Search Results (105)

Deoxynivalenol (DON) is a common mycotoxin that causes immunosuppression in pigs. Its effects on cellular metabolism remain unclear. In this study, we investigate DON-induced metabolic alterations in porcine alveolar macrophage cell line 3D4/21 using non-targeted metabolomics. MTT assays showed DON reduced cell viability in a concentration- and time-dependent manner. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) revealed distinct metabolic profiles between control and DON-treated groups. Metabolomic analysis identified 127 differential metabolites (VIP > 1, p < 0.05), primarily in purine metabolism, glutathione metabolism, and arginine–proline metabolism. Integration with transcriptomic data confirmed that these pathways play key roles in DON-induced immunotoxicity. Specifically, changes in purine metabolism suggested disrupted nucleotide synthesis and energy balance, while glutathione depletion indicated weakened antioxidant defense. These findings provided a systems biology perspective on DON’s metabolic reprogramming of immune cells and identified potential therapeutic targets to reduce mycotoxin-related immunosuppression in swine. Full article

Probiotics play a pivotal role in animal production by promoting growth, enhancing gut health, and modulating immune responses. Bacillus subtilis, a widely utilized probiotic, forms robust spores that exhibit exceptional resistance, making it ideal for feed applications. While B. subtilis spores have been shown to stimulate innate immune signaling, the specific contributions of spore coat proteins to immune modulation remain poorly characterized. In this study, we investigated the immunostimulatory effects of spores deficient in six key coat proteins: SpoIVA, SafA, CotE, CotX, CotZ, and CgeA. These proteins are essential for the assembly and structural integrity of the spore’s multi-layered coat, and are involved in recruiting other coat components. Deletion of these genes result in defects in spore coat architecture, potentially altering spore–host interactions. Using porcine alveolar macrophages (MΦ3D4/2), we assessed cytokine responses to each mutant strain. Our findings demonstrate that the absence of specific structural proteins significantly impacts immune activation, particularly through Toll-like receptor pathways. This work provides novel insights into the immunomodulatory functions of spore coat proteins and lays the foundation for the rational design of next-generation B. subtilis-based probiotics with enhanced immunological properties for agricultural applications. Full article

Glaesserella parasuis (G. parasuis) is the primary pathogen responsible for Glässer’s disease and poses a significant threat to the global pig industry. MicroRNAs are a class of short, endogenous, single-stranded noncoding RNAs that play crucial roles in inflammation, apoptosis, proliferation, differentiation, and invasion in various organisms. This study analyzed the characteristics of porcine alveolar macrophage (PAM) cells infected with G. parasuis through the knockdown and overexpression of ssc-miR-129a-3p. We constructed a cellular model with ssc-miR-129a-3p knockdown invaded by G. parasuis strain XX0306, screening 160 differentially expressed genes via high-throughput sequencing. GO enrichment analysis revealed that 376 GO entries were enriched. KEGG enrichment analysis found that mRNA target genes were enriched in 17 cell signaling pathways, including G protein-coupled components, PPAR, and other signaling pathways that can mediate inflammatory pathways. By examining the expression of relevant inflammatory factors and signaling pathways, we elucidated the molecular mechanisms through which ssc-miR-129a-3p targets the TLR4/NF-κB signaling pathway to regulate inflammatory injury. This study establishes a foundation for further research into the role of miRNA in the pathogenesis of Glässer disease and is highly significant for the prevention and control of bacterial diseases within the pig industry. Full article

