RESUMO
For more than 3 decades, mounting evidence has associated porcine reproductive and respiratory syndrome virus (PRRSV) infection with late-term abortions and stillbirths in sows and respiratory disease in piglets, causing enormous economic losses to the global swine industry. However, to date, the underlying mechanisms of PRRSV-triggered cell death have not been well clarified, especially in the pulmonary inflammatory injury characterized by the massive release of pro-inflammatory factors. Here, we demonstrated that PRRSV infection triggered gasdermin D-mediated host pyroptosis in vitro and in vivo. Mechanistically, PRRSV infection triggered disassembly of the trans-Golgi network (TGN); the dispersed TGN then acted as a scaffold for NLRP3 activation through phosphatidylinositol-4-phosphate. In addition, PRRSV replication-transcription complex (RTC) formation stimulated TGN dispersion and pyroptotic cell death. Furthermore, our results indicated that TMEM41B, an endoplasmic reticulum (ER)-resident host protein, functioned as a crucial host factor in the formation of PRRSV RTC, which is surrounded by the intermediate filament network. Collectively, these findings uncover new insights into clinical features as previously unrecognized mechanisms for PRRSV-induced pathological effects, which may be conducive to providing treatment options for PRRSV-associated diseases and may be conserved during infection by other highly pathogenic viruses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the pathogens responsible for major economic losses in the global swine industry. Characterizing the detailed process by which PRRSV induces cell death pathways will help us better understand viral pathogenesis and provide implications for therapeutic intervention against PRRSV. Here, we showed that PRRSV infection induces GSDMD-driven host pyroptosis and IL-1ß secretion through NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in vitro and in vivo. Furthermore, the molecular mechanisms of PRRSV-induced NLRP3 inflammasome activation and pyroptosis are elucidated here. The dispersed trans-Golgi network (TGN) induced by PRRSV serves as a scaffold for NLRP3 aggregation into multiple puncta via phosphatidylinositol 4-phosphate (PtdIns4P). Moreover, the formation of PRRSV replication-transcription complex is essential for TGN dispersion and host pyroptosis. This research advances our understanding of the PRRSV-mediated inflammatory response and cell death pathways, paving the way for the development of effective treatments for PRRSV diseases.
Assuntos
Inflamassomos , Macrófagos Alveolares , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Proteínas Citotóxicas Formadoras de Poros , Piroptose , Animais , Feminino , Inflamassomos/metabolismo , Macrófagos Alveolares/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose/fisiologia , SuínosRESUMO
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.
Assuntos
Imunidade/genética , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Linfócitos T/imunologia , Animais , Perfilação da Expressão Gênica , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Análise de Sequência de RNA , Transdução de Sinais , Suínos , Transcriptoma , Regulação para CimaRESUMO
Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1ß, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.
Assuntos
Expressão Gênica , Células Caliciformes/virologia , Jejuno/virologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Proteínas de Junções Íntimas/genética , Replicação Viral , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Sus scrofa , Suínos , Proteínas de Junções Íntimas/metabolismoRESUMO
BACKGROUND: Interferon lambda receptor 1 (IFNLR1) is a type II cytokine receptor that clings to interleukins IL-28A, IL29B, and IL-29 referred to as type III IFNs (IFN-λs). IFN-λs act through the JAK-STAT signaling pathway to exert antiviral effects related to preventing and curing an infection. Although the immune function of IFN-λs in virus invasion has been described, the molecular mechanism of IFNLR1 in that process is unclear. OBJECTIVES: The purpose of this study was to elucidate the role of IFNLR1 in the pathogenesis and treatment of porcine reproductive and respiratory syndrome virus (PRRSV). METHODS: The effects of IFNLR1 on the proliferation of porcine alveolar macrophages (PAMs) during PRRSV infection were investigated using interference and overexpression methods. RESULTS: In this study, the expressions of the IFNLR1 gene in the liver, large intestine, small intestine, kidney, and lung tissues of Dapulian pigs were significantly higher than those in Landrace pigs. It was determined that porcine IFNLR1 overexpression suppresses PRRSV replication. The qRT-PCR results revealed that overexpression of IFNLR1 upregulated antiviral and IFN-stimulated genes. IFNLR1 overexpression inhibits the proliferation of PAMs and upregulation of p-STAT1. By contrast, knockdown of IFNLR1 expression promotes PAMs proliferation. The G0/G1 phase proportion in IFNLR1-overexpressing cells increased, and the opposite change was observed in IFNLR1-underexpressing cells. After inhibition of the JAK/STAT signaling pathway, the G2/M phase proportion in the IFNLR1-overexpressing cells showed a significant increasing trend. In conclusion, overexpression of IFNLR1 induces activation of the JAK/STAT pathway, thereby inhibiting the proliferation of PAMs infected with PRRSV. CONCLUSION: Expression of the IFNLR1 gene has an important regulatory role in PRRSV-infected PAMs, indicating it has potential as a molecular target in developing a new strategy for the treatment of PRRSV.
