Studies On The Molecular Mechanism That PDCoV Suppressed IFN-β Production

Porcine deltacoronavirus(PDCoV), an emerging animal coronavirus causing diarrhea, vomiting, dehydration and even death in sows and piglets, belongs to the newly identified Deltacoronavirus genus in the Coronaviridae family. Since early 2014 an outbreak of PDCoV announced in some pig farms in the United States, the detection of PDCoV was reported subsequently in Korea, Canada, and mainland China, which caused economic losses to the pig farms.Interferon(IFN) and the IFN-induced cellular antiviral innate immune response are the primary defense mechanisms against viral infection. To combat the antiviral effects of IFN, coronaviruses have evolved various mechanisms to antagonize the host innate immune responses. The molecular mechanisms of IFN antagonism have been extensively studied for alphacoronaviruses, betacoronaviruses, and gammacoronaviruses, so it is possible that PDCoV have also evolved to evade the host innate immune responses. To reveal the relationship between PDCoV infection and innate immune response as well as its mechanism, we studied the generation of IFN in PDCoV infected cells and the signal pathway in innate immune response during PDCoV infection. Main works are as following: 1. Studies on PDCoV proliferation characteristics in LLC-PK1 cellsFirst we studied the effect of different concentrations of trypsin on PDCoV infection. We found that PDCoV infection on LLC-PK1 cells needed proper concentration of trypsin, and the cytopathic effects(CPEs) were increasingly severe along with the increase concentration of trypsin. When using the trypsin concentration for 10ug/ml, CPEs were observed at 24 h post-infection(hpi) without cell detachment. At the same time we also studied the relationship between PDCoV inoculation doses and the CPEs. The results showed that PDCoV induced CPEs were increasingly severe along with the increase of inoculation dose, and minor CPEs with no cell detachment were observed at 24 hpi with moi of 0.01, which is consistent with the experiment described above. Indirect immunofluorescence assays(IFAs) showed that the number of infected cells were increased as the PDCoV infection progressed and reached a high level at 24 hpi with no cell detachment. A one-step growth curve for PDCoV in LLC-PK1 cells using TCID50 assays showed that virus titers presented a gradually upward tendency as the infection progressed, and at 24 hpi they reached a titer of 107.2 TCID50/ml. Together, these results show that PDCoV infection in LLC-PK1 cells achieves a high infection rate and titer without cell exfoliation at 24 hpi; thus, this time point was selected as the optimal time point for subsequent immunological studies. 2. Studies on the production of IFN-β、NF-κB and IRF3 in PDCoV infected cellsUsing IFN-β promoter luciferase reporter system, we found that PDCoV infection fails to induce and even remarkably inhibits SeV or poly(I:C)-induced IFN-β production in a dose-dependent manner. Moreover, using NF-κB and IRF3 promoter luciferase reporter system, we found that PDCoV infection failed to activate NF-κB and IRF3 promoter activity and blocked the SeV or poly(I:C)-induced promoter activity of NF-κB and IRF3. These results indicate that PDCoV impedes SeV or poly(I:C)-mediated activation of the transcription factors NF-κB and IRF3, which are associated with the suppression of the IFN-β promoter after PDCoV infection. 3. Studies on signaling pathway for IFN-β production during PDCoV infectionSeV and poly(I:C) are critical inducers of the RIG-I-like receptor(RLR)-mediated IFN-β signaling pathway. It is possible that PDCo V interrupts Se V or poly(I:C)-mediated IFN-β production by blocking some of the individual members of the RIG-I signaling pathway. To test this possibility and to identify potential target molecules, we investigated the effect of PDCoV infection on the activity of a series of molecules in the RIG-I signaling pathway: RIG-I/RIG-IN, MDA5, IPS-1, TBK1, IKKε, IRF3 and IRF3(5D). The results showed that the activation of the IFN-β promoter induced by IRF3 and its upstream molecules(RIG-I/RIG-IN, MDA-5, IPS-1, TBK1, and IKKε) was blocked by PDCoV infection. In contrast, the activation of the IFN-β promoter induced by IRF3(5D) was not affected by PDCoV infection. These results indicate IRF3 appears to be the target protein of PDCoV suppression in the RIG-I signaling pathway. 4. Studies on the phosphorylation and nuclear translocation of IRF3 and P65during PDCoV infectionBecause our initial results showed that PDCoV blocked SeV-induced IRF3 and NF-κB promoter activity, and phosphorylation and nuclear translocation are the hallmarks of IRF3 and NF-κB activation. Therefore, we explored the effect of PDCoV infection on the phosphorylation and nuclear translocation of IRF3 and NF-κB. Western blot showed that SeV-mediated increase of IRF3 phosphorylation(p-IRF3) and P65 phosphorylation(p-P65) was significantly reduced in the PDCo V-infected cells. Moreover, IFA showed PDCoV infection blocked the nuclear translocation of IRF3 and P65 otherwise induced by SeV infection. Our data clearly support the hypothesis that PDCoV inhibits SeV-induced IRF3 and NF-κB promoter activity by blocking the phosphorylation and nuclear translocation of IRF3 and P65.