Butyrate and its derivatives may influence inflammatory status and physiology in a variety of organisms and organ systems. Inflammatory conditions of the gastrointestinal tract, such as post-weaning diarrhea, negatively impact swine. Dietary intervention with butyrate-based compounds should be considered a strategy to improve disease resistance in pigs. We aimed to assess the properties of different forms of butyrate treatments using porcine cell culture experiments. This assessment may inform future in vivo feed experiments designed to determine its potential application of the dietary supplements for pigs. An intestinal porcine enterocyte cell line, IPEC-J2, was seeded at 5 × 10³ cells/mL in 96-well plates to confirm cell viability by MTT assay for each dose range used in the current experiments (0, 0.5, 1, 2, 4 mM butyric acid or tributyrin; 0, 1, 2, 4, 8 mM sodium butyrate or monobutyrin). For transepithelial electrical resistance (TEER) analysis, IPEC-J2 was seeded at 5 × 10⁵ cells/mL in 12-well transwell inserts and treated with 5 levels of each butyrate derivative after adherence (n = 5). TEER was measured at 24, 48, and 72 h post-treatment to quantify intestinal barrier integrity of IPEC-J2 monolayers. Butyric acid, sodium butyrate, and monobutyrin significantly increased (p < 0.05) TEER in IPEC-J2 at different time points compared with control. Further, porcine alveolar macrophages (PAMs) were harvested from donor weaned piglets (n = 6) via bronchoalveolar lavage and isolated for primary culture (6 × 10⁵ cells/well, 6-well plates). PAMs were treated with five levels of each butyrate derivative with or without lipopolysaccharide (LPS, 1 μg/mL) challenge. The concentrations of TNF-α and IL-1β in cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Butyric acid and sodium butyrate treatments reduced the production of TNF-α in LPS-challenged PAMs (linear; p < 0.05). Different butyrate derivatives exerted anti-inflammatory properties and improved intestinal barrier integrity. Full article

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious diseases threatening the swine industry worldwide. However, no satisfactory control strategy has existed until now. In this study, the effectiveness of tylvalosin against PRRSV and the underlying mechanism was investigated. The results showed that in porcine alveolar macrophages (PAMs), tylvalosin can inhibit the replication of the NADC30-like and NADC34-like strains in a dose-dependent manner. It is worth noting that pre-incubation with tylvalosin had no significant inhibitory effect on the NADC30-like strain but did inhibit the NADC34-like strain. Co-incubation of both viruses and tylvalosin or post-incubation with tylvalosin after viral infection inhibited PRRSV. We further analyzed the effect of tylvalosin on different stages of PRRSV replication and found that the stages in the PRRSV life cycle could be blocked by tylvalosin. Tylvalosin has an antiviral effect on all four stages of the NADC34-like strain’s infectious cycle but has no effect against the adsorption phase of the NADC30-like strain. These results demonstrated that tylvalosin suppressed PRRSV infection in PAMs and inhibited PRRSV infection at multiple steps of the viral life cycle. This study will contribute to the clinical prevention and control of PRRS and provide a basis for further exploration of the anti-PRRSV effects of tylvalosin. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV) cause serious economic losses to the swine industry worldwide. Both viruses show a tropism for macrophages, based on the use of specific entry mediators (e.g., Siglec-1 and CD163). Identifying additional mediators of viral entry is essential for advancing antiviral and vaccine development. In this context, monoclonal antibodies (mAbs) are valuable tools. This study employed a library of 166 mAbs targeting porcine alveolar macrophages (PAMs) to identify candidates capable of blocking early infection stages, including viral binding, internalization, and fusion. Immunofluorescence analysis revealed 74 mAbs with cytoplasmic staining and 70 mAbs with membrane staining. Fifteen reacted with blood monocytes as determined by flow cytometry. mAb blocking assays were performed at 4 °C and 37 °C to analyze the ability of mAbs to block PRRSV and/or ASFV infections in PAMs. The mAb 28C10 significantly blocked PRRSV (96% at 4 °C and 80% at 37 °C) and ASFV (64% at 4 °C and 81% at 37 °C) infections. The mAb 28G10B6 significantly blocked PRRSV (86% at 4 °C and 74% at 37 °C) and partially blocked ASFV (35% at 4 °C and 64% at 37 °C) infections. mAb 26B8F5-I only partially blocked PRRSV infection (65% at 4 °C and 46% at 37 °C). Western blotting and mass spectrometry identified the corresponding proteins as Siglec-1 (28C10; 250 kDa), MYH9 (28G10B6; 260 kDa), and ANXA1 (26B8F5-I; 37 kDa). Our findings are indicative that Siglec-1, MYH9, and ANXA1 play a role in PRRSV/ASFV entry into macrophages. Full article

