Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Lineage 1 PRRSVs infection induces hemorrhagic injury in intestines of piglets: Effects on complement and coagulation cascades.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly transmissible disease of significant concern in the pig industry. Previous studies have demonstrated that the XM-2020 strain (a lineage 1.8 PRRSV IA/2012/NADC30) can induce special hemorrhagic injury in the small intestines. However, the specific mechanism underlying this injurious effect remains incompletely understood. In this study, we examined the pathogenic properties of XM-2020 and YC-2020 strains (a lineage 1.5 PRRSV IA/2014/NADC34) in piglets. Animal pathogenic tests revealed that with either Lineage 1 PRRSVs strains XM-2020 or YC-2020 demonstrated pronounced intestinal hemorrhage and suppression of peripheral immunological organs, comparing to JXA1 infection. Transcriptome analysis of diseased small intestines unveiled that PRRSV infection stimulated oxidative and inflammatory reactions. Remarkably, we also observed activation of the complement system alongside a notable down-regulation of complement and coagulation cascade pathways in the Lineage 1 PRRSVs infection group. Based on these findings, we propose that the primary mechanism driving the hemorrhagic injury of the small intestine caused by Lineage 1 PRRSVs is the suppression of complement and coagulation cascades resulting from immunosuppression. This discovery deepens our understanding of the pathogenicity of PRRSV in the small intestine and provides promising ways out for the development of innovative strategies aimed at controlling PRRSV.

Evolution Characterization and Pathogenicity of an NADC34-like PRRSV Isolated from Inner Mongolia, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that causes severe abortions in sows and high piglet mortality, resulting in huge economic losses to the pig industry worldwide. The emerging and novel PRRSV isolates are clinically and biologically important, as there are likely recombination and pathogenic differences among PRRSV genomes. Furthermore, the NADC34-like strain has become a major epidemic strain in some parts of China, but the characterization and pathogenicity of the latest strain in Inner Mongolia have not been reported in detail. In this study, an NADC34-like strain (CHNMGKL1-2304) from Tongliao City, Inner Mongolia was successfully isolated and characterized, and confirmed the pathogenicity in pigs. The phylogenetic tree showed that this strain belonged to sublineage 1.5 and had high homology with the strain JS2021NADC34. There is no recombination between CHNMGKL1-2304 and any other domestic strains. Animal experiments show that the CHNMGKL1-2304 strain is moderately virulent to piglets, which show persistent fever, weight loss and high morbidity but no mortality. The presence of PRRSV nucleic acids was detected in both blood, tissues, nasal and fecal swabs. In addition, obvious pathological changes and positive signals were observed in lung, lymph node, liver and spleen tissues when subjected to hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). This report can provide a basis for epidemiological investigations and subsequent studies of PRRSV.

Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication.

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. A growing number of studies have reported that autophagy participates in infection by a variety of viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe financial losses to the global swine industry. Although much research has shown that PRRSV triggers autophagy for its own benefits, the exact molecular mechanisms involved in PRRSV-triggered autophagy remain to be fully elucidated. In the current study, we demonstrated that PRRSV infection significantly induced Golgi apparatus (GA) fragmentation, which promoted autophagy to facilitate viral self-replication. Mechanistically, PRRSV nonstructural protein 2 was identified to interact with and degrade the Golgi reassembly and stacking protein 65 dependent on its papain-like cysteine protease 2 activity, resulting in GA fragmentation. Upon GA fragmentation, GA-resident Ras-like protein in brain 2 was disassociated from Golgi matrix protein 130 and subsequently bound to unc-51 like autophagy activating kinase 1 (ULK1), which enhanced phosphorylation of ULK1 and promoted autophagy. Taken together, all these results expand the knowledge of PRRSV-triggered autophagy as well as PRRSV pathogenesis to support novel potential avenues for prevention and control of the virus. More importantly, these results provide the detailed mechanism of GA fragmentation-mediated autophagy, deepening the understanding of autophagic processes.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious swine disease affecting pig farming worldwide. Despite that numerous studies have shown that PRRSV triggers autophagy for its self-replication, how PRRSV induces autophagy is incompletely understood. Here, we identify that PRRSV Nsp2 degrades GRASP65 to induce GA fragmentation, which dissociates RAB2 from GM130 and activates RAB2-ULK1-mediated autophagy to enhance viral replication. This work expands our understanding of PRRSV-induced autophagy and PRRSV replication, which is beneficial for anti-viral drug development.

