Porcine Alveolar Macrophage Cells Research Articles

Antiviral Activity of Plant-Based Additives Against African Swine Fever Virus (ASFV) in Feed Ingredients.

African swine fever (ASF) is one of the deadliest swine diseases with haemorrhagic symptoms and a high mortality rate. Plant-derived additives are potential antiviral agents against viruses due to their environmental and user-friendly properties. This study aims to evaluate the efficacy of plant-based additives (Phyto.A04 and Phyto.B) compared to an organic acid blend (OAB) in inactivating ASF virus (ASFV) in cell culture and feed. ASFV-spiked feed was treated with individual or combined additives such as OAB, Phyto.A04 and Phyto.B. The viability of ASFV after treatment of ASFV-spiked feed with additives was then confirmed by both methods, real-time PCR and cell culture. The results of the in vitro test with cell cultures showed that all three additives (OAB, Phyto.A04 and Phyto.B) exerted a strong virucidal effect on ASFV in porcine alveolar macrophage cells. OAB at a concentration of 0.3% reduced the virus concentration from 4.48 log10 HAD50/mL after 1 day of treatment (day 1) to 3.29 log10 HAD50/mL after 3 days of treatment (day 3) and remained undetected after 7 days of treatment (day 7). In Phyto.A04 with 1%, the virus was only detectable on day 1 (3.53 log10 HAD50/mL). Phyto.B with 0.01% and 0.05% both showed good efficacy in completely inhibiting virus presence on days 3 and 7. All additives, OAB, Phyto.A04 and Phyto.B, were able to inactivate ASFV in a dose-dependent manner, as confirmed by cell culture and PCR methods. The combination of additives at different concentrations consistently improved the virucidal results.

A Non-Hemadsorbing Live-Attenuated Virus Vaccine Candidate Protects Pigs against the Contemporary Pandemic Genotype II African Swine Fever Virus.

African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to a 100% mortality rate, which leads to a tremendous socio-economic loss worldwide. The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF. In this study, we generated a safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 by serially passaging a virulent genotype II strain (VNUA-ASFV-L2) in an immortalized porcine alveolar macrophage cell line (3D4/21, 50 passages). VNUA-ASFV-LAVL3 lost its hemadsorption ability but maintained comparable growth kinetics in 3D4/21 cells to that of the parental strain. Notably, it exhibited significant attenuation of virulence in pigs across different doses (103, 104, and 105 TCID50). All vaccinated pigs remained healthy with no clinical signs of African swine fever virus (ASFV) infection throughout the 28-day observation period of immunization. VNUA-ASFV-LAVL3 was efficiently cleared from the blood at 14-17 days post-infection, even at the highest dose (105 TCID50). Importantly, the attenuation observed in vivo did not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicited robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (103, 104, and 105 TCID50). Furthermore, a single vaccination (104 TCID50) provided protection for up to 2 months. These findings suggest that VNUA-ASFV-LAVL3 can be utilized as a promising safe and efficacious LAV candidate against the contemporary pandemic genotype II ASFV.

Effects of Isochlorogenic Acid A on Mitochondrial Dynamics Imbalance and RLR Damage in PAM Cells Induced by Combined Mycotoxins

Mycotoxins have strong immunotoxicity and can induce oxidative stress and mitochondrial dynamics imbalance. Mitochondrial antiviral signaling protein (MAVS) in the RIG-I like receptor (RLR) pathway of innate immunity is located on mitochondria, and whether it is affected by mycotoxins has not been reported yet. This experiment used porcine alveolar macrophages (PAM) to evaluate the antagonism of three isomers of chlorogenic acid (chlorogenic acid, isochlorogenic acid A, and neochlorogenic acid) against combined mycotoxins (Aflatoxin B1, Deoxynivalenol, and Ochratoxin A) induced mitochondrial damage and their effects on the RLR pathway, providing assistance for further elucidating the mechanism of mycotoxin immunotoxicity. Western blotting, enzyme linked immunosorbent assay (ELISA), and flow cytometry were used to detect relevant indicators. All three types of chlorogenic acid treatment can antagonize the cytotoxicity induced by combined mycotoxins, especially isochlorogenic acid A, which can protect cells from mycotoxins damage by maintaining mitochondrial dynamic homeostasis and improving innate immune function related to the RLR pathway.

