A dicoumarol-graphene oxide quantum dot polymer inhibits porcine reproductive and respiratory syndrome virus through the JAK-STAT signaling pathway.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) causes substantial economic losses in the global swine industry. The current vaccine options offer limited protection against PRRSV transmission, and there are no effective commercial antivirals available. Therefore, there is an urgent need to develop new antiviral strategies that slow global PRRSV transmission. Methods: In this study, we synthesized a dicoumarol-graphene oxide quantum dot (DIC-GQD) polymer with excellent biocompatibility. This polymer was synthesized via an electrostatic adsorption method using the natural drug DIC and GQDs as raw materials. Results: Our findings demonstrated that DIC exhibits high anti-PRRSV activity by inhibiting the PRRSV replication stage. The transcriptome sequencing analysis revealed that DIC treatment stimulates genes associated with the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway. In porcine alveolar macrophages (PAMs), DIC-GQDs induce TYK2, JAK1, STAT1, and STAT2 phosphorylation, leading to the upregulation of JAK1, STAT1, STAT2, interferon-ß (IFN-ß) and interferon-stimulated genes (ISGs). Animal challenge experiments further confirmed that DIC-GQDs effectively alleviated clinical symptoms and pathological reactions in the lungs, spleen, and lymph nodes of PRRSV-infected pigs. Discussion: These findings suggest that DIC-GQDs significantly inhibits PRRSV proliferation by activating the JAK/STAT signalling pathway. Therefore, DIC-GQDs hold promise as an alternative treatment for PRRSV infection.

PCV2 Induced Endothelial Derived IL-8 Affects MoDCs Maturation Mainly via NF-κB Signaling Pathway.

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.

Porcine Circovirus 2 Increases the Frequency of Transforming Growth Factor-ß via the C35, S36 and V39 Amino Acids of the ORF4.

Porcine circovirus 2 (PCV2) is one of the most important endemic swine pathogens, inducing immunosuppression in pigs and predisposing them to secondary bacterial or viral infections. Our previous studies show that PCV2 infection stimulated pig intestinal epithelial cells (IPEC-J2) to produce the secretory transforming growth factor-ß (TGF-ß), which, in turn, caused CD4+ T cells to differentiate into regulatory T cells (Tregs). This may be one of the key mechanisms by which PCV2 induces immunosuppression. Here, we attempt to identify the viral proteins that affect the TGF-ß secretion, as well as the key amino acids that are primarily responsible for this occurrence. The three amino acids C35, S36 and V39 of the ORF4 protein are the key sites at which PCV2 induces a large amount of TGF-ß production in IPEC-J2 and influences the frequency of Tregs. This may elucidate the regulatory effect of PCV2 on the Tregs differentiation from the perspective of virus structure and intestinal epithelial cell interaction, laying a theoretical foundation for improving the molecular mechanism of PCV2-induced intestinal mucosal immunosuppression in piglets.

TGF-ß from the Porcine Intestinal Cell Line IPEC-J2 Induced by Porcine Circovirus 2 Increases the Frequency of Treg Cells via the Activation of ERK (in CD4+ T Cells) and NF-κB (in IPEC-J2).

Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut's immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4+ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4+ T cells among piglets' peripheral blood mononuclear cells (PBMCs) and caused CD4+ T cells to undergo a transformation into Foxp3+ regulatory T cells (Treg cells) via activating CD4+ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-ß induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-ß, which enhanced the differentiation of CD4+ T cells into Treg cells through the activation of ERK in CD4+ T cells. This information sheds light on PCV2's function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection.

The changes of immune-related molecules within the ileal mucosa of piglets infected with porcine circovirus type 2.