Activation of T-bet, FOXP3, and EOMES in Target Organs From Piglets Infected With the Virulent PRRSV-1 Lena Strain.

Transcription factors (TFs) modulate genes involved in cell-type-specific proliferative and migratory properties, metabolic features, and effector functions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogen agents in the porcine industry; however, TFs have been poorly studied during the course of this disease. Therefore, we aimed to evaluate the expressions of the TFs T-bet, GATA3, FOXP3, and Eomesodermin (EOMES) in target organs (the lung, tracheobronchial lymph node, and thymus) and those of different effector cytokines (IFNG, TNFA, and IL10) and the Fas ligand (FASL) during the early phase of infection with PRRSV-1 strains of different virulence. Target organs from mock-, virulent Lena-, and low virulent 3249-infected animals humanely euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi) were collected to analyze the PRRSV viral load, histopathological lesions, and relative quantification through reverse transcription quantitative PCR (RT-qPCR) of the TFs and cytokines. Animals belonging to both infected groups, but mainly those infected with the virulent Lena strain, showed upregulation of the TFs T-bet, EOMES, and FOXP3, together with an increase of the cytokine IFN-γ in target organs at the end of the study (approximately 2 weeks post-infection). These results are suggestive of a stronger polarization to Th1 cells and regulatory T cells (Tregs), but also CD4+ cytotoxic T lymphocytes (CTLs), effector CD8+ T cells, and γδT cells in virulent PRRSV-1-infected animals; however, their biological functionality should be the object of further studies.

Inhibition of endocytosis of porcine reproductive and respiratory syndrome virus by rottlerin and its potential prophylactic administration in piglets.

Owing to several limitations of porcine reproductive and respiratory syndrome virus (PRRSV) control procedures, the importance of antiviral agents is increasing; however, limited studies have been done on the development of anti-PRRSV agents. Herein, we explored the antiviral effect and mechanism of rottlerin against PRRSV. We demonstrated that treatment of rottlerin at an early stage of PRRSV infection significantly inhibited the viral replication. PRRSV infection induced protein kinase C-δ phosphorylation, which was specifically downregulated by rottlerin. The treatment of rottlerin led to disrupting the PRRSV entry pathway by blocking endocytosis of the virions. Further, to evaluate the anti-PRRSV effect of the rottlerin in vivo, we administrated rottlerin loaded liposome to pigs infected with PRRSV LMY or FL12 strain. The treatment of rottlerin-liposome reduced the blood viral load, interstitial pneumonia and clinical scores compared to untreated pigs. These results provide an evidence of anti-PRRSV effect of rottlerin in vitro via inhibiting PRRSV internalization and in vivo, all of which strongly suggest the applicability of rottlerin as a potential PRRSV prophylactic treatment.

Fetal hypoxia and apoptosis following maternal porcine reproductive and respiratory syndrome virus (PRRSV) infection.