Acquisition of different transcriptional shear mRNA and biological function of porcine interleukin 18 binding protein in PRRSV infection.

Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

208 The antiviral and immunomodulating properties from glycerides of lauric acid in PRRSV-infected PAM cells

Abstract The objective of this study was to assess the ability of a commercially available blend of glycerides of C12 to inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in porcine alveolar macrophages (PAM) and to investigate the immunomodulatory effect during PRRSV infection in vitro. PAM were collected by lung lavage from 4-wk-old piglets, free of PRRSV. PRRSV strain 13V091 was used in this study and a commercially available mixture of mono-, di- and tri-glycerides of lauric acid (GC12). The product concentrations tested were 0.022% (T1), 0.044% (T2) and 0.088% (T3). Three different infection assays were used: (1) Co-incubation; PRRSV was inoculated together with GC12 for 1 h, (2) Pre- and Post-infection; GC12 was added to PAM cells for 24 h before infection and after 1 h of incubation for another 24 h, and (3) Post-infection; GC12 was added only after PRRSV infection for 24 h. Titration of the progeny virus was performed on PAM from all three infection assays. Cytokine analysis was only performed for infection assay 2 and 3. Collected supernatants were subjected to multiplex ELISA to measure the level of TNF-α and IL-8. Two-tailed t test P value was used to determine statistically significant differences. P < 0.05 was considered to be statistically significant. Addition of GC12 in the Co-incubation assay did not reduce PRRSV infectivity at 24-h post infection (hpi; Table 1). In contrast, the Pre- and Post-infection assay showed significantly reduced PRRSV titer from 6 hpi onwards at concentration 0.044%. At concentration 0.088% a significant reduction was observed at 24 hpi. Addition of GC12 after PRRSV inoculation (post-infection) also showed significantly reduced PRRSV titer from 6 hpi onwards at concentration 0.044% and 0.088%. In the Pre- and Post-infection assay, all treatments with GC12 numerically increased the production of TNF-α at 24 hpi. A dose response effect was observed at 24 hpi with increasing concentrations of GC12 resulting in increasing levels of TNF-α. However, in the Post-infection assay only treatment with 0.088% GC12 numerically increased the production of TNF-α by 1.5-fold. Compared with the control (PAM+PRRSV), a significantly greater concentration of IL-8 was observed with 0.044% (P = 0.019) and 0.088% (P = 0.007) GC12 24 hpi in the Pre- and Post-infection assay (1,806 ± 376; 3,751 ± 636; 3,439 ± 420 pg/mL respectively). These two concentrations resulted in numeric increase of IL-8 24 hpi in the Post-infection assay and no effect was observed with 0.022% GC12 (Control 2,332 ± 998; T1 2,079 ± 964; T2 3,525 ± 344; T3 4,475 ± 295). From this study it can be concluded that GC12 exhibited antiviral activity against PRRSV strain 13V091 and influences the cytokine production of PAM during PRRSV infection, potentially neutralizing the suppression of the immune response by PRRSV.

Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-β. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Long non-coding RNA LOC103222771 promotes infection of porcine reproductive and respiratory syndrome virus in Marc-145 cells by downregulating Claudin-4

Porcine reproductive and respiratory syndrome (PRRS) is an important swine disease caused by infection of porcine reproductive and respiratory syndrome virus (PRRSV), which leads to huge loss in swine industry. How to effectively control PRRS is challenging. Long non-coding RNA (lncRNA) are key regulator of viral infections and anti-virus immunological responses, therefore, further understanding of lncRNAs will aid to identification of novel regulators of viral infections and better design of prevention and control strategies to viral infection related diseases and immune disorders. We demonstrated that PRRSV infection upregulated the expression of lncRNA LOC103222771 in Marc-145 cells and porcine alveolar macrophage cells (PAMs) and that LOC103222771 is mainly located in cytoplasm. Knockdown of LOC103222771 could inhibit the PRRSV infection in Marc-145 cells. RNA-seq analysis and subsequent validation revealed increased expression of Claudin-4 (CLDN4) in Marc-145 when LOC103222771 was specifically downregulated，suggesting that LOC103222771 might be an upstream regulator of CLDN4, an important component of tight junctions for establishment of the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. We and others showed that Downregulation of CLDN4 could boost the infection of PRRSV. Collectively, LOC103222771/CLDN4 signal axis might be a novel mechanism of PRRSV pathogenesis, implying a potential therapeutic target against PRRSV infection.

