Understanding Differential Gene Expression In Swine Umbilical Vein Endothelial Cell And Macrophage In Response To Classical Swine Fever Virus Shimen-strain And C-strain Infections

Classical swine fever (CSF) is a highly contagious infection of swine, caused by classicalswine fever virus (CSFV). Over the past few decades, the epidemic of CSF has beeneffectively controlled by takingC-strain vaccine and culling. But CSF begins to be increasingagain in our countryin recent years, especially being a chronic and atypical CSF, which bringsgreat difficult to prevention and control work. Although CSFV pathogenesis research hasmade some progress, previous research work mainly aimed on the whole virus or viral geneswhich could produce pathogenic to some genes of host cell, and lacked systems analysis ontranscription regulatory response of host cell. Meanwhile, it is unclear what response iscaused by virulent strain of CSFV. For this purpose, this paper carried out study ondifferentially expressed genes in swine umbilical vein endothelial cell (SUVEC) andmacrophages infected by CSFV Shimen strain and C strain, which could find out the generegulation characteristic on transcriptome of host cell in response to CSFV infection, furtherto provide scientific data and new starting point for the study on molecular mechanisms ofacute CSF and persistent infection of CSFV.This work has obtained the following results.(1) To the best of our knowledge, this work offered a first report regarding the successfulapplication of high resolution melting (HRM) analysis for simultaneous, one-step detectionand differentiation of CSFV C and Shimen strains. As a single-tube, probe-free, closeddetection system being superior to previous PCR technology, this method not only detects butalso identify CSFV C and Shimen strains. It could provide a detection method to obtain singlevirus infection or no virus infection cell sample for pathogenesis studies and also to screenlarge number of clinical samples in CSF epidemiological study of CSFV.(2) By the application of digital gene expression tag profiling (DGE), a kind ofhigh-throughput sequencing technology, a comparative study first was carried out thatdifferences of the gene expression of SUVEC were showed on CSFV Shimen strain infection24hours and the uninfected group. Data analysis results indicated that CSFV Shimen strainbrought out anti-apoptotic, anti-inflammatory and altering cell cycle to achieve their ownproliferation. So, host cell could be failed to effectively kill virus, and ultimately resulted in damages of tissues and organs. Research further identified PDIA3, AXL were up-regulatedand PDGF, NAMPT was down-regulated in early stages of CSFV Shimen infection,suggesting that these genes were involved in the acute infection mechanism caused byvirulent strains of CSFV.(3)Three groups of DGE detection database, CSFV Shimen strain infection, CSFV ShimenC strain infection, uninfected control group, were comparatively analyzed each other to findresponse differences of SUVEC presented at the mRNA level by using GO and KEGGenrichment analysis. The analysis results showed a possible mechanism of CSFV Shimenstrain72h infection to SUVEC in which virus altered the mechanism of RNA splicing, proteinsynthesis and degradation, and regulated normal cell function, especially in cytoskeleton, celladhesion and cell cycle, in order to achieve virus replication. GO gene function analysis andcluster analysis showed that SRPX2, SOD2, HMGB1and TYMS transcription were inhibitedand VEGFC and CAV1were unregulated after CSFV Shimen strain72h infection in SUVEC.The study provides a new research idea for CSFV to illustrate the pathogenesis of acute CSFin the future.(4) In this work, DGE detection and analysis techniques identified VEGFC expression waseffected after CSFV Shimen strain infection in SUVEC. For validating this result, molecularbiology tests were carried out on mRNA transcription and protein synthesis of VEGFC. Thefinal result showed VEGFC were significantly up-regulated as a result of CSFV Shimen straininfection. The result could give a better understanding for the pathogenesis of the virulentstrain of CSFV.(5) Another three groups of DGE detection database from the macrophage, CSFV Shimenstrain infection, CSFV Shimen C strain infection, uninfected control group, werecomparatively analyzed each other to find different response to CSFV infection by using GOand KEGG enrichment analysis. The result showed that p53signaling pathway played animportant role on the infection of CSFV Shimen strain in macrophages. Further tests showedthat the CSFV Shimen strain infection in macrophages promoted phospho-p53(Ser15) proteinphosphorylation process, increased p21protein expression, and decreased14-3-3proteinexpression. CSFV Shimen strain could activate p53signaling pathway to arrest cell cycle ofmacrophages for achieving their replication, eventually resulting in persistent infection ofCSFV in pigs.