| Porcine reproductive and respiratory syndrome (PRRS),which is caused by PRRS virus (PRRSV) and characterized by respiratory disease in piglets and severe reproductive failure in sows, is a highly contagious disease in pigs, leading to significant economic losses in the swine industry worldwide. It is urgent and necessary to prevent and control the spread and persistence of PRRSV. Thus, study of the role of signal translocates networks including the protein kinase C (PKC) signaling pathways on PRRSV replication and development of novel and effective vaccines are of great significance. This thesis mainly focuses on these two issues.Viruses exploit signaling pathways of their target cells to their advantage during multiple stages of life cycle. Protein kinase C (PKC) plays a central role in diverse cellular processes and signal transduction pathways that control cell proliferation, migration, differentiation and apoptosis. Recently, an increasing number of studies have shown that a lot of viruses can employ PKC signaling pathways to advance their infections. However, the role of protein kinase C (PKC) in PRRSV replication remains elusive. The first part of the thesis, we revealed that inhibition of PKC with the PKC inhibitor GF109203X significantly impairs PRRSV replication, which is associated with decreased viral RNA synthesis, lower virus protein expression and progeny virus titers. Furthermore, this inhibitory effect occurs at the early stage of PRRSV infection, which may be exerted at the entry and postentry stages of the virus life cycle as revealed by the virus internalization assay and treatment of cells with GF109203X at various time points postinfection, implying that PKC signaling pathway plays a necessary role in virus early infection. Consistent with this observation, this study also identified novel PKCδ as an important factor for PRRSV replication. PRRSV infection activates PKCδ in PAMs and treatment of PAMs with PKCδ specific inhibitor rottlerin significantly suppresses PRRSV replication, which is in line with siRNA-mediated knockdown of PKCδ. Moreover, cytokine production in porcine alveolar macrophages (PAMs) infected with PRRSV was shown to be altered by inhibiting PKC.Treatment with GF109203X significantly reduced the mRNA expressing levels of infection-induced antiviral cytokine including IFN-α, IFN-β, IL-1α, IL-1β and TNF-α. In contrast, inhibition of PKC resulted in a slightly elevation of mRNA expressing level of IL-10at the early time after HV infection. Therefore, based on these results, we propose that GF109203X-impaired PRRSV replication is not due to the up-regulation of antiviral cytokine expression. Taken together, these results suggest that PKC is involved in PRRSV infection and beneficial to PRRSV replication, extending our understanding of PRRSV infection.In the second part of the thesis, the study and development of novel PRRSV vaccine were performed. Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control PRRS. However, both types of vaccines have inherent drawbacks and development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities and has been used as an expression vehicle to elicit immune responses to foreign antigens. The recombinant Kluyveromyces lactis (K. lactis) expressing GP5of HP-PRRSV (Yeast-GP5) was generated and the immune responses were analyzed in BALB/c mice. The results showed that intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-γ in spleen CD4+T and CD8+T cells were induced by oral administration of Yeast-GP5. Yeast-GP5administrated subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-y secretion were detected in splenocytes of mice. These results suggest that Yeast-GP5has the potential to be a vaccine candidate for PRRSV in the future. |
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