PRRS is one of the most important diseases in swine industry. Current PRRS inactivated vaccine provides only a limited protection and cannot induce sufficient cell-mediated immune responses. In this study, we first found that the mRNA and protein levels of Th1-type cytokines (IFN-γ, IL-12) and Th2-type cytokines (IL-6, IL-10) were significantly increased through TRIF/MyD88-NF-κB signaling pathway when porcine peripheral blood monocyte-derived dendritic cells (MoDCs) were treated with poly (I: C) of TLR3 ligand and imiquimod of TLR7 ligand, along with inactivated PRRSV antigen. Meanwhile, the ability of catching PRRSV antigen was also significantly enhanced. In mice experiment, it was found that the PRRSV-specific T lymphocyte proliferation, the percentages of CD4(+), CD8(+) T lymphocytes and PRRSV-specific CD3(+) T cells producing IFN-γ and IL-4, the levels of Th1- and Th2-type cytokines and the titers of neutralization antibody were significantly enhanced in poly (I: C), imiquimod along with inactivated PRRSV group. Taken together, results of our experiments described for the first time that synergy of TLR3 and 7 ligands could significantly enhance the function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway and be used as adjuvant candidate for the development of novel PRRS inactivated vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) activates NF-κB during infection. We examined the ability of all 22 PRRSV genes for NF-κB regulation and determined the nucleocapsid (N) protein as the NF-κB activator. Protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1) (PIAS1) was identified as a cellular protein binding to N. PIAS1 is known to bind to p65 (RelA) in the nucleus and blocks its DNA binding, thus functions as a repressor of NF-κB. Binding of N to PIAS1 released p65 for NF-κB activation. The N-terminal half of PIAS1 was mapped as the N-binding domain, and this region overlapped its p65-binding domain. For N, the region between 37 and 72 aa was identified as the binding domain to PIAS1, and this domain alone was able to activate NF-κB. A nuclear localization signal (NLS) knock-out mutant N did not activate NF-κB, and this is mostly likely due to the lack of its interaction with PIAS1 in the nucleus, demonstrating the positive correlation between the binding of N to PIAS1 and the NF-κB activation. Our study reveals a role of N in the nucleus for NF-κB activation and proinflammatory cytokine production during infection.