Assuntos
Macrófagos Alveolares/virologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Receptores de Interferon/metabolismo , Animais , Linhagem Celular , Macrófagos Alveolares/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Sus scrofa , SuínosRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. Pulmonary alveolar macrophages (PAMs) are the main cells supporting PRRSV replication, with CD163 as the essential receptor for viral infection. Although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for PRRSV virulent strains. This research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by PRRSV-1 strains of different virulence. Conventional pigs were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6 and 8 dpi. Lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of IL-6 and IFN-γ in sera compared to 3249-infected pigs. Extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in Lena-infected pigs. Lung viral load and PRRSV-N-protein+ cells were always higher in Lena-infected animals. PRRSV-N-protein+ cells were linked to a marked drop of CD163+ macrophages. The number of CD14+ and iNOS+ cells gradually increased along PRRSV-1 infection, being more evident in Lena-infected pigs. The frequency of CD200R1+ and FoxP3+ cells peaked late in both PRRSV-1 strains, with a strong correlation between CD200R1+ cells and lung injury in Lena-infected pigs. These results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response.
Assuntos
Broncopneumonia/imunologia , Inflamação/imunologia , Pulmão/imunologia , Pulmão/patologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Doença Aguda , Fatores Etários , Animais , Anticorpos Antivirais/sangue , Broncopneumonia/virologia , Citocinas/sangue , Feminino , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Masculino , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Suínos , Carga Viral , Viremia/imunologia , Viremia/patologia , VirulênciaRESUMO
To better understand the host response to porcine reproductive and respiratory virus-2 (PRRSV2) we evaluated circulating thyroid hormone and associated gene expression in a late gestation challenge model. Pregnant gilts were inoculated at gestation day 85 and fetal samples collected at either 12 or 21 days post-infection (dpi). A subset of fetuses was selected for analysis based on viability and viral load categorized as either uninfected-viable (UNIF), high viral load viable (HV-VIA) or high viral load meconium stained (HV-MEC) and were compared with gestational age matched controls (CON). In dams, circulating levels of total T3 and T4 decreased in the acute period following infection and rebounded by 21 dpi. A similar effect was observed in fetuses, but was largely restricted to HV-VIA and HV-MEC, with minimal decrease noted in UNIF relative to CON at 21 dpi. Gene expression in fetal heart at 12 dpi showed significant decompensatory transcription of thyroid hormone transporters (SLC16A2) and deiodinases (DIO2, DIO3), which was not observed in brain. Correspondingly, genes associated with cell cycle progression (CDK1,2,4) were downregulated in only the heart of highly infected fetuses, while expression of their inhibitor (CDKN1A) was upregulated in both tissues. Finally, expression of genes associated with cardiac stress including CAMKD and AGT were upregulated in the hearts of highly infected fetuses, and a shift in expression of MYH6 to MYH7 was observed in HV-MEC fetuses specifically. Collectively, the results suggest PRRSV2 infection causes a hypothyroid state that disproportionally impacts the fetal heart over the brain.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Glândula Tireoide/fisiologia , Animais , Feminino , Doenças Fetais/fisiopatologia , Doenças Fetais/veterinária , Doenças Fetais/virologia , Exposição Materna , Síndrome Respiratória e Reprodutiva Suína/virologia , SuínosRESUMO
Previous studies have demonstrated evidence for a relationship between the vaginal microbiome and reproductive performance, suggesting the vaginal microbiota may serve as a tool to predict farrowing outcomes in commercial pigs. In this study, we compared the vaginal microbiome in sows with low and high farrowing performance and used it to classify animals with contrasting reproductive outcomes in commercial sows following immune challenge with porcine respiratory and reproductive syndrome (PRRS) vaccination. Eighteen microbes were differentially abundant (q-value < 0.05) between the Low and High farrowing performance groups. Among them, Campylobacter, Bacteroides, Porphyromonas, Lachnospiraceae unclassified, Prevotella, and Phascolarctobacterium were also selected in the discriminant and linear regression analyses, and could be used as potential biomarkers for reproductive outcomes. The correct classification rate in the two groups was 100%. In conclusion, this study demonstrates that vaginal microbiota collected after PRRS vaccination could be potentially used to classify sows into having low or high farrowing performance in commercial herds.
