| Innate immune is the first line of host to defense against viral evasion.The host cell can detect viruses through pattern-recognition receptors and then induce the production of antiviral cytokines,such as type I interferons,pro-inflammatory cytokines and chemokines,to inhibit viral infection.Type I IFNs paly a critical role in innate immune.Previous study has shown that the piglets which were infected with PCV2 had low levels of IFN in plasma.But the mechanism of IFN production inducded by PCV2 is still unkown.The host cell can produce IFN to inhibit viral infection.In parallel,viruses also use several methods to subvert the function of IFN.PCV2 infection can inhibit pDC secreting IFN-a.However,the mechanism of PCV2 inhibiting IFN production remains elusive.So it is necessary to know the regulation mechanism of PCV2 on IFN production during PCV2 infection.In this study,to better' understand the pathogenic mechanism of PCV2 and provide a theoretical foundation for the prevention and treatment of PCV2,PK-15 cells were used as a model to explore the expression of IFN-? and signaling pathways of IFN production,and then the inhibition function of PCV2 on IFN-? production was also investigated.1 Signaling Pathways of Interferon-? Induced by PCV2 in PK-15 CellsIn order to investigate the signaling pathways of the production of IFN-? induced by PCV2,PK-15 cells were infected with PCV2 and then were collected at 0 h,3 h,12 h,24 h,48 h and 72 h after infection.The result showed that the mRNA level of IFN-? which was detected by real time PCR in PCV2-infected groups was significantly higher than the control at 48 h and 72 h(P<0.01).The production of IFN-? can be induced by the signaling pathways of IRF3/7 or NF-?B.BX795 and BAY 11-7082 inhibitors were used in this study to inhibit the signal pathway of IRF3 and NF-?B respectively,the results showed that the mRNA level of IFN-? in BX795-treated group was obviously lower than the control group(P<0.01),while no significant difference was found between BAY 11-7028-treated group and the control group.These above results showed that the production of IFN-? induced by PCV2 in PK-15 cells were mainly through the IRF3 signaling pathway.In order to confirm whether the signal molecules of IRF3 signling pathway are activated,the mRNA levels of pattern recognition receptors(RIG-1,MDA-5 and DAI),adaptor proteins(Sting and MAVS),interferon regulatory factors(IRF3 and IRF7)and the protein expressions of MDA-5,RIG-1,DAI,IRF3,p-IRF3 and IRF7 were detected.The results showed that the mRNA levels and protein expressions of RIG-1,MDA-5 and DAI were increased obviously at 72 h(P<0.01).The mRNA levels of adaptor proteins(MAVS and Sting)were up-regulated at 48 h(P<0.01).The protein expressions of p-IRF3 and IRF7 were also up-regulated at 72 h(P<0.01),while no change was foundin the protein expression of IRF3.In conclusion,these' resuts demoinstrated.that PCV2 induced the production of IFN-? in PK-15 cells by recognization of DAI,RIG-1 and MDA-5,and then activation of IRF3.2 Mechanism of PCV2 Inhibiting the Production of Interferon-? Induced by Poly(I:C)in PK-15 CellsIn order to investigate whether PCV2 could inhibit the production of interferon-?,PK-15 cells were infected with PCV2 and then treated with poly(I:C)which was the inducer of IFN.The mRNA levels of IFN-?,pattern recognition receptors(RIG-1 and MDA-5)and antiviral proteins(Mx1,OAS and ISG15)were detected by real time PCR.The protein expressions of RIG-1 and p-IRF3 were detected by western blot.The results showed that PCV2 could inhibit the mRNA expression of IFN-? and down-regulated the mRNA levels of RIG-1,Mx1,OAS and ISG-15,while no change was found in the mRNA expression of MDA-5.Moreover,the protein expressions of RIG-1 and p-IRF3 were also reduced.In conclusion,these results demonstrated that PCV2 could inhibit IFN-?production through RIG-1/IRF3 signaling pathway. |
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