小反芻獸疫是一種由小反芻獸疫病毒（PPRV）引起的家養(yǎng)和野生小反芻動(dòng)物高度傳染性疾病。有關(guān)PPRV復(fù)制的分子機(jī)制及其與宿主的相互作用研究仍不清楚。本研究為了更好地理解PPRV復(fù)制的分子機(jī)制及病毒與宿主細(xì)胞相互作用。利用分子生物學(xué)技術(shù),研究PPRV感染對(duì)Vero細(xì)胞的影響,以及PPRV抵抗宿主細(xì)胞免疫的途徑。我們利用MOI為3的PPRV感染Vero細(xì)胞,對(duì)感染后36h、48h、60h及72h的樣本進(jìn)行分析,結(jié)果表明PPRV對(duì)eIF2α具有去磷酸化的作用,在哺乳動(dòng)物細(xì)胞（如HEK293T）過(guò)表達(dá)PPRV P蛋白能夠誘導(dǎo)宿主細(xì)胞生長(zhǎng)阻滯DNA損傷蛋白（GADD34）的表達(dá),已知GADD34與eIF2α去磷酸化有關(guān)。此外,我們研究發(fā)現(xiàn)PPRV P蛋白能夠抑制PERK/eIF2α磷酸化,使用eIF2α去磷酸化選擇性抑制劑（Salubrinal）處理細(xì)胞,能夠在蛋白水平和mRNA水平抑制PPRV復(fù)制。我們推測(cè)eIF2α去磷酸化有利于PPRV復(fù)制,而且PPRV P蛋白參與調(diào)控這一分子機(jī)制。在病毒感染的過(guò)程中,宿主細(xì)胞特有的信號(hào)通路未折疊蛋白反應(yīng)（UPR）可能被激活或抑制,以對(duì)抗病毒誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激維... 

【文章頁(yè)數(shù)】：91 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
abstract
List of Abbreviations
ChapterⅠLiterature Review
    1.1 Description and the causative agent of peste des petits ruminants
        1.1.1 The PPRV virion structure and genome
        1.1.2 PPRV structural proteins
        1.1.3 PPRV hosts range and susceptibility
        1.1.4 Pathogenesis and clinical symptoms of PPRV
        1.1.5 Diagnosis of PPRV
        1.1.6 Epidemiology,geographic distribution and eradication program of PPRV
        1.1.7 Molecular biology and Virus/host interaction of PPRV
    1.2 The aims of this study
Chapter Ⅱ Case-study of epidemiology and molecular characterization of PPRV in an outbreak in Burundi(2017-2018)
    2.1 Introduction
    2.2 Materials and methods
        2.2.1 Suspicion of PPR outbreak in Burundi and sampling for laboratory diagnosis
        2.2.2 Serology for the detection of antibodies directed against PPR virus
        2.2.3 Reverse transcriptase-PCR for detection of morbillivirus and PPRV RNAs
        2.2.4 Sequencing and phylogenetic analysis
    2.3 Results
        2.3.1 Serological and PCR analysis
        2.3.2 Results of a retroactive study
        2.3.3 Phylogenetic analysis
    2.4 Discussion
    2.5 Conclusion
Chapter Ⅲ Study of the role of PPRV phosphoprotein in virus replication and virus/host cells interaction
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 Plasmids
        3.2.2 Primers design for cloning N and P
        3.2.3 PCR amplification
        3.2.4 Gel electrophoresis and DNA purification
        3.2.5 Restriction enzyme digestion
        3.2.6 DNA ligation
        3.2.7 E.coli transformation
        3.2.8 Plasmid DNA extraction
        3.2.9 Screening of the DNA inserts
        3.2.10 Sequencing and sequences analysis
        3.2.11 Cells culture,virus,antibodies and reagents
        3.2.12 Transfection and transient expression of target proteins
        3.2.13 SDS-PAGE and Western blotting
        3.2.14 Chemical treatment
        3.2.15 Immunofluorescence assay(IFA)
        3.2.16 RT-q PCR assay and PPRV m RNA quantification
        3.2.17 Data analysis
    3.3 Results
        3.3.1 Confirmation of PPRV P and PPRV N plasmids construction
        3.3.2 Analysis of PPRV P and PPRV N proteins expression
        3.3.3 PPRV infection represses PERK/e IF2αphosphorylation in Vero cells
        3.3.4 PPRV infection modulates GADD34 protein expression in infected Vero cells
        3.3.5 PPRV infection represses ER stress-induced e IF2αphosphorylation
        3.3.6 Manipulation of e IF2αphosphorylation differentially regulates PPRV replication
        3.3.7 PPRV P protein is involved in PPRV mediated e IF2αdephosphorylation
        3.3.8 PPRV P protein induces host cellular GADD34
    3.4 Discussion
    3.5 Conclusion
Chapter Ⅳ PPRV modulation of UPR and Stress Granules formation
    4.1 Introduction
    4.2 Materials and methods
        4.2.1 Plasmids
        4.2.2 Cells culture,virus,antibodies and reagents
        4.2.3 Transfection and transient expression of target proteins
        4.2.4 Stress granule induction and assessment
        4.2.5 SDS-PAGE and Western blotting
        4.2.6 Immunofluorescence assay(IFA)
        4.2.7 Co-immunoprecipitation(co-IP)
        4.2.8 Data analysis
    4.3 Results
        4.3.1 PPRV infection induces ATF6 but not IRE1/XBP1 arm of the UPR
        4.3.2 PPRV infection inhibits stress granules formation
        4.3.3 PPRV inhibition of SGs assembly may target G3BP1
        4.3.4 P protein is involved in the control of UPR
        4.3.5 P protein is involved in the control of SGs formation
        4.3.6 P protein alone can inhibit oxidative SG formation
        4.3.7 PPRV P protein may target G3BP1 to control SG formation
    4.4 Discussion
    4.5 Conclusion
Chapter Ⅴ General Conclusions
REFERENCES
Acknowledgements
Author’s Curriculum Vitae


【參考文獻(xiàn)】：
期刊論文
[1]Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses[J]. Alfred Niyokwishimira,Yongxi Dou,Bang Qian,Prajapati Meera,Zhidong Zhang.  Virologica Sinica. 2018(06)



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