The present study aimed to explore if bovine coronavirus nucleocapsid (BCoV N) impacts IFN-β production in the host cells and to reveal further molecular mechanism of BCoV pathogenesis. Human embryonic kidney (HEK) 293 T cells were transiently transfected with pMyc-BCoV-N recombinant plasmids, then infected with the vesicular stomatitis virus (VSV). Expression levels of beta interferon (IFN-β) mRNA were detected using RT-qPCR. The results showed that BCoV N gene was 1347 bp that was consistent with the expected size. pMyc-BCoV-N recombinant protein was 1347 bp which was successfully transcribed and overexpressed in HEK 293 T cells. BCoV-N recombinant protein inhibited dose-dependently VSV-induced IFN-β production ( p < 0.01). MDA5, MAVS, TBK1 and IRF3 could promote transcription levels of IFN-β mRNA. But, BCoV-N protein demoted IFN-β transcription levels induced by MDA5, MAVS, TBK1 and IRF3. Furthermore, expression levels of MDA5, MAVS, TBK1 and IRF3 mRNAs were reduced in RIG-I-like receptor (RLR) pathway. In conclusion, BCoV-N reduced IFN-β levels in RIG-I-like receptor (RLR) pathway in HEK 293 T cells which were induced by MDA5, MAVS, TBK1 and IRF3(5D). BCoV-N protein inhibited IFN-β production and activation of RIG-I-like receptors ( RLRs) signal pathway. Our findings demonstrated BCoV N protein is an IFN-β antagonist through inhibition of MDA5, MAVS, TBK1 and IRF3(5D) in RLRs pathway, also revealed a new mechanism of BCoV N protein to evade host innate immune response by inhibiting type I IFN production, which is beneficial to developing novel prevention strategy for BCoV disease in the animals and humans.

To date, it has not yet been determined if the apoptosis of follicular granulosa cells (FGCs) is mediated by miR- let-7b via MAP3K1. In the present study, FGCs were transfected with a miR-let-7b mimic at different doses (0, 40, 60, 80, 100 and 120 μM), and were allocated into the control group (CG), and MIM-1, MIM-2, MIM-3, MIM-4 and MIM-5, respectively. Expression levels of miR-let-7b and mitogen-activated protein kinase kinase kinase 1 (MAP3K1) mRNAs and proteins were determined using RT-PCR and Western blots. Luciferase report assay was applied to verify the targeting relationship between miR-let-7b and MAP3K1. The results revealed that the proliferation activity of FGCs in the MIM-4 group was significantly lower than that of the CG and MIM-1 groups (P<0.05). The MiR-let-7b mimic obviously reduced expression levels of miR-let-7b of the FGCs. The largest reduction was found in MIM-4. Levels of MAP3K1 mRNAs and proteins in the MIM-3 and MIM-4 groups were lower than that of the CG (P<0.05 or P<0.01). Co-transfection of let-7b mimic significantly inhibited luciferase activity (P<0.05) as compared with the CG. In conclusion, miR-let-7b may obviously depress the cell viability and accelerate apoptosis of ovine FGCs. Higher doses of miR-let-7b mimic (80 μM and 100 μM) could significantly depress expressions of miR-let-7b mRNAs, MAP3K1 mRNAs and protein in ovine FGCs. MiR-let-7b promoted FGCs apoptosis by inhibiting the MAP3K1 gene.

Objective The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-β) production and to reveal further molecular mechanism of BPV immune escape. Method The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-β mRNA were detected using qPCR. ResultsThe expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 10 4 . Expression levels of IFN-β mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-β expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. Conclusion pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-β. Overexpression of pCMV-Myc-BPV-VP decreased IFN-β mRNA expression in HEK 293 T cells and inhibited IFN-β production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.

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