Assuntos
Microbiota , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Reprodução , Suínos/virologia , Vacinação/veterinária , Vagina/microbiologia , Animais , Bactérias/classificação , Biodiversidade , Feminino , Modelos Lineares , Filogenia , Suínos/microbiologiaRESUMO
Pathogen challenges are often accompanied by reductions in feed intake, making it difficult to differentiate impacts of reduced feed intake from impacts of pathogen on various response parameters. Therefore, the objective of this study was to determine the impact of Porcine Reproductive and Respiratory Syndrome virus (PRRSV) and feed intake on parameters of jejunal function and integrity in growing pigs. Twenty-four pigs (11.34 ± 1.54 kg BW) were randomly selected and allotted to 1 of 3 treatments (n = 8 pigs/treatment): 1) PRRSV naïve, ad libitum fed (Ad), 2) PRRSV-inoculated, ad libitum fed (PRRS+), and 3) PRRSV naïve, pair-fed to the PRRS+ pigs' daily feed intake (PF). At 17 days post inoculation, all pigs were euthanized and the jejunum was collected for analysis. At days post inoculation 17, PRRS+ and PF pigs had decreased (P < 0.05) transepithelial resistance compared with Ad pigs; whereas fluorescein isothiocyanate-dextran 4 kDa permeability was not different among treatments. Active glucose transport was increased (P < 0.05) in PRRS+ and PF pigs compared with Ad pigs. Brush border carbohydrase activity was reduced in PRRS+ pigs compared with PF pigs for lactase (55%; P = 0.015), sucrase (37%; P = 0.002), and maltase (30%; P = 0.015). For all three carbohydrases, Ad pigs had activities intermediate that of PRRS+ and PF pigs. The mRNA abundance of the tight junction proteins claudin 2, claudin 3, claudin 4, occludin, and zonula occludens-1 were reduced in PRRS+ pigs compared with Ad pigs; however, neither the total protein abundance nor the cellular compartmentalization of these tight junction proteins differed among treatments. Taken together, this study demonstrates that the changes that occur to intestinal epithelium structure, function, and integrity during a systemic PRRSV challenge can be partially explained by reductions in feed intake. Further, long term adaptation to PRRSV challenge and caloric restriction does reduce intestinal transepithelial resistance but does not appear to reduce the integrity of tight junction protein complexes.
