Porcine γδ T cells express cytotoxic cell-associated markers and display killing activity but are not selectively cytotoxic against PRRSV- or swIAV-infected macrophages.

Background: Gamma-delta (γδ) T cells are a major immune cell subset in pigs. Approximately 50% of circulating T cells are γδ T cells in young pigs and up to 30% in adult sows. Despite this abundance, the functions of porcine γδ T cells are mostly unidentified. In humans and mice, activated γδ T cells exhibit broad innate cytotoxic activity against a wide variety of stressed, infected, and cancerous cells through death receptor/ligand-dependent and perforin/granzyme-dependent pathways. However, so far, it is unknown whether porcine γδ T cells have the ability to perform cytotoxic functions. Methods: In this study, we conducted a comprehensive phenotypic characterization of porcine γδ T cells isolated from blood, lung, and nasal mucosa. To further analyze the cytolytic potential of γδ T cells, in vitro cytotoxicity assays were performed using purified γδ T cells as effector cells and virus-exposed or mock-treated primary porcine alveolar macrophages as target cells. Results: Our results show that only CD2+ γδ T cells express cytotoxic markers (CD16, NKp46, perforin) with higher perforin and NKp46 expression in γδ T cells isolated from lung and nasal mucosa. Moreover, we found that γδ T cells can exhibit cytotoxic functions in a cell-cell contact and degranulation-dependent manner. However, porcine γδ T cells did not seem to specifically target Porcine Reproductive and Respiratory Syndrome Virus or swine Influenza A Virus-infected macrophages, which may be due to viral escape mechanisms. Conclusion: Porcine γδ T cells express cytotoxic markers and can exhibit cytotoxic activity in vitro. The specific mechanisms by which porcine γδ T cells recognize target cells are not fully understood but may involve the detection of cellular stress signals.

Multiplex Assay to Determine Acute Phase Proteins in Modified Live PRRSV Vaccinated Pigs.

Acute phase protein (APP) response to vaccine challenges is an attractive alternative to natural infection for identifying pigs with increased disease resilience and monitoring the productive performance. Currently, the methods used for APP quantification are diverse and often based on techniques that use antibodies that are not necessarily pig specific. The objective of this work is the development of a method based on a UPLC-SRM/MS system for simultaneous determination of haptoglobin, apolipoprotein A1, C-reactive protein, pig-major acute protein, and serum amyloid A and its application in pigs to monitor the effect of a vaccine administered against porcine reproductive and respiratory syndrome virus (PRRSV). With the aim of tracing the complete analytical process for each proteotypic peptide, a synthetic QconCat polypeptide construct was designed. It was possible to develop an SRM method including haptoglobin, apolipoprotein A1, pig-MAP, and serum amyloid A1. The PRRSV vaccine only affected haptoglobin. The pigs with positive viremia tended to show higher values than negative pigs, reaching significant differences in the three haptoglobin SRM-detected peptides but not with the data acquired by immunoenzymatic and spectrophotometric assays. These results open the door to the use of SRM to accurately monitor APP changes in experimental pigs.

PRRSV NSP1α degrades TRIM25 through proteasome system to inhibit host antiviral immune response.

Porcine reproductive and respiratory syndrome (PRRS) is the most economically significant disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Type I interferon (IFN) induces a large number of interferon-stimulated genes (ISGs) expression to inhibit PRRSV infection. To survive in the host, PRRSV has evolved multiple strategies to antagonize host innate immune response. Previous studies have reported that PRRSV N protein decreases the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress IFN-ß production. However, whether other PRRSV proteins inhibit the antiviral function of TRIM25 is less well understood. In this study, we first found that PRRSV NSP1α decreased ISGylation of TRIM25. Meanwhile, NSP1α significantly suppressed TRIM25-mediated IFN-ß production to promote PRRSV replication. Further studies demonstrated that PRRSV NSP1α reduced the protein level of TRIM25 in proteasome system but did not regulate the transcription level of TRIM25. In addition, the function of NSP1α in TRIM25 degradation did not rely on its papain-like cysteine protease activity. Taken together, PRRSV NSP1α antagonizes the antiviral response of TRIM25 by mediating TRIM25 degradation to promote PRRSV replication. Our data identify TRIM25 as a natural target of PRRSV NSP1α and reveal a novel mechanism that PRRSV induces TRIM25 degradation and inhibits host antiviral immune response.

