Proteomics Of Classical Swine Fever Virus Infection

Classical swine fever virus (CSFV), is a small, enveloped virus with a non-segmented positive-stranded RNA genome, belonging to the genus Pestivirus within the family Flaviviridae. The viral genome is approximately 12.5 kb in size and contains a single large open reading frame (ORF). This ORF is translated into a polyprotein which is further cleaved into 11-12 mature proteins : NH2–Npro–C–Erns–E1–E2–p7–NS2–NS3–NS4A–NS4B–NS5A–NS5B–COOH. CSFV is the pathogen of classical swine fever (CSF), a highly contagious swine disease with high morbility and mortality, featuring symptoms of hemorrhagic fever and immune suppression. The disease is a notifiable one of the World organization for animal health (OIE), usually leading to substantial economic losses to the pig industry worldwide. In recent years, CSF with lower morbility and mortality and mild, chronic, atypical symptoms was often observed, even in immune pigs herd.At present, nothing is known about the interaction between CSFV and host cells. To uncover cellular protein responses in CSFV-infected PK-15 cells, a differentially proteomic analysis was conducted using 2D electrophoresis followed by MALDI-TOF-MS/MS identification. Altered expression of 35 protein spots with at least 1.5-fold quantitative differences in expression in infected cells at 48 p.i. were identified in 2D gels, with 21 of these being characterized by MALDI-TOF-MS/MS, including 16 upregulated proteins and 5 downregulated proteins. Western-bloting analysis confirmed the upregulation of annexin 2 and downregulation of GAPDH. The altered proteins could be sorted into 7 groups according to their cellular function: cytoskeleton, energy metabolism, replication/transcription and translation processes, protein processing, anti-oxidant stress, heat shock protein and signal transduction. The altered expression of these proteins provides a response profile of PK-15 host cells to CSFV infection. Further study on these altered proteins may facilitate uncovering the mechanisms of CSFV infection and pathogenesis.According cellular function of differentially expressed proteins, it was speculated that annexin 2, hnRNPs and EF-1δmight be play critical roles in CSFV infection, genome replication, virion assembly and egress. In CSFV infected samples, GAPDH with function of apoptosis activation were down-regulated, and PGAM1 and anti-oxidative proteins (PRDX6 and thioredoxin- like) with function of apoptosis inhibition were up-regulated, which may be one mechanism of CSFV inhibiting CPE of infected PK-15 and establishing persistent infection.CSFV has particular affinity to tissue macrophage and endothelial cell. Studies indicats that CSFV infection lead to leucopenia and immune suppression. To uncover the effects on immune cells following experimental infection with CSFV Shimen strain and the mechanism of response to CSFV infection , changes of kinetics of CD4+ and CD8+ T cell subpopulation and leukocytes apoptosis in peripheral blood was analyzed using flow cytometry. Results showed that CD4+ and CD8+ T cell subpopulations were significantly reduced, the percentage of CD4+ T cell subpopulation decreased from 26.87% prior to infection to 8.83% 4 days post-infection (dpi) and 13.86% 7 dpi, respectively, and CD8+ T cell subpopulation from 28.17% prior to infection to 14.19% 4 dpi and 17.55% 7 dpi, respectively. Numbers of apoptotic leukocytes and early apoptotic leukocytes in peripheral blood of the infected pigs were significantly more than those of the control pigs, which indicated that cells apoptosis induced by CSFV infection is the major cause of leucopenia and immune suppression. Proteomic analysis indicated that 73 differentially expressed protein spots were found in CSFV-infected PBMC samples, with 55 of these(37 upregulated proteins, 18 downregulated proteins) being characterized by MALDI-TOF-MS/MS, reprensentive 34 unique proteins. Western-blot analysis indicated that Annexin A1 was remarkbaly upregulated and cofilin was downregulated, which confirmed the data of 2DE. These altered proteins are involved in cellular function of cytoskeleton, energy metabolism, nucleic acid/protein processing and anti-stress. These data provides the changes in profile of protein expression and physiological functions in PBMC from CSFV-infected porcine, which provides basis for further uncovering mechanism of CSFV pathogenesis.According cellular function of differentially expressed proteins, it was speculated that moesin, EF-1α, Bip protein might be involved in the mechanism of CSFV infection and replication. Derangement and disruption of cytoskeleton of PBMC induced by CSFV infection and upregulation of GAPDH may active apoptosis pathway of PBMC. Apoptosis of PBMC and down-regulation of T lymphocyte immune regulation factor of TSP-1 may be tightly relate to mechanism of immune suppression induced by CSFV infection.To found relevant biomarker and proteins of CSFV infection in serum from infected porcine, differentially proteomic analysis was performed using 2D-DIGE followed by MALDI-TOF-MS and RP-HPLC-MS/MS identification. Results showed that 17 differentially expressed proteins were found in CSFV-infected serum, including upregulated 9 and downregulated 8 spots. 14 of them were identified by MALDI-TOF-MS and RP-HPLC MS/MS. These proteins embodies pathological response of CSFV-infected porcine, further study is needed for potentiality of these proteins as biomarker of CSFV infection.In summary, the present study provided the first proteomic analysis of CSFV infection, and established the most comprehensive differential proteomic index of CSFV-infected cells in vitro and vivo, and CSFV-infected porcine serum. Further functional investigation of these altered proteins may facilitate understanding the pathogenic mechanisms and molecular responses of host cell to CSFV infection. This may also permit identification of biomarkers of CSFV infection and therapeutic targets or development of new diagnostic methods.