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Zeng (2017) - Reverse Genetic System of Bat SARS-like Coronaviruses and Function of ORFX - English
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Zeng (2017) - Reverse Genetic System of Bat SARS-like Coronaviruses and Function of ORFX - English.docx
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Statement of originality and authorization of use of the paper
Acknowledgement
Abstract
SUMMARY
List of abbreviations
Table of Contents
Chapter 1 Introduction
1.1 Overview of SARS and SARS-like coronavirus
1.1.1 SARS coronavirus
1.1.2 Animal traceability of SARS coronavirus
1.1.3 Bat SARS-like coronavirus
1.2 Overview of coronavirus reverse genetics methods
1.2.1 Viral reverse genetics
1.2.2 Targeted RNA recombination technology
1.2.3 Construction of infectious BAC clones
1.2.4 Systematic splicing of viral cDNA fragments
1.2.5 Vaccinia virus vector-based methods
1.2.6 Other methods
1.3 Research content and significance of this thesis
Chapter 2 Establishment of a reverse genetics system for SARS-like coronavirus in bats
2.1 Materials and Methods
2.1.1 Materials and reagents
2.1.2 Extraction of WIV1 virus genomic RNA
2.1.3 Cloning of WIV1 virus cDNA fragments
2.1.4 Identification and conservation of WIV1 virus cDNA fragment T clone
2.1.5 Modification of pBeloBAC11 plasmid
2.1.6 Construction of a full-length infectious BAC clone of WIV1
2.1.7 Identification of BAC-positive clones
2.1.8 Rescue of recombinant viruses
2.1.9 Purification of viruses by the spot-eating method
2.1.10 Determination of viral titers by the phlebotomy method a staining with crystal violet
2.1.11 Identification of recombinant viruses
2.1.12 Construction of a recombinant virus expressing a fluorescent reporter gene
2.1.13 Northern blot
2.1.14 Virus one-step growth curve
2.2 Results and Analysis
2.2.1 Construction scheme of infectious clones
2.2.2 Virological characteristics of recombinant viruses
2.2.3 Successful rescue of reported viruses
2.3 Discussion
Chapter 3 Construction of recombinant virus of bat SARS-like coronavirus and its receptor study
3.1 Materials and methods (refer to Chapter 2 Materials and methods)
3.1.1 Materials and reagents
3.1.2 Cloning of S gene
3.1.3 Construction and rescue of recombinant viruses
3.1.4 Immunofluorescence experiments
3.1.5 Growth curve
3.1.6 SARS-like coronavirus receptor utilization
3.1.7 Antibody neutralization experiment (phage-spot reduction experiment)
3.2 Results and Analysis
3.2.1 Construction scheme of S-gene chimeric recombinant virus
3.2.2 Rescue of SARS-like coronavirus
3.2.3 Replication of SARS-like coronaviruses
3.2.4 Utilization of different animal-derived receptors by SARS-like coronaviruses
3.2.5 Cross-neutralization of SL-CoV by SARS-CoV RBD antibody
3.3 Discussion
Chapter 4 Functional studies of the SL-CoV-specific accessory protein ORFX
4.1 Materials and Methods (refer in part to Chapters 2 and 3 Materials and Methods)
4.1.1 Materials and reagents
4.1.2 RT-PCR amplification of viral subgenomes containing Leader sequences
4.1.3 Construction of ORFX-deficient virus and GFP recombinant virus
4.1.4 Northern blot
4.1.5 Subcellular localization of ORFX
4.1.6 Dual luciferase reporter assay and quantitative PCR
4.1.7 IRF3 nucleation experiments
4.1.8 Effect of ORFX-deficient viruses on cytokines
4.1.9 Interferon sensitivity test
4.2 Results and Analysis
4.2.1 ORFX is an expressed functional gene
4.2.2 Subcellular localization of ORFX
4.2.3 Inhibition of IFN expression by ORFX
4.2.4 ORFX activates NF-kB
4.3 Discussion
Chapter 5 Conclusion and Outlook
5.1 Conclusion
5.2 Problems and outlook
References
Author Biography
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Zeng (2017) - Reverse Genetic System of Bat SARS-like Coronaviruses and Function of ORFX - English.docx
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