The Influence Of CSFV Chronic Infection On The Production Of Cytokines

Classical Swine Fever(CSF) caused by Classical Swine Fever Virus(CSFV) is a highly contagious and hemorrhagic disease of pigs and is classified as a notifiable animal disease by Office International des Epizooties(OIE). Outbreaks of CSF usually lead to significant economic losses in many countries worldwide. Moderate virulent strains can cause chronic infection, while the attenuated vaccine strain could induce immunological protection. By studying the transcription of cytokines after experimental infecting peripheral blood monouclear cells(PBMC) of pigs with moderate virulent strains and the vaccine strain(HCLV) in vitro and vivo, we can systematically analyse these strains'effects on immune systems. The results of this study will supply scientific basement for explaining the mechanism of CSFV chronic infection.In this study, we analyse the effects of CSFV strain HeBHHl/95and HCLV infected PBMC on levels of mRNA accumulation of59genes (interleukin, chemokine, toxicity related molecules, IL-1β-converting enzyme, adhesion molecules, interferon and interferon regulatory factor, antiviral molecules, cytokine receptors)involved in modulation of the host immune response in vitro.Culture supernatants were collected at different hours postinfection (24hpi,48hpi),59cytokines detected by qRT-PCR. At24hpi,59genes showed altered expression upon infection with CSFV strain HeBHH1/95including:9cytokine genes showed up-regulation,50cytokine genes down-regulation. Expression analysis of9cytokine genes showed significant up-regulation of MCP-1, IL-la, IL-1β, IL-8, IL-6at24hpi (RQ>2.1), IL-la expression reached a3.16-fold increase, the highest mRNA accumulation observed in this study, while IFN-a expression was the lowest (RQ=0.54);59genes showed altered expression upon infection with CSFV strain HCLV including:9cytokine genes showed up-regulation,50cytokine genes down-regulation. Expression analysis of9cytokine genes showed significant up-regulation of MCP-1, IL-la, IL-lβ,IL-8, IL-10, TGF-β2(RQ>2), MCP-1expression was the highest (RQ=3.02), while IFN-a expression was the lowest (RQ=0.447). At48hpi,59genes showed altered expression upon infection with CSFV strain HeBHH1/95including:12cytokine genes showed up-regulation,44cytokine genes down-regulation, TLR-4and IFN-a remained unchanged. IL-1α, MCP-1and IL-8showed significantly up-regulation (RQ>2), the highest expression was IL-1α (RQ=2.75), IL-12p35expression was the lowest (RQ=0.427);59genes showed altered expression upon infection with CSFV strain HCLV including:12cytokine genes showed up-regulation,47cytokine genes down-regulation. MCP-1, IL-1α, IL-1βand IL-8showed significantly up-regulation (RQ>2.3), the highest expression was IL-1α (RQ=2.63), level ofTGF-R expression was the lowest (RQ=0.295). Infected with CSFV strain HeBHH1/95or HCLV in vitro, the cytokines involved in modulation of the host immune response showed altered expression, proinflammatory cytokines were significantly up-regulation, such as IL-la, IL-1β, IL-8. While toxicity related molecules, IL-1β-converting enzyme, adhesion molecule were down-regulated significantly. The patterns of cellular gene expression in PBMC induce by HeBHHl/95and HCLV are quite different. Data gathered here suggests that CSFV strain HeBHH1/95and HCLV could induce inflammatory, while the patterns of inducing anti-viral gene expression are quite different.23piglets were divided into3groups randomly as HeBHH1/95infected group, HCLV vaccinated group and control group. PBMC and serum were prepared at different phases(0dpi, ldpi,2dpi,5dpi,7dpi,9dpi,12dpi,16dpi,20dpi,23dpi,28dpi,30dpi,35dpi and43dpi). qRT-PCR, flow cytometry and ELISA were used for detecting mRNA accumulation of10cytokines, analysis of lymphocyte subsets and levels of CSFV antibody.Results showed that the experimental pigs of HeBHHl/95infected group can survived more than43dpi. CSFV chronic infection can reducing T lymphocytes and immunosuppression. The number of уδT cells, killer T cells and helper T cells reduceed from2dpi, those cells have direct killing ability, while activated memory T cells without direct killing ability reduced in9dpi. All detected antiviral factor (IL-1a, IL-2, IL-4, IL-8, IFN-a, IFN-β, TNF-α, Mx1, PKR and OAS) of HeBHH1/95infected group were significant up-regulation. lymphocyte cells of HCLV vaccinated group rose slightly on5-7dpi, reached peak on12dpi, showed significantly different from the control group (P<0.05), then gradually decreased and became stable. Moreover, flow cytometry results showed that the increment of the lymphocyte count mainly due to the helper T cells increase. CSFV antibody of HCLV vaccinated group showed positive (blocking rate>40%) on9-12dpi, IL-8transcription levels were significantly increased in this period. The results revealed that the Th cells induced B lymphocyte maturation and expression of cytokines, produced specific CSFV antibody and form the mechanisms of immune protection by proliferation.Steady state levels of mRNA accumulation were assessed for cytokines involved in modulation of the host immune response, infected with CSFV strain HeBHH1/95and HCLV in vitro and vivo, by using qRT-PCR and flow cytometry. Analyze the dynamic changes of cells and cytokines that play a key role in the process of virus to evade host immune and induce specific antibodies, by comparing each index change of infection before/after and immunity before/after. With purpuse of exploring the role of cytokines and providing a scientific basis to in order to explain the mechanism of CSFV chronic infection and attenuated vaccine strain immunization.