Proteomic Analysis And Proinflammatory Cytokines Expressed Profile Of Primary Porcine Umbilical Vein Endothelial Cells After CSFV Infection

Classical swine fever (CSF) is a highly contagious febrile disease of domestic and wild pigs, which is caused by classical swine fever virus (CSFV). Clinical manifestation of the disease is hemorrhage fever and suppression immunity. Outbreaks can cause heavy losses to the swine industry and hamper international trade of animals and animal products; hence, it is notifiable to the World Organisation for Animal Health (OIE).CSFV is an enveloped, single stranded RNA virus, belonging to the genus Pestivirus within the family Flaviviridae. The viral genome is approximately 12.3 kb in size and contains a single large open reading frame that encodes a 3,898 amino acid polyprotein. The viral polyprotein is co-and post-translationally processed by both viral and host proteases into 12 mature proteins including Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.CSFV has a particular tropism for cells of the immune system including Endothelial Cells (ECs) and peripheral blood leucocyte cells (PBLs) and is known to cause severe leukopenia, in particular lymphopenia, featuring atrophy of primary lymphoid tissue and bone marrow, and depletion of different subsets of leukocytes (T-and B-lymphocytes, monocytes-macrophages and granulocytes) in infected pigs. ECs and PBLs could express and secret the cytokines and chemokines, some of which are potent enhancers of vascular permeability and mediators of pathologic changes such as high fever, coagulation defects, and the bleeding observed in some single-stranded RNA viral infections. One of the main pathological characteristics of CSF is the production of widespread hemorrhages. It is not clear that the molecular mechansim of hemorrhages caused by CSFV infection.To investigate the molecular mechanism of hemorrhages caused by CSFV infection. PUVECs were prepared and infected with CSFV high virulent strain, shimen strain, in vitro, and PBLs were isolated from CSFV infectd pigs. Subsequently. Kinetics of proliferation of CSFV in PUVECs, proteomic analysis of primary porcine endothelial cells after infection by CSFV, cytokines and chemokines expressed profile of CSFV-infected PUVECs, transcriptional kinetics of cytokine induction of PBLs in pigs infected with a CSFV, were studied.PUVECs were prepared by collagenase I treatment successfully, characterization of endothelial cells was identified by IF AT with VIII factor antibody. Monolayer of PUVECs was infected with CSFV Shimen strain, sample of CSFV-infected PUVECs were collected at the indicated time, then IF AT, TCID50 and genomic copies of virus were detecd. Results:The results showed that cytopathic effect (CPE) could not been observed in CSFV-infected PUVECs. Increased numbers of CSFV-infected PUVECs accompanied with the increased times of infecion. Infection rate raised up after CSFV infection, reaching 90%at 48 hours post infection (h p.i) and almost complete infection of PUVECs at 72 h p.i. Kinetics of viral proliferation was coincidenced with the viral genomic repliaction, which peaked at 48 h p.i. Characterization of CSFV cultivated in PUVECs was analyzed in the experiment. Hence, protein samples of two-dimensional difference gel electrophoresis with fluorescent dyes (2D-DIGE) of CSFV and mock-infected PUVECs were prepared at 48 h p.i.2D-DIGE was used to analyze the proteomic profile of PUVECs following CSFV infection. Of 15 protein spots with differential expression,8 were characterized by MALDI-TOF-MS/MS in infected PUVECs at 48 h p.i.:Moesin, peroxiredoxin 6, stathmin-1, a protein similar to nascent polypeptide-associated complex alpha subunit isoform 2, phosphoglycerate kinase 1, glucosidaseⅡ, transketolase and a-tubulin. These could be sorted into 5 functional groups:glycometabolism, cell proliferation, anti-oxidative stress, inflammatory response and cytoskeleton. Western blot and real-time RT-PCR analysis confirmed the down-regulation of phosphoglycerate kinase 1 (PGK1) and up-regulation of Moesin identified by 2D-DIGE. Pathway analysis of these 8 differentially expressed proteins showed that CSFV infection altered the metabolism, cytoskeleton and cell proliferation of PUVECs, and that consequently an inflammatory response was induced.ECs and PBLs could express and secrete the cytokines and chemokines as soon as the antigen stimulate it. Interaction between ECs and PBLs in blood vessel participate the inflammatory and immune response. In the present study, changes of cytokines and chemokines of CSFV-infected PUVECs and PBLs of CSFV stimulated pigs were evaluated by real time RT-PCR and ELISA. The fold increase in proinflammatory cytokines, chemokines and adhesion molecules mRNA was relative to the mock-infected PUVECs at 48 h p.i. Overall, chemokine IP-10 was most highly activated proinflammatory gene products induced in PUVECs. CSFV induced almost 41-fold increases in the expression of IP-10 transcripts at an MOI of 2.0. CSFV induced more than 23-fold increases in IL-la, IL-6, IL-8, MCP-1, TNF-aand 13-fold increases in ICAM-1. However, transcriptional level of VCAM-1 and IFN-y did not change between the CSFV and mock-infected PUVECs. Compared with mock-infectd PUVECs, IFN-y concentration of supernatant in CSFV-infected PUVECs did not change. Concentration of IL-2,IL-4,IL-5,IL-6,TNF-a and IL-8 secreted from infected PUVECs into the supernatants were significant higher than mock-infected PUVECs at 24 h p.i or 48 h p.i. Evaluation of cytokines, chemokines and adhesion molecules expression by real time RT-PCR and ELISA demonstrated enhanced levels of IL-1α, IL-6, IL-8, IP-10, TNF-a and ICAM-1 after viral exposure, which correlated with the appearance of inflammatory response. Real time PCR experiments were performed to determine the kinetics of cytokines, chemokines and adhesion molecules gene expression in PBLs isolated from CSFV-inoculated animals. The fold changes in these molecules mRNA was relative to the PBLs isolated from pre-inoculated animals. CSFV stimulated a rapid and sustained increase in IL-la, IL-4, IL-6 and TNF-amRNA that was detected at 2 d p.i and peaked at 6 or 8 d p.i. For IL-1α, IL-4, TNF-athe peak showed 239,48,46-fold inceease at 8 d p.i, and for IL-6 the peak showed a 10-fold increase at 6 d p.i. Up-regulation of chemokine IP-10 was peaked at 4 d p.i, which showed a 86-fold increase. CSFV infection resulted IL-8 increase range from 20-fold (2 d p.i) to 42-fold (4 d p.i), subsequent decrease range from 1.5-fold (6 d p.i) to 10-fold (8 d p.i) respectively. IFN-yexpression remained stable in the course of pre and post-inoculation. CSFV infection could induce up-regulation of proinflammatory cytokines and chemokines in vitro and in vivo.Compared the proliferation level of CSFV in expressed IP-10 cell lines and PK15. vrirus titer and vrius gene copies of CSFV-infected IP-10/PK-15 cell lines is not significant lower than CSFV-infected PK-15.Based on the above analytic data of proteomic and cytokines, network of inflammatory response following infection was anticipated. These results suggest the involvement of host cellular protein and cytokines in the pathogenesis of CSFV infection. These results characterize proteomic changes, proinflammatory cytokines, chenmokines and adhesion molecules expression pattern during the host response to CSFV in PBLs or PUVECs and provide the foundation to assess the functional significance of these mediators in pathogenesis of hemorrhage.