Since the first isolation of the porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) BJEU06-1 strain from a Beijing pig farm in 2006, more and more PRRSV-1 isolates have been identified in China. In this study, we performed the routine detection of PRRSV-1 using 1521 clinical samples collected in 12 provinces/cities from February 2022 to May 2024. Only three lung samples from severely diseased piglets collected in January 2024 were detected as PRRSV-1-positive (0.197%, 3/1521). A PRRSV-1 strain (AHEU2024-2671) was successfully isolated in primary alveolar macrophages (PAMs) but not in Marc-145 cells. Genome sequencing showed that the AHEU2024-2671 isolate shared the highest genome similarity (90.67%) with the SC2020-1 isolate but only 84.01% similarity with the predominant BJEU06-1 strain. Noticeably, the AHEU2024-2671-like isolates not only contained deletions in nsp2 and the GP3-GP4 overlap region, but also contained a unique 6 nt deletion between nsp12 and the ORF2 gene. Furthermore, a genome-based phylogenetic tree supported that the AHEU2024-2671-like isolates form a novel subgroup within subtype 1. Overall, this study not only supported the idea that PRRSV-1 is rapidly evolving in Chinese swine herds, but also pulled the alarm that novel PRRSV-1 isolates with potentially increased pathogenicity might already exist in China, although they are still rarely detected among Chinese pigs. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major concern for swine health. Isolating PRRSV is essential for identifying infectious viruses and for vaccine formulation. This study evaluated the potential of using tongue fluid (TF) from perinatal piglet mortalities for PRRSV isolation. Four collection protocols were tested: extracting TF from fresh tissues using phosphate-buffered saline (PBS group), extracting TF from fresh tissues using virus transportation medium (VTM group), extracting TF from freeze-thawed tissue (freeze-thaw group), and using tissue homogenates (homogenate group). Two cell lines (ZMAC and MARC-145) and primary alveolar macrophages (PAM) were evaluated for their effect on successful PRRSV isolation. An eligible PRRSV-positive unstable breeding herd in Midwestern USA was chosen for the study. Tongues were collected in 20 batches (~30 mortalities per batch). Within each batch, each tongue tissue was cut into four quarters, with each quarter randomly assigned to one of the four collection protocols and RT-qPCR tested. Virus isolation (VI) was attempted on 10 batches. The mean RT-qPCR cycle threshold (Ct) values for the PBS, VTM, freeze-thaw, and homogenate groups were 21.9, 21.8, 22.6, and 24.8, respectively. The VI success rate was 22.6%, 12.1%, 2.8%, and 2.8% in the PBS, VTM, freeze-thaw, and homogenate groups, respectively. The probability of successful VI was 3.1% and 21.0% in the MARC-145 and ZMAC cell lines, respectively, and 4.8% in the PAM cells. TF from perinatal mortalities is an option for PRRS VI, aiding in PRRSV monitoring and control programs. Full article

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), has a significant impact on the global pork industry. It results in reproductive failure in sows and respiratory issues in pigs of all ages. Despite the availability of vaccines, controlling the PRRSV remains challenging, partly owing to the limitations of cell culture systems. Current methods largely rely on primary porcine alveolar macrophages (PAMs), which must be harvested from piglets and have limited proliferative capacity. Although some simian cell lines support PRRSV replication, their inability to express porcine CD163, which is a key receptor for PRRSV entry, compromises their effectiveness, because the virus replicates differently in these non-target cells. To address these issues, we established an immortalized PAM cell line, PAM-T43, using SV40 large T antigen for immortalization and porcine serum as a culture supplement. PAM-T43 cells maintain essential macrophage functions, including CD163 expression and phagocytic activity, and exhibit high sensitivity to the PRRSV, efficiently supporting viral replication. This novel cell line offers significant potential for advancing PRRSV research, particularly in vaccine development and field strain isolation, by overcoming the limitations of current systems. Full article

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) infection, has been a serious threat to the pork industry worldwide and continues to bring significant economic loss. Current vaccination strategies offer limited protection against PRRSV transmission, highlighting the urgent need for novel antiviral approaches. In the present study, we reported for the first time that betulonic acid (BA), a widely available pentacyclic triterpenoids throughout the plant kingdom, exhibited potent inhibition on PRRSV infections in both Marc-145 cells and primary porcine alveolar macrophages (PAMs), with IC₅₀ values ranging from 3.3 µM to 3.7 µM against three different type-2 PRRSV strains. Mechanistically, we showed that PRRSV replication relies on energy supply from cellular ATP production, and BA inhibits PRRSV infection by reducing cellular ATP production. Our findings indicate that controlling host ATP production could be a potential strategy to combat PRRSV infections, and that BA might be a promising therapeutic agent against PRRSV epidemics. Full article

