蝙蝠研究
2019_利用蝙蝠冠狀病毒的偽病毒系統探討高頭蝠冠狀病毒跨物種傳播_徐筱琹
出版年份：2019
研究生：徐筱琹
分類：碩士論文
題目：利用蝙蝠冠狀病毒的偽病毒系統探討高頭蝠冠狀病毒跨物種傳播
Title：Cell entry of Scotophilus bat coronavirus-512 and severe acute respiratory syndrome coronavirus by using pseudovirus system
摘要：
研究顯示蝙蝠為嚴重急性呼吸道症候群冠狀病毒（Severe acute respiratory syndrome coronavirus, SARS-CoV）的自然宿主，SARS-CoV可進行跨物種感染。冠狀病毒刺棘（Spike, S）蛋白與細胞受體結合是病毒感染細胞的第一步。套膜蛋白質體是將高頭蝠蝙蝠冠狀病毒（Scotophilus kuhlii coronavirus 512, Sco）的S蛋白基因與綠螢光蛋白（Green fluorescent protein, GFP）基因連接後製作成pEGFP-Sco-S，或將SARS-CoV的S蛋白基因製作成pCMV-SARS-spike，或將水疱口炎病毒（Vesicµlar stomatitis virus , VSV）的醣（Glycoprotein, G）蛋白基因製作成pMD.G。將套膜蛋白質體分別與慢病毒偽病毒系統的輔助包裝質體（pCMV△R8.91）和螢光素酶轉移質體（pLAS2w.FLuc.Ppuro） 共同轉染至293T 細胞中可產生高頭蝠蝙蝠冠狀病毒偽病毒（Sco-S），SARS-CoV偽病毒（SARS-S），或VSV偽病毒（VSV-G）。將三種偽病毒對不同細胞（HEK-293T、MDCK、PK15、Vero、Paki、Pabr、Palu、Caco-2、IEC-6、Fcwf-4、MFK）進行感染測試，48小時後用螢火蟲螢光素酶定量。所有細胞都可被三種偽病毒感染，Sco-S對MDCK、Pabr及Palu有高度感染，SARS-S對293T、MDCK、PK15及MFK有高度感染，VSV-G則對大部分細胞有高度感染，除了Palu和Caco-2。結果顯示Sco-S及SARS-S都有跨物種感染的潛力。
Abstract：
Bats have been shown to be the natural reservoir of severe acute respiratory syndrome coronavirus （SARS-CoV）, which can have cross-species transmission. The binding of coronaviral spike （S） protein and cell receptor is the first step of virus infection. To produce pseudotyped Scotophilus bat CoV-512 containing S protein （Sco-S）, SARS-CoV containing S protein （SARS-S） conjugated to green fluorescence protein （GFP）, and vesicµlar stomatitis virus （VSV） containing glycoprotein （VSV-G）, three plasmids were co-transfected into 293T cells: the packaging plasmid （pCMV△R8.91）, the transfer plasmid encoding luciferase gene （pLAS2w.FLuc.Ppuro）, and one of the envelope plasmid encoding surface viral protein （pEGFP-Sco-S, pCMV-SARS-S, or pMD.G）. The infectivity of three pseudoviruses was tested and quantified by luciferase activity level in 11 cell types, including human kidney （293T）、dog kidney （MDCK）、pig kidney （PK15）、monkey kidney （Vero）、fruit bat kidney （Paki）、fruit bat brain （Pabr）、fruit bat lung （PaLu）、human colon （Caco-2）、rat small intestine （IEC-6）、cat whole fetus cell （Fcwf-4）、eastern bent-winged bat kidney （MFK）, after 48 hours post infection. All cells can be infected by three pseudoviruses. Sco-S had the strongest infectivity in MDCK, Pabr and Palu cells, SARS-S had the strongest infectivity in 293T, MDCK, PK15 and MFK cells, and VSV-G can infect strongly in all cells except Palu and Caco-2 cells. It is suggested that both Sco-S and SARS-S have cross-species transmission potential and further studies are required to understand the mechanism.