Nanobody peptide conjugate: a novel CD163 based broad neutralizing strategy against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

Field efficacy of a novel porcine reproductive and respiratory syndrome modified-live virus vaccine with an emphasis on growth performance.

BACKGROUND: This field evaluation was designed to evaluate the efficacy of a new porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) modified live virus vaccine at three independent pig farms. METHODS: Three farms were selected for this study based on their respiratory disease status caused by PRRSV-2 infection in post-weaning and growing pigs. Each farm housed a total of 40, 18-day-old pigs that were randomly allocated to one of two treatment groups. Pigs were administered a 1.0 mL dose of the bivalent vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate buffered saline at the same age. RESULTS: Vaccinated groups were measured and calculated significantly (p < 0.05) higher in body weight and average daily weight gain on all three farms compared with unvaccinated groups. Vaccinated groups elicited PRRS antibodies and PRRSV-2-specific interferon-γ secreting cells, which reduced the amount of PRRSV-2 genomic copies in the blood and reduced macroscopic and microscopic lung lesions severity when compared with unvaccinated groups. CONCLUSIONS: The field evaluation data demonstrated that a new PRRSV-2 modified live virus vaccine was efficacious in swine herds suffering from respiratory diseases caused by PRRSV-2 infection.

Development of a Ferritin Protein Nanoparticle Vaccine with PRRSV GP5 Protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant threat to the global swine industry. The development of highly effective subunit nanovaccines is a promising strategy for preventing PRRSV variant infections. In this study, two different types of ferritin (Ft) nanovaccines targeting the major glycoprotein GP5, named GP5m-Ft and (Bp-IVp)3-Ft, were constructed and evaluated as vaccine candidates for PRRSV. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) demonstrated that both purified GP5m-Ft and (Bp-IVp)3-Ft proteins could self-assemble into nanospheres. A comparison of the immunogenicity of GP5m-Ft and (Bp-IVp)3-Ft with an inactivated PRRSV vaccine in BALB/c mice revealed that mice immunized with GP5m-Ft exhibited the highest ELISA antibody levels, neutralizing antibody titers, the lymphocyte proliferation index, and IFN-γ levels. Furthermore, vaccination with the GP5m-Ft nanoparticle effectively protected piglets against a highly pathogenic PRRSV challenge. These findings suggest that GP5m-Ft is a promising vaccine candidate for controlling PRRS.

Enhancing immunogenicity and antiviral protection of inactivated porcine reproductive and respiratory syndrome virus vaccine in piglets.

OBJECTIVE: Porcine interferon-γ (poIFN-γ) and porcine granulocyte-macrophage colony-stimulating factor (poGM-CSF) are multifunctional cytokines that exhibit robust antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the immunoadjuvant effects of recombinant poIFN-γ-poGM-CSF fusion protein in inactivated PRRSV vaccine administered to piglets were assessed. ANIMALS: Twenty-eight 4-week-old specific pathogen-free piglets. METHODS: The experimental piglets were divided into control, highly pathologic PRRSV, PRRSV killed virus vaccine (KV), poIFN-γ-poGM-CSF, KV + 1.0 mg poIFN-γ-poGM-CSF, KV + 2.0 mg poIFN-γ-poGM-CSF, and KV + 4.0 mg poIFN-γ-poGM-CSF groups. A recombinant poIFN-γ-linker-poGM-CSF fusion gene was constructed via splicing by overlap extension PCR and prepared using an Escherichia coli expression system, after which its adjuvant activity in the context of PRRSV KV administration was assessed. RESULTS: This analysis revealed the successful construction of the poIFN-γ-linker-poGM-CSF fusion gene via splicing by overlap extension PCR, with recombinant poIFN-γ-linker-poGM-CSF successfully being prepared in E coli with a plasmid vector for expressing thioredoxin fusion proteins with an enterokinase site. Importantly, the coadministration of poIFN-γ-linker-poGM-CSF and PRRSV KV significantly increased neutralizing antibody titers, accelerated viral clearance, reduced clinical symptoms, and prevented highly pathogenic PRRSV infection. CLINICAL RELEVANCE: The recombinant poIFN-γ-poGM-CSF fusion protein is a promising candidate adjuvant for use in the context of swine immunization and viral challenge.