Assuntos
Ingestão de Alimentos/fisiologia , Mucosa Intestinal/fisiopatologia , Jejuno/fisiopatologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Animais , Restrição Calórica , Interações Hospedeiro-Patógeno/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Jejuno/citologia , Jejuno/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Sus scrofa , Suínos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismoRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses to the pig industry worldwide over the last 30 years, yet the associated viral-host interactions remain poorly understood. S100A9 is a damage-associated molecular pattern of the S100 protein family. Here, we found that PRRSV infection stimulated S100A9 expression in porcine alveolar macrophages (PAMs) and Marc-145 cells. S100A9 inhibited PRRSV replication via cellular Ca2+ dependent manner. The viral nucleocapsid (N) protein co-localized with S100A9 in the cytoplasm, and directly interacted at amino acid 78 of S100A9 and amino acids 36-37 of N protein. Moreover, we also found that the mutant S100A9 (E78Q) protein exhibited decreased antiviral activity against PRRSV compared with the parent S100A9. Recombinant PRRSV rBB (36/37) with two mutations in amino acid 36-37 in the N protein exhibited greater replication than the parent PRRSV BB0907 in S100A9-overexpressed PAM and Marc-145 cells. Thus, S100A9 may restrict PRRSV proliferation by interacting with the viral N protein.
Assuntos
Calgranulina B/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Mutação , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Proteínas Recombinantes/metabolismo , Suínos , Replicação Viral/fisiologiaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is known to be clinically responsible for reproductive failure in sows and post-weaning respiratory disease in growing piglets. During the last years, highly pathogenic PRRSV isolates have been discovered. In Italy, a PRRSV-1 subtype 1 strain (namely PR40/2014) characterized by high pathogenicity was isolated and experimental infection was characterized in terms of virological/clinical features and immune modulation (Canelli et al., 2017; Ferrari et al., 2018). The present study was performed in 4-week-old pigs experimentally infected with the highly pathogenic PRRSV1_PR40/2014 (HP-PR40) or with the conventional PRRSV1_PR11/2014 (PR11). The aim was to evaluate the interrelation between plasmatic hormones and cytokines in infected pigs compared to uninfected controls in order to address potential effects on the course of an experimental infection. The time-related changes of growth hormone (GH), insulin-like growth factor-1 (IGF-1), adiponectin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels appear to be modulated by the infection depending on the PRRSV isolate (HP-PR40 vs. PR11). In particular, in HP-PR40 infected animals, the association between high GH levels and viremia may testify the need to block the anabolic action of GH in order to shift available energy towards the immune response. This need appeared to be delayed in PR11 animals, given the lower pathogenicity of the isolate. Adiponectin, IL-6 and TNF-α course supports the hypothesis of GH resistance mechanisms to guarantee homeostasis in HP-PR40 animals and underlines the key role of energy availability in events leading to an effective response to the virus.
Assuntos
Citocinas/sangue , Hormônios/sangue , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Feminino , Masculino , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , SuínosRESUMO
The phospholipase C (PLC) is a family of kinases that hydrolyze phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to generate two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which stimulate distinct downstream signaling. Recently, it has been reported that PLC signaling is activated by multiple viruses for efficient replication and the virus-induced inflammatory response. In this study, we demonstrated that PLC-specific inhibitor U73122 strongly suppressed porcine reproductive and respiratory syndrome virus (PRRSV) productive infection in cell cultures. The inhibitor affected both viral post-binding cell entry and post-entry processes. The virus infection led to an early transient activation of PLCγ-1 at 0.5 h post-infection (hpi), and sustained event at a stage from 4 to 16 hpi in MARC-145 cells. In addition, U73122 inhibited the activation of p38 MAPK signaling stimulated by PRRSV infection, suggesting that PLC signaling may be associated with the virus infection-induced inflammatory response. Taken together, these studies suggested that PLC signaling played an important role in PRRSV infection or pathogenesis. Keywords: PRRSV; U73122; phospholipase C; PLCγ-1.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Fosfolipases Tipo C , Animais , Linhagem Celular , Estrenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Pirrolidinonas/farmacologia , Transdução de Sinais , Suínos , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/fisiologia , Internalização do Vírus/efeitos dos fármacosRESUMO
BACKGROUND: The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has been responsible for several viral attacks in the Asian porcine industry, since the first outbreak in China in 2006. During the early stages of the HP-PRRSV infection, high levels of proinflammatory cytokines are noted in the host peripheral blood, which are accompanied by severe lesions in the lungs and immune system organs; these are considered as the greatest contributors to the overall disease burden. We hypothesized that the anti-PRRSV response in piglets might be mediated by the hypothalamus-pituitary-adrenal (HPA) axis, which led to a decrease in the psycho-neuroendocrinological manifestation of HP-PRRSV etiology via immune response regulation. RESULTS: We investigated the regulation of the HPA axis in HP-PRRSV-infected piglets that were treated with 1 mg/kg body weight (b. w.)/day mifepristone (RU486) or 2 mg/kg b.w./day dexamethasone (DEX). Both RU486 and DEX enhanced the disease status of the piglets infected by the HP-PRRSV HuN4 strain, resulting in high mortality and more severe pathological changes in the lungs. CONCLUSIONS: HP-PRRSV infection activates the HPA axis, and artificial regulation of the immune-endocrine system enhances disease severity in HP-PRRSV-infected piglets. Thus, DEX and RU486 should be avoided in the clinical treatment of HP-PRRS.
