Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ying Fang / Porcine reproductive and respiratory syndrome virus (PRRSV) employs multiple functions to modulate host’s innate immune response, and several viral nonstructural proteins (nsps) are major players. In this dissertation, the research was mainly focused on identification and functional dissection of ORF1a-encoded nsps. PRRSV replicase polyproteins encoded by ORF1a region are predicted to be processed into at least ten nonstructural proteins. In chapter 2, these predictions were verified by using a panel of newly established antibodies specific to ORF1a-encoded nsps. Most predicted nsps (nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8) were identified, and observed to be co-localized with de novo-synthesized viral RNA in the perinuclear region of the cell. Among all PRRSV proteins screened, nsp1β is the strongest type I interferon antagonist. In chapter 3, mutagenesis analysis of nsp1β was performed to knock down nsp1β’s IFN antagonist function. A highly conserved motif, GKYLQRRLQ, was determined to be critical for nsp1β’s ability to suppress IFN-β and reporter gene expression. Double mutations introduced in this motif, K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV), improved PRRSV’s ability to stimulate the expression of IFN-α, IFN-β and ISG15. In addition to its critical roles involving in modulating host innate immune response, in the studies of Chapter 4, we demonstrated that PRRSV nsp1β functions as a transactivator to induce the -2/-1 ribosomal frameshifting in nsp2, which results in expression of two novel PRRSV proteins, nsp2TF and nsp2N. The conserved motif GKYLQRRLQ is also determined to be critical for the transactivation function of nsp1β. In chapter 5, the interferon antagonist, de-Ub and de-ISGylation activity of newly identified nsp2TF and nsp2N were evaluated. In vitro and in vivo characterization of three nsp2TF-deficient recombinant viruses indicated that all mutant viruses have improved ability to stimulate the innate immune response and provide improved protection in mutant virus-vaccinated animals. In summary, this study verified the previously predicted PRRSV pp1a processing products, further evaluated the function of nsp1β and nsp2-related proteins. These data obtained here will provide basic knowledge for future development of vaccines and control measurements.

Since late 1980s, porcine reproductive and respiratory syndrome virus (PRRSV) has emerged as the most economically important swine pathogen affecting pig industries worldwide. Vaccination is the principal means that have been used for prevention of PRRSV infection. However, the currently available vaccines for PRRSV are generally considered as not very effective. One of the major obstacles for developing an effective modified live-attenuated vaccine (MLV) with broad protection is the delayed and insufficient immune responses mounted by PRRSV, and the problem is further exacerbated by the antigenic variations of the constantly-evolving field strains of PRRSV. In order to boost the immune response induced by the MLV vaccine virus, we evaluated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs expressing porcine IL-15 or IL-18 as adjuvants. The cytokine genes were fused with a GPI modification signal so that they are anchored onto the cell surface upon infection with the recombinant MLV. Both cytokines are successfully expressed on the cell membrane of porcine alveolar macrophage (PAMs) after recombinant MLVs infection in vitro. Subsequently, pigs vaccinated with cytokine-expressing recombinant PRRSV MLVs had an improved antiviral response of cytotoxic lymphocytes including natural killer (NK) cells and T cells, characterized by increased IFN-γ secretion and/or enhanced CD107a expression. The results offer a novel strategy to incorporate cytokine genes into PRRSV genome as potent bio-active adjuvants expressed by the vaccine virus itself. Since we showed that PRRSV VR2385 down-regulated swine leukocyte antigen class I surface expression, naturally the next logical question is which viral protein is responsible for this down-regulation. To answer the question, we cloned and expressed all known PRRSV structural and non-structural proteins and examined which protein(s) is involved in SLA-I downregulation. Our results identified the newly-discovered nonstructural protein Nsp2TF of PRRSV as the main mediator in down-regulating SLA-I expression. We also demonstrated that the Nsp2TF-knockout mutant virus lost its function of negatively modulating SLA-I presentation compared to the wild-type virus. The results suggest that disruption of the Nsp2TF's ability to down-regulate SLA-I expression may improve the existing PRRSV vaccines towards a better CMI response against the virus. / PHD / Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen, causing enormous economical losses in the pork industry worldwide. However, the vaccine program is not satisfactory, with the insufficient protection against genetically divergent strains and newly emerged strains. One of the most important reasons is that PRRSV is able to suppress immune responses in the host, but the underlying mechanisms are not well known. Therefore, the first dissertation study is to investigate novel strategies of developing live-attenuated vaccines with improved efficacy against PRRSV. In this study, we successfully generated recombinant PRRSV live vaccines that are able to express immuno-activating cytokines as adjuvants. Subsequently, pigs vaccinated with cytokine-expressing PRRSVs had significantly improved anti-PRRSV immune repsonses when compared to pigs vaccinated with unmodified PRRSV. Those recombinant PRRSVs also provided cross-protection against a heterologous PRRSV challenge. The second part of disseration research is to understand the mechanism of immune modulation by PRRSV. Our results showed that one of PRRSV proteins- Nsp2TF contributes to the PRSV-induced down-regulation of swine leukocyte antigen (SLA) class I expression. Since SLA class I molecules are essential in the activation of the immune response and required for the clearance of viruses, Our study suggested that knocking-out Nsp2TF could be of great value to generate PRRSV vaccines with a better immune response.