Journal of Cell Signaling
Open Access

Porcine Circo Virus 2: Immunosuppressive Pathogen

Umar Zafar Khan^{* *}Correspondence: Umar Zafar Khan, Department of Sciences, Superior University, Lahore, Pakistan, Email:

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Description

Porcine CircoVirus 2 (PCV2) has been recognized as an immunosuppressive virus. However, the crosstalk between this virus and its host cells in related signaling pathways remains poorly understood. In this study, the expression profiles of 84 genes involved in Transforming Growth Factor-beta (TGF-β) signaling pathway were probed in PCV2b-infected Primary porcine Alveolar Macrophages (PAMs) by using an RT2 profiler PCR array system. The protein expression levels of cytokines involved in the TGF- β signaling pathway were determined with a Ray Biotech fluorescent Quantibody porcine cytokine array system [1]. A large number of genes analyzed by a co-expression network and implicated in transcriptional regulation and apoptosis were differentially expressed in PCV2b-infected PAMs. Among these genes, TGF- β, interleukin-10, CCAAT/ Enhancer-Binding Protein Beta (C/EBPB), growth arrest, and DNA-Damage-Inducible 45 Beta (GADD45B), and BCL2 were up-regulated. By contrast, SMAD family member 1 (SMAD1) and SMAD3 were down regulated. These results suggested that the TGF-β signalling pathway was repressed in PAMs at the early onset of PCV2 infection. The inhibited apoptosis was indicated by the up-regulated C/EBPB, GADD45B, and BCL2, and by the down regulated smad1 and SMAD3, which possibly increased the duration of PCV2 replication-permissive conditions and caused a persistent infection.

Porcine CircoVirus 2 (PCV2), a non-enveloped, single-stranded circular DNA virus, is the primary contributing pathogen of PCV2 Systemic Disease (PCV2-SD) and other PCV2 associated diseases (PCVADs). PCVADs are the most economically important diseases affecting the global swine industry [2]. Pigs with PCV2-SD show severe depletion in lymphocytes in their lymphoid tissues and a remarkable decrease in T and B cells in their peripheral blood, suggesting that lymphoid depletion largely contributes to immunosuppression in PCV2-affected pigs. The PCV2-induced decrease in the proliferation and lysis of lymphocytes and apoptosis of primary lymphoid organs or precursor cells are potential causes of lymphocyte depletion. Cytokines, secreted by cells undergoing cellular stress, are essential for the development of the immune response. The differential expressions of some cytokines, such as Interleukin (IL)-1/8/10/12, and TNF- α, in PCV2-infected porcine monocytes and macrophages, have been reported. IL-10 has been suggested to play an important role in PCV2-induced systemic immunosuppression [3]. Transforming Growth Factorbeta (TGF- β) and its signalling pathway participates in cell growth, apoptosis, differentiation, migration, and metastasis in a context-dependent manner in various cell types. Dysregulation of TGF- β signalling pathway has been implicated in numerous human diseases. In addition, it is a pleiotropic cytokine and has regulatory activity on multiple types of immune cells. T cells are established as critical targets of TGF- β, which regulates T cell development, homeostasis, tolerance, and differentiation. TGF- β plays its biological role primarily through the canonical Smads signalling pathway which has three isoforms that are involved in several developmental processes as TGF- βs, Tβ- Rs, and SMADs.

Animals and isolation of PAM cells

Before the collection of alveolar fluid, piglets were euthanized with intravenous sodium pentobarbital overdose. PAMs were isolated from lungs according to previous methods. The isolated PAMs was adjusted to 1 × 107 cells/ mL and cultured with a growth medium containing RPMI-1640 supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 100 u/mL penicillin, and 100 μg/mL streptomycin and incubated at 37°C with 5% CO₂.

Virus preparation for infection

PK-15 cells monolayers were inoculated at 60% confluence. After 24 h of incubation at 37°C, the monolayer was treated with 200 mM D-Glucosamine for 1 h. After further 72 h incubation, the cells were harvested and lysed three times by freeze-thawing. The genomic copies of the PCV2 in lysed cell mixture were determined by qPCR. PAMs were infected with PCV2b in multiplicity of infection (MOI) 1 (1 viral DNA copy/cell). After infection, cells were cultured in a maintained medium RPMI-1640 supplemented with 2% FBS and incubated at 37°C, and 5% CO2, for 1, 24, and 48 hours respectively. The cells and supernatant were collected respectively at indicated time intervals. The cells were kept in RNA later, RNA stabilization reagent kits to prevent RNA degradation and stored at −70°C until use [4].

Identification of PCV2 in PAMs

PCV2 in PAMs after the infection was detected through Immuno Fluorescence Assay (IFA). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100 in PBS. PCV2 in PAMs was indicated by Quantum dots conjugated specific single domain antibody (QDs-psdAb) probe.

RNA extraction and cDNA preparation

Total RNA was extracted by using an RNA extraction kit according to the instruction. The quantity of RNA was determined with a micro spectrophotometer and the RNA integrity was evaluated through 1.8% agarose gel electrophoresis.

Analysis of TGF- β expressions

The secreted TGF- β in the supernatant of PAMs post PCV2 infection was detected by Quantibody porcine cytokine array system. Monocyte/Macrophage ancestry cells assume significant parts in opposing the infection of microorganisms. PCV2 has been displayed to infect and endure in cells of the immune framework, including PAMs. PCV2 alone possible causes exceptional utilitarian weakness in PAMs, remembering a decrease for phagocytosis and microbicidal ability. Recorded examinations on PCV2-infected monocyte/macrophage genealogy cells have shown that qualities connected with irritation and apoptosis are differentially communicated. In the current review, TGF- β signalling pathway target qualities were investigated in PCV2-infected PAMs by utilizing a PCR exhibit framework. The results of the PCR array showed that 58 of the 84 genes focusing on the TGF- β signalling pathway were differentially communicated in PAM in various phases of post PCV2 disease. These outcomes recommended that the TGF- β signalling pathway was potentially implicated in useful changes in PCV2-contaminated PAMs. Strangely, 48 qualities were differentially controlled at 1 hpi which fulfills the adsorption and disguise for the infection. Our outcomes demonstrated that PAMs showed a solid response towards PCV2 during adsorption and disguise of the infection.

Conclusion

TGF-β was altogether up regulated at mRNA and protein levels in PAM post-PCV2 disease. SMAD1 and SMAD3, in any case, key mediators of the TGF- β signalling pathway for cell apoptosis were down regulated. PCR cluster examination showed that 58 of the downstream qualities of the TGF- β signalling pathway were differentially communicated in PAMs in various stages after PCV2 disease. Among these qualities, IL-10 and TGF- β were up regulated essentially, proposing their cooperation in PCV2 pathogenesis. Be that as it may, the restrained apoptosis in the PAMs demonstrated by the down regulated smad1 and SMAD3 and by the up regulated C/EBPB, GADD45B, and BCL2 might have part of the way delayed PCV2 replication-permissive circumstances and caused tireless disease in clinical cases. The noticed articulation profile of TGF- β signalling pathway-related qualities in PCV2-contaminated PAMs might give a potential component of the practical changes in the invulnerable arrangement of PCV2-impacted pig.

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