Assuntos
Sistema Hipotálamo-Hipofisário/fisiologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Animais Recém-Nascidos/imunologia , Citocinas/sangue , Feminino , Masculino , Sistema Hipófise-Suprarrenal/fisiologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Suínos/imunologia , Carga Viral/veterináriaRESUMO
Highly contagious and emerging diseases cause significant losses in the pig producing industry worldwide. Rapid and exact acquisition of real-time data, like body temperature and animal movement from the production facilities would enable early disease detection and facilitate adequate response. In this study, carried out within the European Union research project RAPIDIA FIELD, we tested an online monitoring system on pigs experimentally infected with the East European subtype 3 Porcine Reproductive & Respiratory Syndrome Virus (PRRSV) strain Lena. We linked data from different body temperature measurement methods and the real-time movement of the pigs. The results showed a negative correlation between body temperature and movement of the animals. The correlation was similar with both body temperature obtaining methods, rectal and thermal sensing microchip, suggesting some advantages of body temperature measurement with transponders compared with invasive and laborious rectal measuring. We also found a significant difference between motion values before and after the challenge with a virulent PRRSV strain. The decrease in motion values was noticeable before any clinical sign was recorded. Based on our results the online monitoring system could represent a practical tool in registering early warning signs of health status alterations, both in experimental and commercial production settings.
Assuntos
Monitorização Fisiológica/veterinária , Sistemas On-Line , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Criação de Animais Domésticos , Animais , Temperatura Corporal , Monitorização Fisiológica/instrumentação , Monitorização Fisiológica/métodos , Movimento , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , SuínosRESUMO
In recent years, several entry mediators have been characterized for porcine reproductive and respiratory syndrome virus (PRRSV). Porcine sialoadhesin [pSn, also known as sialic acid-binding immunoglobulin-type lectin (Siglec-1)] and porcine CD163 (pCD163) have been identified as the most important host entry mediators that can fully coordinate PRRSV infection into macrophages. However, recent isolates have not only shown a tropism for sialoadhesin-positive cells, but also for sialoadhesin-negative cells. This observation might be partly explained by the existence of additional receptors that can support PRRSV binding and entry. In the search for new receptors, recently identified porcine Siglecs (Siglec-3, Siglec-5 and Siglec-10), members of the same family as sialoadhesin, were cloned and characterized. Only Siglec-10 was able to significantly improve PRRSV infection and production in a CD163-transfected cell line. Compared with sialoadhesin, Siglec-10 performed equally effectively as a receptor for PRRSV type 2 strain MN-184, but it was less capable of supporting infection with PRRSV type 1 strain LV (Lelystad virus). Siglec-10 was demonstrated to be involved in the endocytosis of PRRSV, confirming the important role of Siglec-10 in the entry process of PRRSV. In conclusion, it can be stated that PRRSV may use several Siglecs to enter macrophages, which may explain the strain differences in the pathogenesis.
