The Signaling Pathways Of Type ? Interferon Production Induced By Porcine Circovirus Type ? Virus

PCV2 is a small, circular, single-stranded DNA virus and also the smallest mammalian virus yet known. PCV2 as an important pathogeny of postweaning multisystemic wasting syndrome (PMWS) is distributed in current world. It has been associated with other diseases and caused serious economic loss to pig production. This study adopted TMT approach to identify the phosphorylation proteins expressed differentially in PAMs from the PCV2-infected group compared to the uninfected control group. A total of 213 cellular phosphorylation proteins in PAMs were significantly altered at 6 hours post infection.Among them, the phosphorylation of MAVS increased siganificantly. This suggested that PCV2 activated innate immune. Further study showed that MyD88 , DAI and RIG-1 played an important role in the production of type ? interferon induced by PCV2, while NF-?B played a minor role in that process. In addition, preliminary studies suggest that DAI and RIG-1 could influence the replication of PCV2 in PK15. These studies helped to elucidate the mechanism of immune features and infection of PCV2 and laid a theoretical foundation.The concrete research content is as follows:1. TMT labeling approach revealed first phosphoproteomics profiles of pulmonary alveolar macrophages infected with PCV2The major target cells of porcine circovirus type 2 (PCV2) are macrophages. In order to further study the activation of phosphorylation site when virus infected pulmonary alveolar macrophages (PAMs), the differences of the phosphorylated proteins in PAMs were analyzed at 6 hours post infection, using mass spectrometry analysis combined with the relative and absolute quantitative amount of ectopic tag technology (TMT). And 213 phosphorylation proteins differentially expressed, of which 87 phosphorylation proteins significantly increased (including 102 phosphorylation sites), 126 phosphorylation proteins significantly decreased (including 178 phosphorylation sites) were found. These proteins involved in biological combination, cytoskeleton, signal transduction and cell adhesion, etc.These provided clues for further study about the pathogenesis of PCV2.2. The role of NF-?B in PCV2-induced type ? interferon signaling pathways.NF-?B can participate in enhancing type ? interferon induction through cooperative interactions with IRF3 and IRF7. In the experiment, the PAMs were isolated aseptically and divided into four groups, control group, PCV2 group, inhibition group and inhibition-PCV2 group and cultured in vitro.The IFA results show that the PCV2 antigens were observed in cytoplasm of PAMs at 48h by laser confocal microscope. Western Blot results showed that NF-?B inhibitors BAY 11-7082 inhibits the phosphorylated forms of I?B?, inhibited the I?B? degradation and the accumulation of NF-?B in the nucleus during PCV2 infection.Using ELISA method to detect of the dynamic changes of IFN-? and IFN-? in cultural supernatant found PCV2 could induce IFN-?secretion in PAMs. When inhibitors inhibited the NF-?B activation, the ability of PCV2 inducing IFN-? secretion in PAMs did not decline. These suggested that the NF-?B signaling pathways may not participate in the process of PCV2 induced secretion IFN-? in PAMs.3. PCV2 induced the expression of type ? interferon in PAMs through the MyD88 -IRFs signaling pathwaysIn order to further clarify the signal pathway of secreting IFN-? induced by PCV2 induced, we adopted the method of siRNA to make MyD88 gene silence and study the role of MyD88-signaling pathways in IFN-? secretion induced by PCV2.The results showed that the gene transcriptions of IFN-? raised by PCV2 had a great relationship with IKK??IRF7 and IRF3.When interferencing MyD88,the levels of IKK??IRF7 and IRF3 transcription were decreased, and eventually made IFN-? transcription decreased obviously.In addition, the correlation analysis showed that IFN-? had a significant positive correlation with MyD88, IRF3 and IRF7 and so did IFN-?.The results showed that MyD88-IRF(s)signaling pathways affected the transcription of type ? interferon induced by PCV2.4. The preliminary study of the production of type ? interferon induced by PCV2 is impacted by DAI and RIG-1 in PK15 cellsDAI and RIG-1 are upstream signal molecules in a type ? interferon pathway. In order to study the role of DAI and RIG-1 in type ? interferon production pathways induced by PCV2. According to the DAI and RIG genes sequences, siRNA sequences of DAI and RIG were designed, respectively. At the same time, negative control siRNA sequences were also designed. Recombinant lentiviruses were constructed. Recombinant lentivirus infected PK15 cells. By puro resistance screening, positive cells were obtained. After monoclonal screening, interference efficiency was verified by real-time PCR and Western Blotting.PK15-shDAI and PK15-shRIG-lcell lines were succeeded. The expressions of DAI and RIG-1 were in a stable low level in them. Type ? interferon levels decreased significantly in PK15- shRIG-1 and PK15-shDAI infected with PCV2. Replication ability of PCV2 was also reduced significantly in PK15-shRIG-1 and PK15-shDAI cell lines. These rusults showed that RIG-1 and DAI may be involved in type ? interferon production process induced by PCV2, and associated with PCV2 replication in PK15 cells.