The mRNA Vaccine Expressing Single and Fused Structural Proteins of Porcine Reproductive and Respiratory Syndrome Induces Strong Cellular and Humoral Immune Responses in BalB/C Mice.

PRRS is a viral disease that profoundly impacts the global swine industry, causing significant economic losses. The development of a novel and effective vaccine is crucial to halt the rapid transmission of this virus. There have been several vaccination attempts against PRRSV using both traditional and alternative vaccine design development approaches. Unfortunately, there is no currently available vaccine that can completely control this disease. Thus, our study aimed to develop an mRNA vaccine using the antigens expressed by single or fused PRRSV structural proteins. In this study, the nucleotide sequence of the immunogenic mRNA was determined by considering the antigenicity of structural proteins and the stability of spatial structure. Purified GP5 protein served as the detection antigen in the immunological evaluation. Furthermore, cellular mRNA expression was detected by immunofluorescence and western blotting. In a mice experiment, the Ab titer in serum and the activation of spleen lymphocytes triggered by the antigen were detected by ELISA and ICS, respectively. Our findings demonstrated that both mRNA vaccines can significantly stimulate cellular and humoral immune responses. More specifically, the GP5-mRNA exhibited an immunological response that was similar to that of the commercially available vaccine when administered in high doses. To conclude, our vaccine may show promising results against the wild-type virus in a natural host.

PRRSV degrades MDA5 via dual autophagy receptors P62 and CCT2 to evade antiviral innate immunity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically devastating pathogen that has evolved various strategies to evade innate immunity. Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5 (MDA5), a receptor that senses viral RNA. In this study, the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed, and the detailed mechanisms were explored. We found that the interaction between P62 and MDA5 is enhanced due to two factors: the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2α and the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells. As a result of these modifications, the classic P62-mediated autophagy is triggered. Additionally, porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2 (CCT2), which is enhanced by PRRSV nsp3. This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination. In summary, enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways: the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2, leading to intense innate immune suppression. The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies.

The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function.

Porcine reproductive and respiratory syndrome virus (PRRSV) modulates host innate immunity which plays a key role against PRRSV infection. As a RNA virus, PRRSV is mainly sensed by innate immune RNA receptors, whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated. Here, we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages. The results show that in Marc-145 cells, the stable expression of STING with or without stimulations exhibited anti-PRRSV activity, and STING knockout heightened PRRSV infection. In CD163-3D4/21 porcine macrophages, either expression of STING or stimulation of cGAS-STING signaling obviously suppressed PRRSV infection, whereas in STING knockdown macrophages, the PRRSV infection was upregulated. Our results clearly demonstrate that the host cGAS-STING signal exerts an important antiviral role in PRRSV infection.

Swine Dendritic Cell Response to Porcine Reproductive and Respiratory Syndrome Virus: An Update.