A Cell-Adapted Live-Attenuated Vaccine Candidate Protects Pigs against the Homologous Strain VNUA-ASFV-05L1, a Representative Strain of the Contemporary Pandemic African Swine Fever Virus.

African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. Currently, there is no ASF vaccine commercially available. All infected animals must be isolated and culled immediately upon the confirmation of the presence of the virus. Studies leading to the rational development of protective ASF vaccines are urgently needed. Here, we generated a safe and efficacious live-attenuated vaccine (LAV) VNUA-ASFV-LAVL2 by serially passaging a field isolate (VNUA-ASFV-05L1, genotype II) in porcine alveolar macrophages (PAMs, 65 passages) and an immortalized porcine alveolar macrophage cell line (3D4/21, 55 passages). VNUA-ASFV-LAVL2 can efficiently replicate in both PAMs and 3D4/21 cells. It provides 100% protection, even with the low dose of 102 HAD50, to the vaccinated pigs against the challenge of contemporary pandemic ASFV field isolate. Pigs vaccinated with this LAV in a dose range of 102 to 105 HAD50 remained clinically healthy during both the 28-day observation period of immunization and the 28-day observation period of challenge. VNUA-ASFV-LAVL2 was eliminated from blood by 28 days post-inoculation (DPI), and from feces or oral fluids by 17 DPI. Although the vaccine strain in serum remained a safe and attenuated phenotype after five passages in swine, a reversion-to-virulence study using blood or tissue homogenates at peak viremia will be conducted in the future. ASFV-specific IgG antibodies and significant cellular immunity were detected in vaccinated pigs before the ASFV challenge. These results indicate that the VNUA-ASFV-LAVL2 strain is a safe and efficacious LAV against the genotype II ASFV strain responsible for current ASF outbreaks in Asia.

Mitigating the adverse effects of Aflatoxin B1 in LMH, IPEC-J2 and 3D4/21cells by a novel integrated agent.

This study was to evaluate the efficacy of TOXO-XL (XL), an integrated mycotoxin-mitigating agent, on aflatoxin B1 (AFB1)-induced damage in Leghorn male hepatoma (LMH), porcine jejunum epithelial cell line (IPEC-J2) and porcine alveolar macrophages (3D4/21) cells, and to explore its potential mechanisms. The results showed that 30% inhibition concentration (IC30) of AFB1 in LMH, IPEC-J2 and 3D4/21cells was 0.5, 15.0, and 2.5mg/L, respectively. Notably, cell viability, ROS, apoptosis and DNA lesion induced by AFB1 (IC30) could be ameliorated by the supplementation with XL at the dosage of 0.025, 0.025 and 0.005%, respectively. Additionally, the migration and phagocytosis abilities impaired by AFB1 were also restored by XL in 3D4/21. Further experiments revealed that XL supplementation markedly attenuated AFB1-induced inflammatory response by decreasing IL-1β, IL-6 and IL-10 in LMH, IL-6 in IPEC-J2 and IL-1β in 3D4/21cells. Meanwhile, XL supplementation reversed the alterations of BAX, BCL-2 and caspase-3 induced by AFB1 in the three cells, suggesting that AFB1-induced apoptosis may be suppressed via the mitochondria-dependent pathway. Furthermore, XL may have a protective effect on the intestinal barrier through the restoration of occludin protein. Conclusively, these findings indicated that XL could alleviate AFB1-induced cytotoxicity in the three cells, potentially through the regulation of cytokines, ROS, apoptotic and DNA damage signaling.

