Development of Pseudovirus-based Assay to Investigate the Mechanism for Interspecies Transmission of Bat Coronavirus

• Main Authors: Sheng-Wei Wang, 王勝緯
• Other Authors: Yi-Ning Chen
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/30114031090419566753

碩士 === 中原大學 === 生物科技研究所 === 105 === Bats have been proven to be the natural host of severe acute respiratory syndrome (SARS). The preliminary study suggests that Middle-East respiratory syndrome coronavirus (MERS-CoV) is associated with bats. In order to understand the zoonotic risk of bat CoVs, infection studies of bat CoV on cells of different animal species are required. However, bat CoVs are difficult to grow in cell culture system and the experiments using live viruses have biosafety risks. Therefore, this study aims to establish a pseudovirus system that can infect susceptible cells once but have no ability to replicate in the infected cells. By inserting the spike (S) gene of bat CoV into lentivirus-based pseudovirus system, bat CoV pseudovirus can be produced for the infection studies on different cells because S protein determines whether CoV can infect susceptible cells. The full length of S gene amplified from the cDNA of Scotophilus bat CoV 512 strain CYCU-S1/2013 isolated in Taiwan has been cloned into pEGFP-N3 as pEGFP-Sco-S. Pseudovirus expressing S protein of Scotophilus bat CoV 512 and green fluorescent protein (GFP) were produced after co-transfection of pEGFP-N3-Sco-S, packaging plasmid. The production of Scotophilus bat CoV pseudovirus was confirmed by virus infection test on 293T cells and immunofluorescent antibody assay(IFA) against GFP and p24 protein of lentivirus. The Scotophilus bat CoV pseudovirus-infected 293T cells expressed red fluorescence and were tested positive for p24 capsid protein of lentivirus. The morphology of pseudovirus will be examined by Transmission electron microscopy (TEM). The results showed that lentivirus-based pseudovirus system can produce Scotophilus bat CoV pseudovirus for further infection studies on cells of different animal species to understand the cross-species transmission of bat CoV.