BACKGROUND: Enteritis is one of the most frequently reported symptoms in piglets infected with porcine circovirus type 2 (PCV2), but the immunopathogenesis has not been reported. OBJECTIVES: This study examined the effect of a PCV2 infection on the intestinal mucosal immune function through morphological observations and immune-related molecular detection. METHODS: Morphological changes within the ileum of piglets during a PCV2 infection were observed. The expression of the related-molecules was analyzed using a gene chip. The immunocyte subsets were analyzed by flow cytometry. The secretory immunoglobulin A (SIgA) content was analyzed by enzyme-linked immunosorbent assay. RESULTS: The PCV2 infection caused ileal villus damage, intestinal epithelial cells exfoliation, and an increase in lymphocytes in the lamina propria at 21 days post-infection. Differentially expressed genes occurred in the defense response, inflammatory response, and the complement and coagulation cascade reactions. Most of them were downregulated significantly at the induction site and upregulated at the effector site. The genes associated with SIgA production were downregulated significantly at the induction site. In contrast, the expression of the Toll-like receptor-related genes was upregulated significantly at the effector site. The frequencies of dendritic cells, B cells, and CD8⁺T cells were upregulated at the 2 sites. The SIgA content decreased significantly in the ileal mucosa. CONCLUSIONS: PCV2 infections can cause damage to the ileum that is associated with changes in immune-related gene expression, immune-related cell subsets, and SIgA production. These findings elucidated the molecular changes in the ileum after a PCV2 infection from the perspective of intestinal mucosal immunity, which provides insights into a further study for PCV2-induced enteritis.

Porcine circovirus type 2 upregulates endothelial-derived IL-8 production in porcine iliac artery endothelial cells via the RIG-I/MDA-5/MAVS/JNK signaling pathway.

BACKGROUND: Dysfunction of endothelial cells and vascular system is one of the most important pathological changes of porcine circovirus disease (PCVD) caused by porcine circovirus type 2 (PCV2). PCV2-infected endothelial cells can upregulate the production of endothelial-derived IL-8, which can inhibit the maturation of dendritic cells. Endothelial-derived IL-8 has different structural and biological characteristics compared with monocyte-derived IL-8. However, the mechanism of endothelial-derived IL-8 production is still unclear. RESULTS: Key molecules of RIG-I-like signaling pathway RIG-I, MDA-5, MAVS and a key molecule of JNK signaling pathway c-Jun in PCV2-infected porcine iliac artery endothelial cells (PIECs) were upregulated significantly detected with quantitative PCR, Western blot and fluorescence confocal microscopy, while no significant changes were found in NF-κB signaling pathway. Meanwhile, the expression of endothelial-derived IL-8 was downregulated after RIG-I, MDA-5, or MAVS genes in PIECs were knocked down and PIECs were treated by JNK inhibitor. CONCLUSIONS: PCV2 can activate RIG-I/MDA-5/MAVS/JNK signaling pathway to induce the production of endothelial-derived IL-8 in PIECs, which provides an insight into the further study of endothelial dysfunction and vascular system disorder caused by PCV2.

Endothelial IL-8 induced by porcine circovirus type 2 affects dendritic cell maturation and antigen-presenting function.

BACKGROUND: Porcine circovirus (PCV) disease caused by PCV type 2 (PCV2) is mainly attributed to immunosuppression and immune damage. PCV2 can infect vascular endothelial cells and induce high expression of endothelial IL-8. Dendritic cells (DCs), as professional antigen-presenting cells, can not only present antigens but also activate naïve T-cells, causing an immune response. METHODS: To demonstrate whether endothelial IL-8 is the main factor inhibiting the maturation and related functions of dendritic cells during PCV2 infection, monocyte-derived DCs (MoDCs) and porcine iliac artery endothelial cells (PIECs) processed by different methods were co-cultured in two ways. Flow cytometry, molecular probe labeling, fluorescence quantitative PCR, and the MTS assay were used to detect the changes in related functions and molecules of MoDCs. RESULTS: Compared to those in the PIEC-DC group, the endothelial IL-8 upregulation co-culture group showed significantly lower double-positive rates for CD80/86 and MHC-II of MoDCs and significantly increased endocytosis of MoDCs. Meanwhile, the adhesion rate and average fluorescence intensity of MoDCs were significantly downregulated in migration and adhesion experiments. Furthermore, the MHC-I and LAMP7 mRNA levels in MoDCs and the proliferation of MoDC-stimulated T-cells were markedly reduced. However, the changes in MoDCs of the endothelial IL-8 downregulation co-culture group were the opposite. CONCLUSIONS: PCV2-induced endothelial IL-8 reduces the adhesion and migration ability of MoDCs, resulting in a decreased maturation rate of MoDCs, and further inhibits antigen presentation by DCs. These results may explain the immunosuppressive mechanism of PCV2 from the perspective of the interaction between endothelial cells and DCs in vitro.