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

Pseudorabies virus infection activates the NLRP3 and IFI16 inflammasomes to trigger pyroptosis

Pseudorabies virus (PRV) preferably invades neural tissue and various organs, whereupon may result in multisystemic lesions. Pyroptosis mediated by proteolytic cleavage of gasdermin D (GSDMD) by inflammatory caspases (caspase-1/4/5/11), is closely associated with the activation of inflammasomes, a multiprotein proinflammatory complex. However, further studies on the mechanisms regarding PRV-induced pyroptosis in its natural host are required. Herein, it is demonstrated that PRV infection triggered GSDMD- not GSDME-mediated pyroptosis in porcine alveolar macrophage cells, resulting in increased secretion of IL-1β and LDH. During this process, caspase-1 was activated and participated in the cleaving of GSDMD. Interestingly, we found that the viral replication process or protein production is required to induce pyroptotic cell death. Also, our findings showed that PRV triggered NLRP3 inflammasome activation, which was associated with the production of reactive oxygen species (ROS) and potassium efflux. In addition to the NLRP3 inflammasome, the IFI16 inflammasome was also activated. Importantly, the NLRP3- and IFI16- inflammasomes were both involved in pyroptosis during PRV infection. Finally, we observed that the cleaved GSDMD, activated caspase-1, increased IFI16 levels, and elevated NLRP3 protein in PRV-infected tissues (brain and lung), supporting the occurrence of pyroptosis and the activation of NLRP3- and IFI16- inflammasome in PRV-infected pigs. This research advances our understanding of the PRV-mediated inflammatory response and cell death pathways, providing a deeper knowledge of effective treatments for pseudorabies.

An Intracellular Epitope of ASFV CD2v Protein Elicits Humoral and Cellular Immune Responses.

The African swine fever virus (ASFV) causes high mortality in domestic pigs. ASFV encodes an important protein target for subunit vaccine development, CD2v, but its most effective immunological regions are not known. Herein, we generated a monoclonal antibody (mAb) named IF3 by immunizing mice against the intracellular region of the CD2v protein (CD2v-IR). 1F3 specifically recognized CD2v, which is expressed transiently in transfected Sf9 cells and also in inactivated ASFV-infected porcine alveolar macrophage (PAM) cells. The epitope recognized by 1F3 is 264EPSPREP270, which is highly conserved in ASFV genotypes. Immunization of mice with this epitope elicited an increased IgG response, including IgG1 and IgG2a subtypes, and also increased CD8+ T cells and cytokine expression. Overall, these results indicate that this epitope induces both humoral and cellular immune responses that may be used for ASFV-related subunit vaccine design and development.

Development of porcine induced pluripotent stem cells with a CD163 reporter system

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most infectious viral diseases of swine. Although Cluster of differentiation 163 (CD163) is identified as an essential receptor for mediating PRRS virus (PRRSV) infection, the important residues involved in infection on CD163 are still unclear. Therefore, it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs. In this study, we first generated immortalized porcine alveolar macrophage (IPAM) cell lines harboring 40-residues (residues 523–562, including R561 (arginine (R) at position 561)) deletion of CD163. PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection. We then generated cloned pigs carrying CD163-R561A (an arginine (R) to alanine (A) substitution at position 561 of CD163). PRRSV challenge experiments in porcine alveolar macrophages (PAMs) isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs. Through this study, we show that CD163 523–562 contains essential residues for mediating PRRSV infection, and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection. These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection. Furthermore, CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

Comparison of pathogenicity of different subgenotype porcine reproductive and respiratory syndrome viruses isolated in China

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen causing huge economic losses to the swine industry worldwide, because of its rapid evolution and variation in genomes. It is necessary to monitor the newly emerging epidemic strains and compare their virulence diversity in swine. In this study, two strains of PRRSV2 were isolated from clinical samples by using primary porcine alveolar macrophage (PAMs) cells and designated as SD1805 and FJ1805, respectively. They were unable to grow in MARC-145 cells. Full-length genomes sequence analysis revealed that SD1805 strain has the molecular characterization of NADC30 strain from American, and belongs to the branch of NADC30-like PRRSV (NL-PRRSV) (lineagea 1). FJ1805 isolate came from the inter-subgenotype recombinant of NADC30 strain with a highly pathogenic PRRSV (HP-PRRSV) strain HUN4. Moreover, it belongs to a lineagea 3 represented by the Chinese isolate QYYZ. Pathogenicity analysis showed that SD1805 strain had similar mortality and viral loads in lungs to HP-PRRSV BB0907 strain. FJ1805 strain showed milder pathogenicity compared to NL-PRRSV strain FJ1402 that was previously isolated with Marc-145 cells. It provides evidence of the circulation of the different subgenotype PRRSV strains in China with variations in cell adaption and pathogenic abilities.