BACKGROUND: Mechanisms of fetal death following maternal PRRSV2 infection remain uncharacterized, although hypoxia from umbilical cord lesions and/or placental detachment due to apoptosis are hypothesized. We performed two experiments examining hypoxia and apoptosis in PRRSV-infected and non-infected, third-trimester fetuses to elucidate possible associations with fetal death. Fetuses were selected based on four phenotypic infection groups: fetuses from non-challenged control gilts (CTRL); low viral load fetuses (LVL; Exp 1) or uninfected fetuses (UNINF; Exp 2) from inoculated gilts; viable high viral load fetuses (HVL-VIA); and HVL meconium-stained fetuses (HVL-MEC). RESULTS: In experiment 1, paraffin embedded fetal tissues collected 21 days post maternal infection (DPI) were examined for DNA fragmentation associated with apoptosis. Positively stained foci were larger and more numerous (P < 0.05) in heart, liver, and thymus of HVL-VIA and HVL-MEC compared to CTRL and LVL fetuses. In experiment 2, group differences in gene expression within the hypoxia (HIF1a, IDO1, VEGFa, LDHA, NOS2, NOX1) and apoptosis (CASP3, CASP7, CASP8, CASP9, RIPK1, RIPK3) pathways were assessed by RT-qPCR in fetal tissues collected at 12 DPI. High viral load fetuses showed differential expression relative to the CTRL and UNINF (P < 0.05 for all). Brain tissue from HVL-VIA and HVL-MEC fetuses presented increased expression of CASP7, CASP8, RIPK3, HIF1a and IDO1. Fetal heart showed increased expression of CASP8, HIF1a, IDO and NOX1 and a decrease in NOS2 expression in infected groups. CASP7, CASP9, RIPK1 and RIPK3 were only increased in the heart of HVL-VIA while VEGFa was only increased for HVL-MEC fetuses. Thymus from HVL-MEC had decreased expression of CASP9 and there was increased IDO1 in all infected fetuses. CONCLUSIONS: There is strong evidence of apoptosis occurring in the heart, liver and thymus of highly viral load fetuses at 21 DPI. Furthermore, there was clear upregulation of apoptotic genes in the heart of high viral load infected fetuses and less prominent upregulation in the brain of PRRSV-infected fetuses, whereas thymus appears to be spared at 12 DPI. There was no strong evidence of hypoxia at 12 DPI in brain and thymus but some indication of hypoxia occurring in fetal heart.

NSP2 Is Important for Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus to Trigger High Fever-Related COX-2-PGE2 Pathway in Pigs.

In 2006, atypical porcine reproductive and respiratory syndrome (PRRS) caused by a highly pathogenic PRRSV (HP-PRRSV) strain broke out in China. Atypical PRRS is characterized by extremely high fever and high mortality in pigs of all ages. Prostaglandin E2 (PGE2) derived from arachidonic acid through the activation of the rate-limiting enzyme cyclooxygenase type 1/2 (COX-1/2) plays an important role in fever. Here, we showed that HP-PRRSV infection increased PGE2 production in microglia via COX-2 up-regulation depending on the activation of MEK1-ERK1/2-C/EBPß signaling pathways. Then, we screened HP-PRRSV proteins and demonstrated that HP-PRRSV nonstructural protein 2 (NSP2) activated MEK1-ERK1/2-C/EBPß signaling pathways by interacting with 14-3-3ζ to promote COX-2 expression, leading to PGE2 production. Furthermore, we identified that the amino acid residues 500-596 and 658-777 in HP-PRRSV NSP2 were essential to up-regulate COX-2 expression and PGE2 production. Finally, we made mutant HP-PRRS viruses with the deletion of residues 500-596 and/or 658-777, and found out that these viruses had impaired ability to up-regulate COX-2 and PGE2 production in vitro and in vivo. Importantly, pigs infected with the mutant viruses had relieved fever, clinical symptoms, and mortality. These data might help us understand the molecular mechanisms underlying the high fever and provide clues for the development of HP-PRRSV attenuated vaccines.

Immunity against a Japanese local strain of porcine reproductive and respiratory syndrome virus decreases viremia and symptoms of a highly pathogenic strain.

