Replication-Competent Recombinant Porcine Reproductive and Respiratory Syndrome (PRRS) Viruses Expressing Indicator Proteins and Antiviral Cytokines

Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more effective adaptive immunity against PRRSV infection

Post-Transcriptional Control of Type I Interferon Induction by Porcine Reproductive and Respiratory Syndrome Virus in Its Natural Host Cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is not only a poor inducer of type I interferon but also inhibits the efficient induction of type I interferon by porcine transmissible gastroenteritis virus (TGEV) and synthetic dsRNA molecules, Poly I:C. However, the mechanistic basis by which PRRSV interferes with the induction of type I interferon in its natural host cells remains less well defined. The purposes of this review are to summarize the key findings in supporting the post-transcriptional control of type I interferon in its natural host cells and to propose the possible role of translational control in the regulation of type I interferon induction by PRRSV

Interplay between Interferon-Mediated Innate Immunity and Porcine Reproductive and Respiratory Syndrome Virus

Innate immunity is the first line of defense against viral infection, and in turn, viruses have evolved to evade host immune surveillance. As a result, viruses may persist in host and develop chronic infections. Type I interferons (IFN-α/β) are among the most potent antiviral cytokines triggered by viral infections. Porcine reproductive and respiratory syndrome (PRRS) is a disease of pigs that is characterized by negligible induction of type I IFNs and viral persistence for an extended period. For IFN production, RIG-I/MDA5 and JAK-STAT pathways are two major signaling pathways, and recent studies indicate that PRRS virus is armed to modulate type I IFN responses during infection. This review describes the viral strategies for modulation of type I IFN responses. At least three non–structural proteins (Nsp1, Nsp2, and Nsp11) and a structural protein (N nucleocapsid protein) have been identified and characterized to play roles in the IFN suppression and NF-κB pathways. Nsp’s are early proteins while N is a late protein, suggesting that additional signaling pathways may be involved in addition to the IFN pathway. The understanding of molecular bases for virus-mediated modulation of host innate immune signaling will help us design new generation vaccines and control PRRS

Studies on the Interaction between Porcine Plasmacytoid Dendritic Cells and Porcine Reproductive and Respiratory Syndrome Virus

For over 20 years, porcine reproductive and respiratory syndrome (PRRS) has been the most troubling disease afflicting the pork industry. The main reason for this dissertation is that the pathogen responsible for this disease, PRRS virus (PRRSV), has developed the ability to subvert the immune response of its host. Evidence available at the outset of this project hinting that subversion of the immune system was a possibility, were the observations that PRRSV infection provoked a rapid humoral response characterized by the production of ineffective, non-immunizing antibodies and a cell-mediated immune response that is weak and protracted. The later was proposed to be result of a lack of adequate IFN-<;t production by the host, which is necessary to promote the development of protective anti-viral cell mediated immunity. Although this deficiency was attributed to non-translation of IFN-a gene transcripts produced in PRRSV-infected alveolar macrophages (AM), the most potent cell responsible for IFN-a. production in response to a viral infection, the plasmacytoid dendritic cell (PDC), had been largely ignored. Conceivably, this dismissal was based on the fact that a subset of AM is the principal target cell of PRRSV and that these cells readily support its replication in vitro. Because of the exuberant ability of PDC to produce IFN-a, the focus of this dissertation was directed instead to examine the porcine PDC. First, methodology was created that deployed the use of cell sorting instruments to allow isolation of a relatively homogeneous group of cells (CD4+CD17ztow) that phenotypically were likely to represent porcine PDC. Subsequently, these cells were indeed shown to be PDC based on their characteristic ability to rapidly secrete copious amounts of IFN-a. upon activation by the exposure to the coronavirus, transmissible gastroenteritis virus (TGEV) or to type A CpG-ODN, followed by a maturational change in the appearance of these cells from plasmacytoid to dendritic cell morphology. Based on their established phenotype, electronic gating on flow cytometric histograms in conjunction with multiple antibody stainings were used to further analyze these cells and reveal the existence of novel surface proteins including CD18, CD29, and CD44. However, the more impressive results were obtained with the purified PDC. For the first time, secretion of a variety of cytokines including IL-2, IL-8, and IFN-y, by activated porcine PDC could be demonstrated with a degree of certainty. Moreover, simple transcriptome patterns of resting and activated PDC could be compared and by doing so exemplify the tremendous induction of type I IFN gene expression by TGEV and a TLR9 agonist, type A CpG­ ODN (ODN19). In terms of immunobiology, probably the most important discovery was that in general PRRSV not only did not elicit an IFN-a response from PDC, but, actually impeded the typical induction of IFN-a expression by TGEV- or ODNl 9- stimulated PDC. Confirming that the inhibitory effect of PRRSV on PDC function also occurred upon in vivo exposure of PDCs to PRRSV validated the potential biological significance of these observations. Furthermore, the maturation of PDC was also impaired by PRRSV, as manifested by the lack of transformation of the PDC into dendritic cells and the de novo expression of CD80/86 upon to exposure to activating agents. Experiments aimed at determining the mechanism of inhibition suggest that the inhibitory effect PRRSV on PDC function is probably mediated by preventing the translocation of STAT-1 to the nucleus, thus preventing an increase in the level IRF-7 expression, which is required for optimal IFN-a production by PDC. The extent of the subversive effect of PRRSV on the activation of PDC was further appreciated by transcriptional analyses of PDC exposed to PRRSV as compared to their exposure to the porcine coronavirus TGEV. These studies revealed that the expression of a number of IFN-inducible genes was not stimulated by PRRSV. The unique nature of the subversive effect of PRRSV on porcine PDC was illustrated by the distinct transcriptional profile and cytokine production pattern exhibited by PDC exposed to PRRSV as compared to porcine AM exposed to the same virus. In the case of AM, the cells were not inhibited by PRRSV in their ability to produce IFN-a in response to TGEV. Finally, a ray of hope was obtained by the analyses of the outcome of the interaction between PDC and a mutant of PRRSV that does not inhibit IFN-a production. It is expected that this virus will provide information for the development of an effective second generation PRRSV vaccine