Assuntos
Clonagem Molecular , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Receptores Virais/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Endocitose , Dados de Sequência Molecular , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Virais/química , Receptores Virais/genética , Alinhamento de Sequência , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/química , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , SuínosRESUMO
A large challenge experiment using North American porcine reproductive and respiratory virus (PRRSV-2) provided new insights into the pathophysiology of reproductive PRRS. Deep phenotyping of dams and fetuses identified maternal and fetal predictors of PRRS severity and resilience. PRRSV infection resulted in dramatic decreases in all leukocyte subsets by 2days post inoculation. Apoptosis in the interface region was positively related to endometrial vasculitis, viral load in endometrium and fetal thymus, and odds of meconium staining. Viral load at the maternal-fetal interface was a strong predictor of viral load in fetal thymus and odds of fetal death. However, interferon-alpha suppression, a consequence of PRRSV infection, was protective against fetal death. Although the prevalence of fetal lesions was low, their presence in fetal organs and umbilical cord was strongly associated with fetal compromise. Fetal death and viral load clustered in litters suggesting inter-fetal transmission starting from a limited number of index fetuses. Factors associated with index fetal infection are unclear, but large fetuses appear at greater risk. Disease progression in fetuses was associated with an up-regulation of genes associated with inflammation, innate immunity, and cell death signaling, and down-regulation of genes associated with cell cycle and lymphocyte quality. A number of maternal transcriptomic responses were associated with PRRS resilience including higher basal gene expression correlated with platelet function, interferon and pro-inflammatory responses. Twenty-one genomic regions across 10 chromosomes were associated with important traits including fetal viral load, fetal death and viability suggesting that selection for reproductive PRRS resilience may be possible.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Animais , Genoma/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/mortalidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Seleção Genética , Suínos/genéticaRESUMO
CD169 and CD163 have been involved in the process of PRRS virus attachment and infection in macrophages, although recent studies have challenged the requirement for CD169. In addition to CD169, macrophages express other siglecs, whose role in PRRS virus infection is so far unknown. Splenic CD163+ macrophages express Siglec-3 and Siglec-5 but almost undetectable levels of CD169. Hence, we considered this cell population appropriate for analysing the role of these siglecs in the attachment and internalization of PRRS virus into macrophages. PRRS virus replicated efficiently in these macrophages, yielding even higher titres than in alveolar macrophages. Besides, a recombinant protein consisting in the ectodomain of porcine Siglec-3 fused to the Fc fragment of human IgG1 (Siglec3-Fc) was able to bind PRRS virus, while binding to Siglec-5-Fc was inconsistent. Antibodies to CD169 but not to Siglec-3 or Siglec-5 blocked the binding and infection of PRRS virus on alveolar macrophages. Unexpectedly, our antibody to CD169 also blocked the binding of PRRS virus to splenic CD163+ macrophages, whereas antibodies to Siglec-3 or Siglec-5 had no effect. These results show that very low levels of CD169 expression are enough to support the attachment and internalization of PRRS virus into macrophages, whereas Siglec-3 and Siglec-5 do not seem to contribute to the virus entry in these cells.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Receptores de Superfície Celular/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Anticorpos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Ligação Proteica , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Suínos , Ligação Viral , Internalização do Vírus , Replicação ViralRESUMO
Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is activated by myriad cytokines, which are involved in regulation of cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. Because of its significance in immune response, JAK-STAT pathway is often targeted by pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes reproductive failure in sows and respiratory disease in pigs of all ages. A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response. One of the possible reasons for the weak protective immune response is that PRRSV interferes with cytokine-mediated JAK-STAT signaling. PRRSV inhibits interferon-activated JAK-STAT signaling by blocking nuclear translocation of STAT1 and STAT2. The mechanism is that PRRSV non-structural protein 1ß (nsp1ß) induces degradation of karyopherin α1 (KPNA1), a critical adaptor in nucleo-cytoplasmic transport. PRRSV also antagonizes IL6-activated JAK-STAT3 signaling via inducing degradation of STAT3. In this review, we briefly introduce JAK-STAT signaling, summarize the PRRSV interference with it, and provide perspective on the perturbation in the context of PRRSV-elicited immune response.
