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Abstract
At the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan, which subsequently caused an unprecedented pandemic around the world. As of March 2023, the coronavirus disease 2019 (COVID-19) pandemic has led to more than 670 million people infected and over 6.8 million deaths globally. The COVID-19 pandemic severity has highlighted the urgent need of understanding SARS-CoV-2 virology and identifying novel antiviral mechanisms. The current paradigm of SARS-CoV-2 research mostly employs biosafety level 2-compatible systems such as SARS-CoV-2 spike-pseudoviruses or SARS-CoV-2 virus-like particles, which fail to resemble the complete viral structure or deliver exogeneous genes upon infection. This dissertation project is designed to develop a novel SARS-CoV-2 viral vector composed of all four SARS-CoV-2 structural proteins, the packaging signal sequence of SARS-CoV-2, a reporter gene, and an RNA amplification component of Venezuelan equine encephalitis virus (VEEV). This VEE-SARS-CoV-2 viral vector transduces target cells in an ACE2-dependent manner, and the incorporation of the VEEV self-amplification mechanism generates a significantly higher and extended gene expression profile in transduced cells. This dissertation consists of four chapters. Chapter 1 is a literature review of the characteristics of SARS-CoV-2 and COVID-19 and a summary of current model systems of SARS-CoV-2 research. Chapter 2 describes the details regarding the materials, instruments and methods used to develop the VEE-SARS-CoV-2 vector system. Chapter 3 shows the data of design, optimization, and characterization of the vector system and demonstrates its applications in neutralizing antibody quantification and antiviral drug testing. Chapter 4 discusses the advantages, applications, limitations and future perspectives of utilizing this novel vector system for studying SARS-CoV-2 and developing antiviral strategies against COVID-19. The results presented provide direct evidence in support of successful establishment of SARS-CoV-2 viral vectors and clear demonstration of their broad applications in studying virology of SARS-CoV-2, drug and vaccine development against COVID-19, and probing the underlying mechanisms of various symptoms associated with COVID-19.