Detection of Differential Expression of Porcine IFN- α Subtypes by Reverse Transcription Polymerase Chain Reaction
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China.Journal of Interferon & Cytokine Research (Impact Factor: 2). 08/2007; 27(7):579-87. DOI: 10.1089/jir.2006.0126
The porcine interferon-alpha (IFN-alpha) multigene family is a new IFN-alpha system in recent research. Characterization of the PoIFN-alpha multigene family has been described in our previous work, and 14 functional PoIFN-alpha genes were detected in the porcine genome. In this study, we designed subtype-specific primers and consensus primers for PoIFN-alpha. The expression of PoIFN-alpha was detected using the two PCR strategies in three systems, namely, poly(I).poly(C)-DEAE-dextran-induced PK15 cells, pseudorabies virus-infected PK15 cells, and infected PK15 cells with an attenuated strain of swine fever virus, respectively. In poly(I).poly(C)-DEAE-dextran-induced PK15 cells, the expression of IFN-alpha2, -alpha3, -alpha4, -alpha8, and -alpha9 after 6-h/24-h inducement in PK15 cells were observed. In pseudorabies virus-infected PK15 cells, the expression of PoIFN-alpha2, -alpha3, -alpha8, -alpha9, -alpha10, and -alpha13 was observed after 6-h/24-h infection, and in the attenuated strain of swine fever virus-infected PK15 cells, upregulation of PoIFN-alpha2, -alpha3, -alpha4, -alpha8, -alpha9, and -alpha10 was detected. The results of realtime quantitative PCR analysis suggested that the expression was time-dependent in pseudorabies virus/poly(I).poly(C)-DEAE-dextran-induced PK15 cells, but in the attenuated swine fever virus-infected PK15 system, the expression level of IFN-alpha subtypes was not obviously time dependent.
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ABSTRACT: During the acute phase of the viral hemorrhagic disease, classical swine fever (CSF), a severe hematologic depletion in primary lymphoid organs and depletion of peripheral blood T and B lymphocytes are observed. The onset of these pathologic events is before viremia and independent of leukocyte infection, indicating a host-mediated effect possibly through a cytokine storm. Here, we show that high serum levels of interferon- alpha (IFN-alpha) were found during this phase of CSF, detectable as early as 2 days postinfection and reaching maximum levels 3-5 days postinfection (250-1300 U/mL). This IFN-alpha response was related to the virulence of the viral strain used, with avirulent virus not inducing any detectable serum IFN-alpha. A progressive depletion of natural IFN-producing cells/plasmacytoid dendritic cells (pDC), the likely in vivo source of IFN-alpha, was also induced by the viral infection. An important finding was that the onset of severe lymphopenia was concomitant with the IFN-alpha responses, and all animals with serum IFN-alpha had depleted B and T lymphocytes. A statistically significant correlation between lymphocyte depletion and serum IFN-alpha indicates a relationship between the two events, which is supported by the known hematologic effects of high IFN-alpha doses in vivo.Journal of Interferon & Cytokine Research 05/2006; 26(4):248-55. DOI:10.1089/jir.2006.26.248 · 2.00 Impact Factor
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ABSTRACT: The coding sequences of porcine interferon-stimulated gene 15 (ISG15) and the interferon-stimulated gene (ISG43) were cloned from swine spleen mRNA. The amino acid sequences deduced from porcine ISG15 and ISG43 genes coding sequence shared 24-75% and 29-83% similarity with ISG15s and ISG43s from other vertebrates, respectively. Structural analyses revealed that porcine ISG15 comprises two ubiquitin homologues motifs (UBQ) domain and a conserved C-terminal LRLRGG conjugating motif. Porcine ISG43 contains an ubiquitin-processing proteases-like domain. Phylogenetic analyses showed that porcine ISG15 and ISG43 were mostly related to rat ISG15 and cattle ISG43, respectively. Using quantitative real-time PCR assay, significant increased expression levels of porcine ISG15 and ISG43 genes were detected in porcine kidney endothelial cells (PK15) cells treated with poly I:C. We also observed the enhanced mRNA expression of three members of dsRNA pattern-recognition receptors (PRR), TLR3, DDX58 and IFIH1, which have been reported to act as critical receptors in inducing the mRNA expression of ISG15 and ISG43 genes. However, we did not detect any induced mRNA expression of IFNalpha and IFNbeta, suggesting that transcriptional activations of ISG15 and ISG43 were mediated through IFN-independent signaling pathway in the poly I:C treated PK15 cells. Association analyses in a Landrace pig population revealed that ISG15 c.347T>C (BstUI) polymorphism and the ISG43 c.953T>G (BccI) polymorphism were significantly associated with hematological parameters and immune-related traits.Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 04/2009; 153(4):301-9. DOI:10.1016/j.cbpb.2009.03.006 · 1.55 Impact Factor
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ABSTRACT: Interferon-omega is a member of the type I interferon family. In this work, 8 functional porcine interferon-omega genes and 4 pseudogenes present on porcine chromosome 1 were identified in the porcine genome database by BLAST scanning. Their genetic and genomic characteristics were investigated using bioinformatics tools. Then the PoIFN-omega functional subtype genes were isolated and expressed in BHK-21 cells. The PoIFN-omega subtypes possessed about 10(4) to 10(5) units of antiviral activity per milliliter. PoIFN-omega 7 had the highest antiviral activity, about 20 times that of PoIFN-omega 4, which had the lowest antiviral activity. Differential expression of the subtypes was detected in PK15 cells and porcine peripheral blood mononuclear cells (PBMCs) in response to pseudorabies virus and poly(I).poly(C). Expression of PoIFN-omega 2/-omega 6 was up-regulated to the greatest extent by virus infection.Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 10/2009; 29(10):687-93. DOI:10.1089/jir.2008.0060 · 2.00 Impact Factor
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