Dendritic cells (DCs) are the most potent antigen-presenting cells, unique to initiate and coordinate the adaptive immune response. In pigs, conventional DCs (cDCs), plasmacytoid DCs (pDCs), and monocyte-derived DCs (moDCs) have been described in blood and tissues. Different pathogens, such as viruses, could infect these cells, and in some cases, compromise their response. The understanding of the interaction between DCs and viruses is critical to comprehend viral immunopathological responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important respiratory pathogen in the global pig population. Different reports support the notion that PRRSV modulates pig immune response in addition to their genetic and antigenic variability. The interaction of PRRSV with DCs is a mostly unexplored area with conflicting results and lots of uncertainties. Among the scarce certainties, cDCs and pDCs are refractory to PRRSV infection in contrast to moDCs. Additionally, response of DCs to PRRSV can be different depending on the type of DCs and maybe is related to the virulence of the viral isolate. The precise impact of this virus-DC interaction upon the development of the specific immune response is not fully elucidated. The present review briefly summarizes and discusses the previous studies on the interaction of in vitro derived bone marrow (bm)- and moDCs, and in vivo isolated cDCs, pDCs, and moDCs with PRRSV1 and 2.

Development of a monoclonal antibody against PRRSV glycoprotein 3 using an immuodominant peptide as immunogen.

Glycoprotein 3 (GP3), a highly glycosylated membrane protein, is a protective antigen and minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV), and plays a crucial role in virus assembly and infection. In the present study, we synthesized 23 overlapping peptides span GP3 protein sequence and used pig anti-PRRSV serums to identify immunodominant peptides by indirect ELISA. Five immunodominant peptides GP3-P3, P4, P5, P6 and P7 were identified and GP3-P4 (P55LCPTRQAAAEILEPGKS72) was conjugated to carrier protein BSA. One mAb 1E5 against GP3 was generated from BALB/c mice immunized with the conjugates BSA-P4. The Characterization of mAb was identified by Western blot, Dot-ELISA, IPMA and IFA. We found that mAb 1E5 can specifically react with HP-PRRSV strains but not C-PRRSV or NADC30-like PRRSV strains tested in this study. Site-directed alanine substitution analysis revealed that 8 amino acid residues were involved in antibody binding, among them E65, L67 and P69 were critical residue recognized by mAb 1E5. Taken together, this study provided a novel strategy for generating specific mAbs against virus proteins by using immunodominant peptides as targets, and the mAb 1E5 may be useful for development of rapid differential detection method differentiating HP-PRRSV from C-PRRSV and NADC30-like PRRSV.

Porcine Reproductive and Respiratory Syndrome Virus Infection Upregulates Negative Immune Regulators and T-Cell Exhaustion Markers.

Porcine alveolar macrophage (PAM) is one of the primary cellular targets for porcine reproductive and respiratory syndrome virus (PRRSV), but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystander PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP), and GFP+ (PRRSV infected) and GFP- (bystander) cells were sorted for RNA sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP+ and 0.06% reads from GFP- PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP+ compared to GFP- PAMs. Importantly, negative immune regulators, including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3) and T-cell exhaustion markers (programmed death ligand-1 [PD-L1], PD-L2, interleukin-10 [IL-10], IDO1, and transforming growth factor ß2 [TGFB2]) were highly upregulated in GFP+ cells compared to GFP- cells. By using an in situ hybridization assay, RNA transcripts of tumor necrosis factor (TNF) and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is widespread in many swine-producing countries, causing substantial economic losses to the swine industry. Porcine alveolar macrophage (PAM) is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAMs from acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV strain expressing green fluorescent protein to infect pigs and sorted infected and bystander PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV-infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.

Rutin, α-tocopherol, and l-ascorbic acid up-regulate type I interferon-regulated gene and type I and II interferon expressions and reduce inflammatory cytokine expressions in monocyte-derived macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus.