BACKGROUND: The type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has spread throughout countries of southeast Asia, where it has caused severe economic losses. Even countries presently free of PRRSV are at high risk for infection and spread of this virus. Some of these countries, including Japan, have broad epidemics of the local type 2 PRRSV, creating chronic pathogenicity in the domestic pig population. The present study aimed to evaluate the protective efficacy of immunity by infection with a Japanese field isolate, EDRD1, against heterologous challenge with a Vietnamese HP-PRRSV field strain. To this end, four groups of PRRSV-negative crossbreed piglets were used for a challenge study. Groups 1 and 2 were inoculated with EDRD1 via the intranasal route. After 26 days, Groups 2 and 3 were inoculated with HP-PRRSV via the same route. Group 4 served as an uninfected control. Blood and oral fluid samples were taken every 3-4 days after HP-PRRSV challenge; on day 16 post-challenge, all pigs were euthanized, and examined pathologically. RESULTS: The nucleotide sequence analysis of nonstructural protein 2 gene of EDRD1 and comparison with Vietnamese HP-PRRSV showed that the 39 amino acid deletion sites of EDRD1 was nearly in the same region as the 29 amino acid deletion sites of HP-PRRSV. Immunity conferred by inoculation with EDRD1 dramatically reduced viral load in the sera and tissues besides viral shedding (Group 2) compared with those in pigs infected only with HP-PRRSV (Group 3). The clinical signs and rectal temperature were significantly reduced, and the average daily weight gain was significantly improved in the EDRD1-inoculated pigs (Group 2) compared with the Group 3 pigs. Notably, no viral RNA was detected in various organs of the Group 2 pigs 16 days post-infection with HP-PRRSV, except in one pig. Therefore, the immunity induced by EDRD1 and its genetically close field isolates may play a role in reducing viremia caused by HP-PRRSV. CONCLUSIONS: The results of the present study demonstrate that pigs are highly protected against heterologous Vietnamese HP-PRRSV challenge by immunity against a Japanese local strain, EDRD1.

Palmitoylation of the envelope membrane proteins GP5 and M of porcine reproductive and respiratory syndrome virus is essential for virus growth.

Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped positive-strand RNA virus in the Arteiviridae family, is a major pathogen affecting pigs worldwide. The membrane (glyco)proteins GP5 and M form a disulfide-linked dimer, which is a major component of virions. GP5/M are required for virus budding, which occurs at membranes of the exocytic pathway. Both GP5 and M feature a short ectodomain, three transmembrane regions, and a long cytoplasmic tail, which contains three and two conserved cysteines, respectively, in close proximity to the transmembrane span. We report here that GP5 and M of PRRSV-1 and -2 strains are palmitoylated at the cysteines, regardless of whether the proteins are expressed individually or in PRRSV-infected cells. To completely prevent S-acylation, all cysteines in GP5 and M have to be exchanged. If individual cysteines in GP5 or M were substituted, palmitoylation was reduced, and some cysteines proved more important for efficient palmitoylation than others. Neither infectious virus nor genome-containing particles could be rescued if all three cysteines present in GP5 or both present in M were replaced in a PRRSV-2 strain, indicating that acylation is essential for virus growth. Viruses lacking one or two acylation sites in M or GP5 could be rescued but grew to significantly lower titers. GP5 and M lacking acylation sites form dimers and GP5 acquires Endo-H resistant carbohydrates in the Golgi apparatus suggesting that trafficking of the membrane proteins to budding sites is not disturbed. Likewise, GP5 lacking two acylation sites is efficiently incorporated into virus particles and these viruses exhibit no reduction in cell entry. We speculate that multiple fatty acids attached to GP5 and M in the endoplasmic reticulum are required for clustering of GP5/M dimers at Golgi membranes and constitute an essential prerequisite for virus assembly.

Dexamethasone enhances CD163 expression in porcine IPKM immortalized macrophages.

In our previous study, we established a unique porcine macrophage cell line, immortalized porcine kidney-derived macrophages (IPKM). The purpose of the present study was to further elucidate the characteristics of IPKM. CD163 is a scavenger receptor for the hemoglobin-haptoglobin complex and is used as a phenotypic marker of anti-inflammatory M2 macrophages. The expression of CD163 is enhanced by dexamethasone (DEX), a potent steroidal anti-inflammatory drug, in human and rodent macrophages in vitro. Therefore, we investigated the effects of DEX on CD163 expression in porcine IPKM. Treatment with DEX markedly enhanced CD163 expression in the IPKM. In addition, we found that SB203580, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), blocked the effects of DEX, suggesting that the p38 MAPK signaling pathway is involved in the regulation of the DEX-induced enhancement of CD163 expression. Since CD163 is considered to be a putative receptor for the porcine reproductive and respiratory syndrome virus (PRRSV), the effects of DEX on the infection of IPKM by PRRSV were evaluated. Although the IPKM were susceptible to infection by the Fostera PRRSV vaccine strain, DEX treatment did not affect the propagation of the virus in the IPKM. This suggests that the DEX-induced enhancement of CD163 expression alone is not sufficient to facilitate the infection of IPKM by PRRSV.

Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in neonatal pigs using different adjuvants.

Vaccination of neonatal pigs could be supportive to prevent porcine reproductive and respiratory syndrome virus (PRRSV), which is an important porcine pathogen causing worldwide welfare and health problems in pigs of different age classes. However, neonatal immunity substantially differs to adults, thus different vaccines may be required in neonateal pigs. We examined if the immunogenicity and efficacy of inactivated PRRSV (iPRRSV) vaccines in neonatal pigs could be improved with adjuvants containing oil-in water (O/W) emulsions with or without Toll-like receptor (TLR) agonists and by altering the delivery route from intramuscular (i.m.) to the skin. Three-day-old PRRSV-naïve piglets (n = 54, divided in 6 groups) received a prime vaccination and a booster vaccination four weeks later. The vaccine formulations consisted of different O/W emulsions (Montanide™ ISA28RVG (ISA28)), a squalene in water emulsion (SWE) for i.m. or a Stable Emulsion (SE) with squalene for skin vaccination) and/or a mixture of TLR1/2, 7/8 and 9 agonists (TLRa) combined with iPRRSV strain 07V063. These vaccines were delivered either i.m. (ISA28, SWE, TLRa or SWE + TLRa) or into the skin (skiSE + TLRa) with dissolving microneedle (DMN)-patches. All animals received a challenge with homologous PRRSV three weeks after booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, while lung pathology was studied at necropsy. After booster vaccination, only ISA28 induced a specific antibody response while a specific T-cell IFN-γ response was generated in the SWE group, that was lower for ISA28, and absent in the other groups. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the emulsion formulation, but not dependent on the presence of the TLRa or delivery route. Despite the measured immune responses none of the vaccines showed any efficacy. Further research focused on the early immune response in draining lymph nodes is needed to elucidate the potential of TLR agonists in vaccines for neonatal pigs.

Upregulation of HMGB1 secretion in lungs of pigs infected by highly pathogenic porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) remains a major driver for substantial economic losses to the swine industry across the world. Pulmonary inflammatory injury is a common manifestation in infected pigs. Previous studies reported that PRRS virus (PRRSV) induces secretion of high mobility group box 1 (HMGB1), a proinflammatory factor, in cultured cells. The objective of this study was to evaluate whether HMGB1 secretion is associated with PRRSV-induced pulmonary inflammatory responses in the early stage of infection in vivo. Three-week-old piglets were inoculated with either HuN4, a highly pathogenic PRRSV (HP-PRRSV) strain, or CH1R, an avirulent PRRSV vaccine strain. Necropsy was performed at 7 days post-infection. The results showed that HuN4 significantly induced the secretion of HMGB1 and inflammatory cytokines (IL-1ß, IL-6) into the bronchoalveolar lavage fluid (BALF). HuN4 infection induced severe interstitial pneumonia in the pigs. In contrast, pigs infected by CH1R had mild lung inflammation with minimal HMGB1 secretion. In addition, high viral load of HuN4 was detected in both pulmonary alveolar macrophages (PAMs) and lung tissue, whereas viral RNA of CH1R was confined to PAMs. In consistent with the pneumonia development, HuN4 induced inflammatory cytokines in both PAMs and lung tissue, while their expression in CH1R-infected pigs confined only to PAMs. These results indicate that the HuN4-induced HMGB1 secretion into BALF may enhance the pulmonary inflammatory response and exacerbate the lung injury. This finding provides insights to the inflammatory response and pathogenesis of the HP-PRRSV infection.