Streptococcus suis is an important bacterial pathogen that affects the global pig industry. The immunosuppressive nature of S. suis infection is recognized, and our previous research has confirmed thymus atrophy with a large number of necrotic cells. In this current work, we aimed to uncover the role of pyroptosis in cellular necrosis in thymic cells of S. suis-infected mice. Confocal microscopy revealed that S. suis activated the M1 phenotype and primed pyroptosis in the macrophages of atrophied thymus. Live cell imaging further confirmed that S. suis could induce porcine alveolar macrophage (PAM) pyroptosis in vitro, displaying cell swelling and forming large bubbles on the plasma membrane. Meanwhile, the levels of p-p38, p-extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) were increased, which indicated the mitogen-activated protein kinase (MAPK) and AKT pathways were also involved in the inflammation of S. suis-infected PAMs. Furthermore, RT-PCR revealed significant mRNA expression of pro-inflammatory mediators, including interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor (TNF)-α and chemokine CXCL8. The data indicated that the inflammation induced by S. suis was in parallel with pro-inflammatory activities of M1 macrophages, pyroptosis and MAPK and AKT pathways. Pyroptosis contributes to necrotic cells and thymocyte reduction in the atrophied thymus of mice. Full article

African Swine Fever (ASF) is a devastating viral hemorrhagic disease that causes high morbidity and mortality in domestic pigs and wild boars, severely impacting the swine industry. The etiologic agent, African Swine Fever virus (ASFV), mainly infects myeloid cells of the swine mononuclear phagocytic system (MPS). For other porcine viruses, in vitro culture models with primary cells are widely used as they mimic the in vivo viral replication behavior better compared to continuous cell lines. Our study validates this possible correlation for ASFV using cell culture models established for three different porcine macrophages, isolated from the lungs (porcine alveolar macrophages), blood (monocyte-derived macrophages) and spleen (spleen macrophages). The cells were infected with two genotype I and two genotype II strains with different pathogenic potential in vivo. The highly virulent strains replicated better in general than the low-virulent strains. This was most pronounced in monocyte-derived macrophages, although only statistically significant 18 h post-infection (hpi) in the intracellular genomic ASFV copies between E70 and the low-virulent strains. For this reason, we conclude that the different replication characteristics between the strains with different virulence do not proportionally represent the differences in pathology seen between the strains in vivo. Additionally, ASFV-positive cells were observed earlier in monocyte-derived macrophages (MDMs) compared to the alveolar and spleen macrophages, subsequently leading to an earlier rise in extracellular virus, and, ultimately, more MDMs were infected at the end of sampling. For these reasons, we propose MDMs as the best-suited cell type to study ASFV. Full article

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. Full article

One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus’ assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein–protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus–host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines. Full article

African swine fever (ASF), caused by the African swine fever virus (ASFV), is one of the most important infectious diseases that cause high morbidity and mortality in pigs and substantial economic losses to the pork industry of affected countries due to the lack of effective vaccines. The need to develop alternative robust antiviral countermeasures, especially anti-ASFV agents, is of the utmost urgency. This study shows that fangchinoline (FAN), a bisbenzylisoquinoline alkaloid found in the roots of Stephania tetrandra of the family Menispermaceae, significantly inhibits ASFV replication in porcine alveolar macrophages (PAMs) at micromolar concentrations (IC₅₀ = 1.66 µM). Mechanistically, the infection of ASFV triggers the AKT/mTOR/NF-κB signaling pathway. FAN significantly inhibits ASFV-induced activation of such pathways, thereby suppressing viral replication. Such a mechanism was confirmed using an AKT inhibitor MK2206 as it inhibited AKT phosphorylation and ASFV replication in PAMs. Altogether, the results suggest that the AKT/mTOR pathway could potentially serve as a treatment strategy for combating ASFV infection and that FAN could potentially emerge as an effective novel antiviral agent against ASFV infections and deserves further in vivo antiviral evaluations. Full article