV QL/12 infection in SPF chicken nearly completely blocked the several PPRs genes expression including chMDA5 and chTLR3, which accompanied with slow increase of chLGP2 gene expression, the expression of other genes, such as PKR, OAS, and Mx, were both increased. These results indicated that chicken PRRs ( chMDA5,chTLR3 and chLGP2) playing a key role in recognizing IBDV infection, virulent strain of IBDV infection was able to block chMDA5 and chTLR3 expression, and also,our results demonstrated that there are likely some different antivial immune responses existed for in vivo and in vitro IBDV infection.5. gga-miR-142-5P inhibits the replication of IBDV through chMAD5: the molecular mechanismInfectious Bursal Disease (IBD) is high risky infectious diseases for chicken breeding caused by Infectious Bursal Disease Virus (IBDV). chMDA5 plays an important role in the activation of innate immunity and recognition of pathogen-associated molecular patterns of hosts, but the molecular mechanisms for chMDA5 to recognize IBDV remain unknown. In the present study, the expression level of chMDA5 in DT40 cells infected by IBDV was significantly increased in an in vitro infection experiment with IBDV as a model virus. An over-expression of chMDA5 could inhibit the expression of IBDV and activate the activity of IFN-(3 promoter and Mx promoter, while a contrary result was observed when the expression of chMDA5 was inhibited. chMDA5 activated the activity of IFN-? promoter mainly through IRF7 pathway, suggesting that chMDA5 can prevent IBDV infection by activating the innate antiviral immunity of chicken. Results of bioinformatics analysis and dual-luciferase reporter system revealed that gga-miR-142-5p could act on the 3'UTR of chMDA5. qRT-PCR and immune-blotting analysis indicated that gga-miR-142-5p could decrease the expression level of chMDA5, but the expression level of mRNA remained unchanged. The over-expression of gga-miR-142-5p could decrease the activity of IFN-? promoter and Mx promoter, inhibit IFR7 pathway,increase the level of IBDV replication, but had no effect on NF-Kb pathway. Our results suggested that gga-miR-142-5p could prevent IBDV infection through its regulation on the expression of chMDA5 and the consequent effect on the activity of IFN-? promoter and Mx promoter by acting on the 3'UTR of chMDA5. |
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