Iron Deficiency And Overload Attenuate Inflammatory Response and Modulate Metabolism in Alveolar Macrophage in Vitro

ObjectiveThe study aimed to characterize immunometabolic signatures of porcine alveolar macrophage (PAM) associated with cellular iron deficiency (ID) and iron overload (IE). We hypothesized that cellular iron imbalance modulates LPS‐induced inflammatory and metabolic responses in PAM cells.MethodsAlveolar macrophage cells were collected from 6‐week old donor piglets (N= 5‐6) and were cultured in complete media at 37 ˚C with 5% CO2 until attachment. Cells were treated with complete media (control, CON) or complete media supplemented with 500 µM deferiprone (DFP) or 200 µM ferric ammonium citrate (FAC) to induce iron deficiency (ID) or iron excess (IE), respectively. At the end of the 36‐hour treatment, cells were challenged with 100 ng/mL LPS (L) or sterile saline (S) for another 6 hours. Cells were tested for viability using XTT assay or harvested for analysis of gene, protein expression, and untargeted metabolome using RT‐qPCR, western blot, and GC time‐of‐flight MS, respectively. Cell culture media were collected for analysis of inflammatory cytokines (TNFα, IL‐6, and IL‐10) using ELISA kits. Data were analyzed for two‐way ANOVA using PROC MIXED of SAS (v.9.4). Statistical significance was declared when P‐value or FDR is less than 0.05 or 0.1, respectively.ResultsLPS challenge reduced cell viability by 10 – 20% regardless of the cellular iron status (P < 0.001). Ferritin H was markedly increased in IE cells compared to CON or ID cells (P < 0.001), suggesting cellular iron overload. The mRNA expression of transferrin receptor 1 was the highest in ID‐S cells and was significantly higher in ID and CON compared to IE cells, irrespective of the LPS challenge (P < 0.001). LPS significantly increased mRNA expression of divalent metal transporter 1 (DMT1)and zinc transporter (ZIP14) by 10‐300 fold (P< 0.001), while iron treatment had no significant effect. Unsurprisingly, LPS drastically induced (P < 0.001) genes encoding pro‐ (TNFA and IL1B) and anti‐(TGFB1 and IL10) inflammatory cytokines. Unexpectedly, both ID and IE diminished the induction of those genes caused by the LPS challenge (P < 0.05). The response of TNFA gene was mirrored by changes in TNFα concentration in culture media. Twenty‐nine, 14, and 5 metabolites were altered by cellular iron status, LPS challenge, and their interaction (FDR < 0.1), respectively. A total of 28 altered metabolites were mapped in the user database. Interestingly, metabolites involved in the glycolysis and TCA cycle (e.g. glucose‐6‐phosphate, citric acid, and aconitic acid) were most abundant in ID cells, whereas, in comparison with CON, both ID and IE decreased several amino acids in PAM (e.g. glycine, alanine, leucine, and lysine). LPS increased metabolites involved in glycolysis (fructose‐1‐phosphate) and pentose phosphate pathway (ribose‐5‐phosphate and fructose‐6‐phosphate). Cellular iron status modulated changes in itaconic acid, glucose, fumaric acid, and putrescine in response to LPS challenge; ID enhanced the LPS‐induced increase in itaconic acid, a metabolite marker of macrophage activation.ConclusionPresence of iron chelator and iron overexposure altered cellular iron metabolism of PAM cells and its inflammatory and metabolic responses to the invasion of bacterial pathogens.

Senecavirus A Entry Into Host Cells Is Dependent on the Cholesterol-Mediated Endocytic Pathway.

Senecavirus A (SVA), an important member of the Picornaviridae family, causes vesicular disease in pigs. Here, we generated an EGFP-expressing recombinant SVA re-SVA-EGFP, which exhibited similar growth kinetics to its parental virus. The reporter SVA was used to study the role of pig ANTXR1 (pANTXR1) in SVA infection in a porcine alveolar macrophage cell line (PAM-Tang cells). Knockdown of the pANTXR1 significantly reduced SVA infection and replication in PAM-Tang cells, while re-expression of the pANTXR1 promoted the cell susceptibility to SVA infection. The results indicated that pANTXR1 is a crucial receptor mediating SVA infection. Subsequently, the viral endocytosis pathways for SVA entry into pig cells were investigated and the results showed that cholesterol played an essential role in receptor-mediated SVA entry. Together, these results demonstrated that SVA entered into host cells through the pANTXR1-mediated cholesterol pathway. Our findings provide potential targets to develop antiviral drugs for the prevention of SVA infection in the pig population.