Transcriptional Immune Signatures of Alveolar Macrophages and the Impact of the NLRP3 Inflammasome on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Replication.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a contagious viral (PRRSV) disease in pigs characterized by poor reproductive health, increased mortality, and reductions in growth rates. PRRSV is known to implement immuno-antagonistic mechanisms to evade detection and mute host responses to infection. To better understand the cellular immunosignature of PRRSV we have undertaken transcriptome and immunomodulatory studies in PRRSV-infected porcine alveolar macrophages (PAMs). We first used genome-wide transcriptome profiling (RNA-seq) to elucidate PRRSV-induced changes in the PAM transcriptome in response to infection. We found a number of cellular networks were altered by PRRSV infection, including many associated with innate immunity, such as, the NLRP3 inflammasome. To further explore the role(s) of innate immune networks in PRRSV-infected PAMs, we used an NLRP3-specific inhibitor, MCC950, to identify the potential functionality of the inflammasome during PRRSV replication. We found that PRRSV does quickly induce expression of inflammasome-associated genes in PAMs. Treatment of PAMs with MCC950 suggests NLRP3 inflammasome activation negatively impacts viral replication. Treatment of PAMs with cell culture supernatants from macrophages subjected to NLRP3 inflammasome activation (via polyinosinic-polycytidylic acid (poly I:C) transfection), prior to PRRSV infection resulted in significantly reduced viral RNA levels compared to PAMs treated with cell culture supernatants from macrophages subjected to NLRP3 inflammasome inhibition (MCC950 treatment/poly I:C transfection). This further supports a role for NLRP3 inflammasome activation in the innate macrophagic anti-PRRSV immune response and suggests that PRRSV is sensitive to the effects of NLRP3 inflammasome activity. Taken together, these transcriptome and immunoregulatory data highlight the complex changes PRRSV infection induces in the molecular immune networks of its cellular host.

Histopathological Lesions Accompanied with First-Time Isolation of a PRRSV-2 Strain in Greece.

Genotype 2 strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) have been reported sporadically in Europe. Even if, PRRSV-2 reported to be genetically homogenous in Europe due to the introduction of an MLV vaccine strain, independent introductions of PRRSV-2 field strains have been reported. The aim of the present study was to report the complete genome sequence and evaluate the histopathological lesions of a PRRSV-2 strain, isolated for the first time in Greece. During a routine blood sampling in a commercial pig farm, the results revealed positive samples in weaners of 40-60 days for the PRRSV-2, using real-time polymerase chain reaction. The clinical picture was characterized from respiratory symptoms in weaners, as well as coughing and poor performance at finishing stage and less than 3% mortality rate from weaning stage to finishing stage. The use of ORF5 for PRRSV phylogenetic analysis of the isolated PRRSV strain, named "x1544-1 strain", was successfully determined, belonging to the genotype PRRSV-2. Comparison of the obtained sequence revealed nucleotide sequence identity >98% with PRRSV-2 strain VR2332 and other related strains from Denmark and China. The histopathological evaluation revealed diffuse interstitial pneumonia, multifocal interstitial nephritis, while in the lymphoid organs, follicular and paracortical hyperplasia, coexisting with necrosis and depletion of germ cells were detected. The results of current study undersign the importance for veterinary practitioners to have up-to-date access to phylogenetic data linked to phenotypic information to follow-up the control and prevention strategies against PRRSV.

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.

Matrine exhibits antiviral activity in a PRRSV/PCV2 co-infected mouse model.

BACKGROUND: PRRSV and PCV2 co-infection is very common in swine industry which results in huge economic losses worldwide. Although vaccination is used to prevent viral diseases, immunosuppression induced by PRRSV and PCV2 leads to vaccine failure. PURPOSE: Our previous results have demonstrated that Matrine possess antiviral activities against PRRSV/PCV2 co-infection in vitro. This study aims to establish a PRRSV/PCV2 co-infected KM mouse model and evaluate the antiviral activities of Matrine against PRRSV/PCV2 co-infection. STUDY DESIGN: A total of 144 KM mice were randomly divided into six groups with 24 mice in each group, named as: normal control, PRRSV/PCV2 co-infected group (PRRSV/PCV2 group), Ribavirin treatment positive control (Ribavirin control) and Matrine treatment groups (Matrine 40 mg/kg, Matrine 20 mg/kg and Matrine 10 mg/kg). METHODS: Except normal control group, all mice in other five groups were inoculated with PRRSV, followed by PCV2 at 2 h later. At 7 days post-infection (dpi), mice in the treatment groups were intraperitoneally administered with various doses of Matrine and Ribavirin, twice a day for 5 consecutive days. RESULTS: PRRSV N and PCV2 CAP genes were detected by PCR in multiple tissues including heart, liver, spleen, lungs, kidneys, thymus and inguinal lymph nodes. The viral load of PCV2 was the highest in liver followed by thymus and spleen. Although PRRSV were detected in most of tissues, but the replication of PRRSV was not significantly increased, as shown by qPCR analysis. Comparing with PCV2 infection alone, PRRSV infection significantly elevated PCV2 replication and exacerbated PCV2 induced interstitial pneumonia. qPCR analysis demonstrated 40 mg/kg Matrine significantly attenuated PCV2 replication in liver and alleviated virus induced interstitial pneumonia, suggesting Matrine could directly inhibit virus replication. In addition, Matrine treatment enhanced peritoneal macrophages phagocytosis at 13 and 16 dpi, and 40 mg/kg of Matrine increased the proliferation activity of lymphocytes. Body weight gain was continuously promoted by administrating Matrine at 10 mg/kg. CONCLUSION: Matrine possessed antiviral activities via inhibiting virus replication and regulating immune functions in mice co-infected by PRRSV/PCV2. These data provide new insight into controlling PRRSV and PCV2 infection and support further research for developing Matrine as a new possible veterinary medicine.