Assuntos
Citocinas/metabolismo , Janus Quinases/metabolismo , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , SuínosRESUMO
Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) was first characterized in 2006 in China, and it causes great economic losses to the Chinese swine production industry. A China Landrace pig, the Tibetan pig, which has striking phenotypic and physiological differences from lowland pigs, is mainly distributed in the Tibetan highlands of China. The susceptibility of the Tibetan pig to HP-PRRSV has not been reported. In this study, 15 4-week-old Tibetan piglets were divided into three groups, and their susceptibility to HP-PRRSV was examined in the highland region. Five pigs in group 1 were inoculated intranasally with HP-PRRSV strain BB0907. At 2days post-inoculation, five other pigs were introduced into this group and then removed to a separated room to serve as contact group 2. Meanwhile, five pigs in group 3 were mock infected and used as controls. The results showed that the pigs in the inoculated and contact groups showed high fevers and clear clinical signs, including depression, anorexia, lethargy, sticky eye secretions, and hind limb paralysis, with high mortality. The main symptom was interstitial pneumonia. Viremia appeared on days 4 to 14 post-infection. HP-PRRSV infection resulted in inflammatory responses within the first week of infection, as evidenced by the expression of tumor necrosis factor alpha, interleukin (IL)-1ß, IL-6, and IL-10. All the data indicate that the Tibetan pig is susceptible to HP-PRRSV infection. Thus, it is necessary to investigate and prevent PRRSV infections in the highland region in China.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Doenças dos Suínos/virologia , Administração Intranasal/veterinária , Animais , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Suínos , Doenças dos Suínos/fisiopatologia , Tibet , Viremia/fisiopatologia , Viremia/veterinária , Viremia/virologiaRESUMO
The causative agent of porcine reproductive and respiratory syndrome is the PRRS virus (PRRSV), an enveloped, single-stranded and positive-sense RNA virus. The host factors and mechanisms that are involved in PRRSV entry are still largely unknown. In our present studies, we found that syndecan-4, one of the heparan sulfate proteoglycans, plays a critical role in PRRSV entry, especially in PRRSV attachment. Moreover, EGFR interacts with syndecan-4 in MACR-145 cells and disruption of their interaction impaired PRRSV entry. Furthermore, EGFR inhibitor AG1478 or syndecan-4 derived peptide SSTN87-131 inhibited syndecan-4 endocytosis induced by PRRSV entry. Altogether, syndecan-4, a PRRSV attachment factor, mediated PRRSV entry by interacting with EGFR.
Assuntos
Receptores ErbB/metabolismo , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Suínos/virologia , Sindecana-4/metabolismo , Animais , Linhagem Celular , Endocitose , Interações Hospedeiro-Patógeno , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Mapas de Interação de Proteínas , Suínos/metabolismo , Ligação Viral , Internalização do VírusRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) exists as two distinct viruses, type 1 (PRRSV-1) and type 2 (PRRSV-2). Atrophy of the thymus in PRRSV-2 infected piglets has been associated with a loss of thymocytes. The present study aimed to evaluate the impact of PRRSV-1 strains of differing virulence on the thymus of infected piglets by analysing the histomorphometry, the presence of apoptotic cells and cells producing cytokines. Thymic samples were taken from animals experimentally infected (with LV, SU1-bel, and 215-06 strains) or mock inoculated animals at 3, 7 and 35days post-infection (dpi) and processed for histopathological and immunohistochemical analyses. PRRSV antigen was detected in the thymus from 3dpi until the end of the study in all virus-infected animals with the highest numbers of infected cells detected in SU1-bel group. The histomorphometry analysis and counts of CD3(+) thymocytes in the thymic cortex displayed significant differences between strains at different time-points (p≤0.011), with SU1-bel group showing the most severe changes at 7dpi. Cell death displayed statistically significant increase in the cortex of all infected animals, with SU1-bel group showing the highest rate at 3 and 7dpi. The number of cells immunostained against IL-1α, TNF-α and IL-10 were predominantly detected in the medulla (p≤0.01). An increase in the number of TNF-α and IL-10 positive cells was observed in LV and SU-1bel groups. Our results demonstrate that different PRRSV-1 strains induced depletion of the thymic cortex due to apoptosis of thymocytes and that the most severe depletion was associated with the highly virulent SU1-bel strain.