This study evaluated the immunomodulatory effect of two types of phytochemicals, i.e. rutin and ß-carotene, and two types of vitamins, i.e. α-tocopherol and l-ascorbic acid on improving innate immune responses to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Monocyte-derived macrophages (MDM) from eight PRRSV-seronegative pigs were inoculated with HP-PRRSV and subsequently stimulated with rutin, ß-carotene, α-tocopherol, and l-ascorbic acid in the absence or presence of either polyinosinic:polycytidylic acid or lipopolysaccharide. The mRNA expression levels of myxovirus resistance 1, interferon regulatory factor 3 (IRF3), IRF7, 2'-5'-oligoadenylatesynthetase 1, stimulator of interferon genes (STING), osteopontin (OPN), interferon alpha (IFNα), IFNß, IFNγ, interleukin-10 (IL-10), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFß) were evaluated by real-time PCR. Compared with control MDM, HP-PRRSV significantly suppressed mRNA expressions of all immune-related genes except IL-10 and TGFß. Compared with HP-PRRSV-inoculated MDM, stimulation with rutin, α-tocopherol, and l-ascorbic acid, but not ß-carotene significantly enhanced mRNA expression levels of IRF3, IRF7, STING, OPN, IFNα, IFNß, and IFNγ in HP-PRRSV-inoculated MDM. Stimulation with rutin also significantly reduced mRNA expression levels of TNFα and TGFß, whereas stimulation with ß-carotene and α-tocopherol significantly reduced TNFα mRNA expression in HP-PRRSV-inoculated MDM. Our findings demonstrate the potentials of rutin, α-tocopherol, and l-ascorbic acid in enhancing type I interferon-regulated genes and type I and II IFN expressions, and in reducing pro- and/or anti-inflammatory cytokine expressions in HP-PRRSV-inoculated MDM. Our findings suggest that rutin, α-tocopherol, and l-ascorbic acid may serve as effective immunomodulators for improving innate immune response to HP-PRRSV.

Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines.

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.

Wnt/ß-catenin signaling pathway inhibits porcine reproductive and respiratory syndrome virus replication by enhancing the nuclear factor-κB-dependent innate immune response.

The Wnt/ß-catenin signaling pathway is an evolutionarily highly conserved signaling pathway related to the replication of various viruses. However, the interaction between the Wnt/ß-catenin pathway and porcine reproductive and respiratory syndrome virus (PRRSV) is unknown. In the present study, we showed that PRRSV-infected Marc-145 and PAM cells expressed high levels of c-myc and cyclinD1 mRNA and accumulation of ß-catenin in the nucleus. PRRSV nonstructural proteins (Nsps) 1α, 1ß, 3, 4, 7, 10, and 12, and proteins encoded by open reading frames (ORFs) 2b, 3, and 5 induced the activation of the Wnt pathway according to TOP/FOP luciferase reporter assay. But, Nsp5 inhibited the activation of the Wnt pathway. Pre-treatment with Wnt3a inhibited PRRSV replication in Marc-145 cells in a dose-dependent manner. Over-expression of ß-catenin also inhibited PRRSV replication, while silencing of ß-catenin by small hairpin RNA increased its replication in Marc-145 cells. Over-expression of ß-catenin increased interferon regulatory factor (IRF)-3 expression and nuclear factor (NF)-κB phosphorylation, NF-κB and interferon-stimulated response element promoter activities, and interferon-ß, DExD/H-box helicase 58 (DDX58), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-8 mRNA expression. Conversely, silencing ß-catenin decreased phosphorylated IRF-3 and NF-κB, NF-κB and IFIT1 promoter activities, and IFN-ß, DDX58, IFIT1, IL-1ß, TNF-α, and IL-8 mRNA levels in Marc-145 cells. Co-immunoprecipitation and immunofluorescence colocalization analyses confirmed that ß-catenin interacted with NF-κB in Marc-145 cells. In conclusion, PRRSV infection activates the Wnt/ß-catenin signaling pathway via Nsps 1α, 1ß, 3, 4, 7, 10, and 12, and proteins encoded by ORFs 2b, 3, and 5. The Wnt/ß-catenin pathway then inhibits PRRSV replication by enhancing the NF-κB-dependent innate immune response. These findings further our understanding of the role of the Wnt/ß-catenin signaling pathway in regulating PRRSV replication and provide new insights into virus-host interactions.