Novel lineage 1 recombinants of porcine reproductive and respiratory syndrome virus isolated from vaccinated herds: genome sequences and cytokine production profiles.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely disseminated, macrophage-tropic arterivirus that exhibits profound genetic and pathogenic heterogeneity. The present study was conducted to determine the complete genome sequences of two novel Korean lineage 1 PRRSV-2 strains, KNU-1901 and KNU-1902, which were isolated from vaccinated pig farms experiencing unusually high morbidity and mortality. Both isolates contained notable discontinuous 423-nucleotide deletions (DELs) within the genes encoding nonstructural protein 2 (nsp2) and GP3 when compared with the prototype strain VR-2332. In particular, the nsp2 DEL viruses had unique quadripartite discontinuous DEL signatures (111-1-19-9) in nsp2; this is an expanded version of the tripartite 111-1-19 DEL previously identified in virulent lineage 1 PRRSV-2 strains. Phylogenetic analysis revealed that both novel nsp2 DEL viruses belong to the Korean clade (KOR C) of lineage 1 isolates based on ORF5 but cluster with lineage KOR A strains based on the nsp2 or complete genome sequence. Recombination detection analysis suggested that both novel isolates are recombinants and may have evolved via natural inter-lineage recombination between circulating KOR A and KOR C strains. Interestingly, compared with the prototype VR-2332 virus, the novel nsp2 DEL variants were less efficient at promoting the expression of immune response genes in porcine alveolar macrophage culture. Taken together, we conclude that KNU-1901 and KNU-1902 are recently evolved recombinant variants of the virulent lineage 1 family that caused the regional severe PRRS outbreaks.

Pathogenicity of NADC34-like PRRSV HLJDZD32-1901 isolated in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a substantial economic loss to the swine industry. Recently, NADC34-like PRRSV was reported in the USA, China and Peru and consistently attributed to a large number of abortions in the clinic. In the USA, the pathogenicity of NADC34-like PRRSV in piglets is highly variable. However, the pathogenicity of NADC34-like PRRSV in China is unclear. In this study, an NADC34-like PRRSV strain, HLJDZD32-1901, was isolated in primary alveolar macrophage (PAM) cells from a sow blood sample collected from an abortive farm in China. HLJDZD32-1901, with no recombination, has a 100-aa deletion in the NSP2 protein corresponding to positions 328-427 in the VR2332 strain. Phylogenetic analysis based on open reading frame 5 (ORF5) indicated that HLJDZD32-1901 belongs to sublineage 1.5. Animal experiments showed that the weight loss of HLJDZD32-1901-infected piglets was significantly different from that of control piglets at 8-14 dpi. In addition, the challenge group had obvious histopathological lesions, including interstitial pneumonia and enlarged lymph nodes, and increased viremia and viral loads in three tissues. However, piglets in the challenge group had only mild clinical symptoms, with no death or fever. Our results showed that NADC34-like PRRSV HLJDZD32-1901 is a mildly pathogenic strain in piglets. However, we speculate that HLJDZD32-1901 may be a highly pathogenic strain in pregnant sows based on clinical morbidity.