Interferon regulatory factor 5 activation in monocytes of systemic lupus erythematosus patients is triggered by circulating autoantigens independent of type I interferons

OBJECTIVE: Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFN) believed to be involved in SLE pathogenesis. The aim of this study was to determine the activation status of IRF5 by assessing its nuclear localization in immune cells of SLE patients and healthy donors, and to identify SLE triggers of IRF5 activation. METHODS: IRF5 nuclear localization in subpopulations of peripheral blood mononuclear cells (PBMC) from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. IRF5 activation and function were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream and cytokine expression by Q-PCR and ELISA. RESULTS: IRF5 was activated in a cell type-specific manner; monocytes of SLE patients had constitutively elevated levels of nuclear IRF5 compared to NK and T cells. SLE serum was identified as a trigger for IRF5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens comprised of apoptotic/necrotic material triggered IRF5 nuclear accumulation in monocytes. Production of cytokines IFNα, TNFα and IL6 in monocytes stimulated with SLE serum or autoantigens was distinct yet correlated with the kinetics of IRF5 nuclear localization. CONCLUSION: This study provides the first formal proof that IRF5 activation is altered in monocytes of SLE patients that is in part contributed by the SLE blood environment

Genome-wide analysis of antiviral signature genes in porcine macrophages at different activation statuses

Macrophages (MФs) can be polarized to various activation statuses, including classical (M1), alternative (M2), and antiviral states. To study the antiviral activation status of porcine MФs during porcine reproductive and respiratory syndrome virus (PRRSV) infection, we used RNA Sequencing (RNA-Seq) for transcriptomic analysis of differentially expressed genes (DEGs). Sequencing assessment and quality evaluation showed that our RNA-Seq data met the criteria for genome-wide transcriptomic analysis. Comparisons of any two activation statuses revealed more than 20,000 DEGs that were normalized to filter out 153–5,303 significant DEGs [false discovery rate (FDR) ≤0.001, fold change ≥2] in each comparison. The highest 5,303 significant DEGs were found between lipopolysaccharide- (LPS) and interferon (IFN)γ-stimulated M1 cells, whereas only 153 significant DEGs were detected between interleukin (IL)-10-polarized M2 cells and control mock-activated cells. To identify signature genes for antiviral regulation pertaining to each activation status, we identified a set of DEGs that showed significant up-regulation in only one activation state. In addition, pathway analyses defined the top 20–50 significantly regulated pathways at each activation status, and we further analyzed DEGs pertinent to pathways mediated by AMP kinase (AMPK) and epigenetic mechanisms. For the first time in porcine macrophages, our transcriptomic analyses not only compared family-wide differential expression of most known immune genes at different activation statuses, but also revealed transcription evidence of multiple gene families. These findings show that using RNA-Seq transcriptomic analyses in virus-infected and status-synchronized macrophages effectively profiled signature genes and gene response pathways for antiviral regulation, which may provide a framework for optimizing antiviral immunity and immune homeostasis