Construction and efficacy evaluation of novel swine leukocyte antigen (SLA) class I and class II allele-specific poly-T cell epitope vaccines against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease. Here we report the development of subunit PRRSV-2 vaccines by expressing swine leucocyte antigen (SLA) class I and class II allele-specific epitope antigens in a robust adenovirus vector. SLA I-specific CD8 and SLA II-specific CD4 T cell epitopes of PRRSV-2 NADC20 were predicted in silico. Stable murine leukaemia cell lines (RMA-S), which are TAP-deficient and lacking endogenous class I epitope loading, were established to express different SLA I alleles. The binding stability of PRRSV T cell epitope peptides with SLA I alleles expressed on RMA-S cells was characterized. Two PRRSV poly-T cell epitope peptides were designed. NADC20-PP1 included 39 class I epitopes, consisting of 8 top-ranked epitopes specific to each of 5 SLA I alleles, and fused to 5 class II epitopes specific to SLA II alleles. NADC20-PP2, a subset of PP1, included two top-ranked class I epitopes specific to each of the five SLA I alleles. Two vaccine candidates, Ad-NADC20-PP1 and Ad-NADC20-PP2, were constructed by expressing the polytope peptides in a replication-incompetent human adenovirus 5 vector. A vaccination and challenge study in 30 piglets showed that animals vaccinated with the vaccines had numerically lower gross and histopathology lung lesions, and numerically lower PRRSV RNA loads in lung and serum after challenge compared to the controls, although there was no statistical significance. The results suggested that the Ad-NADC20-PP1 and Ad-NADC20-PP2 vaccines provided little or no protection, further highlighting the tremendous challenges faced in developing an effective subunit PRRSV-2 vaccine.

Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo.

The humoral immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection is characterized by a rapid induction of nonneutralizing antibodies (non-NAbs) against nonstructural proteins (NSPs). Here, we systematically investigated the potential mechanism for the induction of PRRSV NSP-specific non-NAbs. Our data suggested that PRRSV NSP-specific antibodies appeared within 10 days after PRRSV infection in vivo In the in vitro model, functional upregulation of swine leukocyte antigen (SLA)-DR was observed in bone marrow-derived dendritic cells (BMDCs) and porcine alveolar macrophages (PAMs), whereas remarkable inhibition at the mRNA level was observed after infection by both PRRSV-1 and PRRSV-2 isolates. Notably, the inconsistency in SLA-DR expression between the mRNA and protein levels resulted from deubiquitination of SLA-DR via the ovarian tumor (OTU) domain of PRRSV NSP2, which inhibited ubiquitin-mediated degradation. Moreover, mass spectrometry-based immunopeptidome analysis identified immunopeptides originating from multiple PRRSV NSPs within SLA-DR of PRRSV-infected BMDCs. Meanwhile, these PRRSV NSP-derived immunopeptides could be specifically recognized by serum from PRRSV-infected piglets. Notably, certain NSP-derived immunopeptides characterized in vitro could be identified from PAMs or hilar lymph nodes from PRRSV-infected piglets. More importantly, an in vitro neutralizing assay indicated that serum antibodies against NSP immunopeptides were unable to neutralize PRRSV in vitro Conversely, certain structural protein (SP)-derived immunopeptides were identified and could be recognize by pig hyperimmune serum against PRRSV, which further indicates that the NSP-derived antibody response is nonprotective in vivo In conclusion, our data suggested that PRRSV infection interferes with major histocompatibility complex class II (MHC-II) molecule-mediated antigen presentation in antigen-presenting cells (APCs) via promoting SLA-DR expression to present immunopeptides from PRRSV NSPs, which contributes to the induction of non-NAbs in vivoIMPORTANCE PRRSV has haunted the swine industry for over 30 years since its emergence. Besides the limited efficacy of PRRSV modified live vaccines (MLVs) against heterogeneous PRRSV isolates, rapid induction of nonneutralizing antibodies (non-NAbs) against PRRSV NSPs after MLV immunization or wild-strain infection is one of the reasons why development of an effective vaccine has been hampered. By using in vitro-generated BMDCs as models to understand the antigen presentation process of PRRSV, we obtained data indicating that PRRSV infection of BMDCs promotes functional SLA-DR upregulation to present PRRSV NSP-derived immunopeptides for evoking a non-NAb response in vivo Our work not only uncovered a novel mechanism for interference in host antigen presentation by PRRSV but also revealed a novel insight for understanding the rapid production of nonneutralizing antibodies against PRRSV NSPs, which may have benefit for developing an effective vaccine against PRRSV in the future.