The Competitive Endogenous RNA (ceRNA) Regulation in Porcine Alveolar Macrophages (3D4/21) Infected by Swine Influenza Virus (H1N1 and H3N2).

H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.

Deoxynivalenol aggravates the immunosuppression in piglets and PAMs under the condition of PEDV infection through inhibiting TLR4/NLRP3 signaling pathway

Mycotoxins are toxic metabolites produced by fungi, which are ubiquitous in cereals and feed worldwide and threaten human and animal health. Deoxynivalenol (DON) is one of the most prevalent mycotoxins and causes a series of toxicities, especially enterotoxicity and immunotoxicity. Porcine epidemic diarrhea virus (PEDV) is a destructive enteropathogenic animal coronavirus, is often accompanied with DON contamination in the swine herd. Previous studies have shown that PEDV infection leads severe immunosuppression in pigs. However, whether DON exposure aggravates the PEDV-induced immunosuppression remains unclear. In this study, weaned piglet and porcine alveolar macrophage cell (PAM) models were established to explore the effects of DON on the PEDV-induced immunosuppression and to clarify its underlying mechanism. The in vivo results showed that 2.25 mg/kg feed DON significantly exacerbated the immunosuppressive effects on the PEDV-infected piglets, as demonstrated by the decreases in growth performance, the numbers of goblet cells and CD3+T cells, as well as the protein expressions of ZO-1, Claudin1 and Muc2, in addition to the increases in anti-inflammatory factors levels and the intestinal injury. Similarly, the in vitro results demonstrated that 3–4 μM DON markedly aggravated apoptosis, enhanced the expressions of anti-inflammatory factors, but reduced the migration and phagocytosis abilities of the PEDV-infected PAMs. Furthermore, DON significantly suppressed the expressions of TLR4/NLRP3 in vivo and in vitro. To contrast, lipopolysaccharide (LPS), the corresponding activator, obviously alleviated the DON-exacerbated immunosuppression. Our findings suggest that DON could aggravate host immunosuppression under the condition of PEDV infection through inhibiting TLR4/NLRP3 signaling pathway, and provide novel theoretical insights into the further studies on the immunotoxicity of DON contamination and PEDV-induced immunosuppression.

Comprehensive Analysis of G-Quadruplexes in African Swine Fever Virus Genome Reveals Potential Antiviral Targets by G-Quadruplex Stabilizers.

African Swine Fever Virus (ASFV), a lethal hemorrhagic fever of the swine, poses a major threat to the world’s swine population and has so far resulted in devastating socio-economic consequences. The situation is further compounded by the lack of an approved vaccine or antiviral drug. Herein, we investigated a novel anti-ASFV approach by targeting G-Quadruplexes (G4s) in the viral genome. Bioinformatics analysis of putative G-quadruplex-forming sequences (PQSs) in the genome of ASFV BA71V strain revealed 317 PQSs on the forward strand and 322 PQSs on the reverse strand of the viral genome, translating to a density of 3.82 PQSs/kb covering 9.52% of the entire genome, which means that 85% of genes in the ASFV genome have at least 1 PQS on either strand. Biochemical characterization showed that 8 out of 13 conserved PQSs could form stable G4s in the presence of K+, and 4 of them could be stabilized by G4 ligands, N-Methyl Mesoporphyrin (NMM), and pyridostatin (PDS) in vitro. An enhanced green fluorescent protein (EGFP)-based reporter system revealed that the expression of two G4-containing genes, i.e., P1192R and D117L, could be significantly suppressed by NMM and PDS in 293T cells. In addition, a virus infection model showed that NMM could inhibit the replication of ASFV in Porcine Alveolar Macrophages (PAM) cells with an EC50 value of 1.16 μM. Altogether, the present study showed that functional PQSs existent in the promoters, CDS, 3′ and 5′ UTRs of the ASFV genome could be stabilized by G4 ligands, such as NMM and PDS, and could serve as potential targets for antivirals.