Reduced antigen presentation capability and modified inflammatory/immunosuppressive cytokine expression of induced monocyte-derived dendritic cells from peripheral blood of piglets infected with porcine circovirus type 2.

The efficiency of immune responses and host defense against pathogens largely depends on the function of dendritic cells (DCs). Porcine circovirus type 2 (PCV2) infection causes viremia and extensive modulation of immune activities in the blood. The objective of the present study was to investigate the effects of PCV2 infection in vivo on the immunological function of DCs induced from peripheral blood monocytes (MoDCs). At different points after infection with PCV2, peripheral blood monocytes from PCV2-infected pigs were used to induce differentiation of DCs in vitro. Flow cytometry and quantitative real-time reverse transcription PCR were conducted to detect mRNA expression of surface markers related to antigen presentation and inflammatory/immunosuppressive cytokines of the induced MoDCs. The ability of induced MoDCs to stimulate T cells was measured using an MTS assay. In the early phase of infection at 3 days post-inoculation (DPI), IL-10, IL-8 and MIP-1ß in MoDCs were upregulated significantly. By the peak of virus proliferation at 7 DPI, antigen presentation molecules SLA-DR (MHC II) and CD80/86 together with cytokines IL-12 and IL-10 had decreased, accompanied by a rapid reduction of IL-8 and MIP-1ß. The T cell stimulation index of induced MoDCs in PCV2 groups after different infection times declined to some extent, with a significant difference at 7 DPI. PCV2 infection in vivo functionally reduced the antigen presentation capability of induced MoDCs from peripheral blood and modified expression of inflammatory/immunosuppressive cytokines that may be related to PCV2-induced immunosuppression.

Change in the immune function of porcine iliac artery endothelial cells infected with porcine circovirus type 2 and its inhibition on monocyte derived dendritic cells maturation.

Porcine circovirus-associated disease is caused by porcine circovirus type 2 (PCV2) infection, which targets iliac artery endothelial cells (PIECs); it leads to severe immunopathologies and is associated with major economic losses in the porcine industry. Here, we report that in vitro PCV2 infection of PIECs causes cell injury, which affects DC function as well as adaptive immunity. Specifically, PCV2 infection downregulated PIEC antigen-presenting molecule expression, upregulated cytokines involved in the immune and inflammatory response causing cell damage and repair, and altered the migratory capacity of PIECs. In addition, PCV2-infected PIECs inhibited DC maturation, enhanced the endocytic ability of DCs, and weakened the stimulatory effect of DCs on T lymphocytes. Together, these findings indicate that profound functional impairment of DCs in the presence of PCV2-infected PIECs may be a potential pathogenic mechanism associated with PCV2-induced porcine disease.

A DNA aptamer efficiently inhibits the infectivity of Bovine herpesvirus 1 by blocking viral entry.

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of domestic and wild cattle responsible for major economic losses in dairy and beef industries throughout the world. Inhibition of viral entry plays a crucial role in the control of BoHV-1 infection and aptamers have been reported to inhibit viral replication. In this study, nine DNA aptamers that target BoHV-1 were generated using systemic evolution of ligands by exponential enrichment. Of the nine candidates, aptamer IBRV-A4 exhibited the highest affinity and specificity for BoHV-1, which bound to BoHV-1 with a Kd value of 3.519 nM and demonstrated the greatest virus binding as shown by fluorescence imaging. The neutralizing ability of aptamer IBRV-A4 was determined using neutralization assays and real time PCR in BoHV-1 infected Madin-darby bovine kidney cells. Virus titration, immunofluorescence and confocal laser scanning microscopy showed virus replication significantly decreased when aptamer IBRV-A4 was added to BoHV-1 infected MDBK cells at 0 and 0.5 hours post-infection, whereas no change was seen when IBRV-A4 was added 2 hours post-infection. This concludes that aptamer IBRV-A4 efficiently inhibits viral entry of BoHV-1 in MDBK cells and is therefore a novel tool for diagnosis and treatment of BoHV-1 infection in cattle.