Replacing the decoy epitope of PCV2 capsid protein with epitopes of GP3 and/or GP5 of PRRSV enhances the immunogenicity of bivalent vaccines in mice.

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure and causes economic losses in the domestic swine industry. The decoy epitope (169-180 amino acid (aa)) of the PCV2 capsid (Cap) protein is an immunodominant epitope and diverts the immune response away from protective epitopes. The mixed infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most common co-infections in the pig industry and shows more severe clinical symptoms. Linear B-cell antigenic epitopes of PRRSV GP3 epitope Ⅰ (61-72aa) and PRRSV GP5 epitope Ⅳ (187-200aa) efficiently elicited neutralizing antibodies against PRRSV. The recombinant baculovirus expressing the Cap protein (Bac-Cap) was modified by replacing the decoy epitope of the Cap protein with either the PRRSV GP3 epitope Ⅰ, the PRRSV GP5 epitope Ⅳ, or the PRRSV GP3 epitope Ⅰ- GP5 epitope Ⅳ to produce the recombinant baculoviruses Bac-Cap-GP3, Bac-Cap-GP5 and Bac-Cap-GP35. The four recombinant baculoviruses were successfully established and characterized as demonstrated with western blot analysis and immunofluorescence assay. Immunogenicities of the four recombinant baculoviruses in mice were tested in sera harvested at 21 and 42 days post-primary immunization. The titers of antibodies in the sera were determined by a PCV2-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The serum IFN-γ levels were measured by indirect ELISA. The results showed that Bac-Cap-GP3, Bac-Cap-GP5, and Bac-Cap-GP35 elicited higher GP3/GP5 and Cap antibody titers than the Bac-Cap. Virus neutralization test also confirmed that the serum from the Bac-Cap-GP3 immunized mice had high levels of the both PCV2 and PRRSV neutralization antibodies. These findings collectively demonstrated that substituting the decoy epitope of the PCV2 capsid substituted with PRRSV epitopes could be developed into an effective vaccine against PCV2.

The IFN-γ-induced immunoproteasome is suppressed in highly pathogenic porcine reproductive and respiratory syndrome virus-infected alveolar macrophages.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) evades cytotoxic T lymphocyte (CTL) responses through interactions between viral Nsp1α and Nsp4 and ß2 M heavy and light chains, respectively, of swine leukocyte antigen class (SLA)-I. However, whether the immunoproteasome (i-proteasome) complex, which is an important component of the antigen delivery pathway that functions by mediating peptide production, is also affected by viral infection is unknown. In this study, we investigated the effects of HP-PRRSV (HuN4-F5) infection on IFN-γ-induced i-proteasome expression using a cell culture system (alveolar macrophages, AMs). We found that this virus inhibited the expression of IFN-γ-induced i-proteasome subunits LMP2, LMP7, and MECL-1 at the mRNA and protein level. In addition, expression levels of the i-proteasome regulatory subunits PSME1 and PSME2 in the HP-PRRSV HuN4-F5-infected group were also significantly decreased compared to those in the uninfected group. However, there was no significant difference in the expression of proteasome subunits PSMB5, PSMB6, and PSMB7 between HP-PRRSV HuN4-F5-infected and uninfected groups. This study provides insight into the mechanisms underlying immune regulation by HP-PRRSV; specifically, this virus affects the antigen-processing machinery by suppressing IFN-γ-induced i-proteasome expression in infected AMs.