蝙蝠研究
2020_台灣蝙蝠麗莎病毒之分子與血清學調查_葉鉌怡
出版年份：2020
研究生：葉鉌怡
分類：碩士論文
題目：台灣蝙蝠麗莎病毒之分子與血清學調查
Title：Molecular and serological detection of lyssavirus in the bat populations of Taiwan
摘要：
麗莎病毒(Lyssavirus)是一群可在人畜間共同傳染的病毒，其中包括會造成狂犬病的狂犬病病毒(Rabies virus)。攜帶狂犬病病毒的宿主會透過咬傷來傳播，引發致命的急性腦炎，而狂犬病病毒以外的其他麗莎病毒在國外有造成人類死亡案例，但目前發病與造成的病徵還不是很清楚。台灣在過去並未在蝙蝠中檢測出狂犬病病毒，但在2016年及2018 年於東亞家蝠(Pipistrellus abramus)及絨山蝠(Nyctalus noctula)檢測出兩種新型的蝙蝠麗莎病毒。為了瞭解台灣蝙蝠族群中麗莎病毒的感染狀況，本研究使用偵測麗莎病毒屬基因型1~6 型的專一性引子對去檢測麗莎病毒N 基因與P 基因的一部分，檢測68 糞便樣本、108 個口水拭子與29 個腦組織，結果皆為陰性。為了檢測蝙蝠血清中麗莎病毒的特異性抗體，本實驗利用桿狀病毒表現系統來表達台灣蝙蝠麗莎病毒醣蛋白(Glycoprotein, G)，並使用不同的洗滌劑與變性劑處理後純化，結果使用Triton X-100 細胞裂解液所得到的可溶性醣蛋白較RIPA 與CHAPs 細胞裂解液來得多，使用濃度越高的尿素回溶細胞團塊的效果越好，在純化細胞裂解上清液時發現使用含有3mM濃度DTT 的緩衝液效果最佳，純化細胞團塊時則是使用含有10mM 濃度DTT 的緩衝液的效果最佳。使用免疫螢光法檢測台灣蝙蝠血清中麗莎病毒的特異性抗體，檢測121個血清結果皆為陰性。藉此核酸檢測與血清學調查，可了解台灣蝙蝠麗莎病毒在台灣蝙蝠族群中的感染狀況。
Abstract：
Lyssavirus is a group of viruses that can be transmitted between humans and animals, including rabies virus. Rabies virus is transmitted through bites and causes fatal acute encephalitis. Some other Lyssavirus was known of causing human deaths abroad even though the symptoms of these infections cases are not clear. Rabis virus has not been detected in the bat populations of Taiwan previously. In 2016 and 2018, two new types of bat Lyssavirus were detected in Pipistrellus abramus and Nyctalus noctula. In order to understand the infection status of Lyssavirus in the bat population of Taiwan, this study used specific primer pairs for detecting Lyssavirus genotypes 1 to 6 to detect part of the N gene and P gene of Lyssavirus. In the detected 68 fecal samples, 108 oral saliva swabs and 29 brain tissues were all negative. Baculovirus expression vector system was then used to express the glycoprotein (G) of Taiwan bat Lyssavirus. Different conditions of protein expressions and purification methods were tested. More protein was produced from the solution of cells lysed by Triton X-100 than those by RIPA or CHAPs. The insoluble cell pellets were dissolved more by using higher concentrations of Urea. The best quality of protein was purified from the solution of lysed cells by using 3mM of DTT in the buffers for protein purification. The best quality of protein was purified from the dissolved solutions of insoluble cell pellets by using 10mM of DTT in the buffers for protein purification. Immunofluorescence assay (IFA) was used to detect the antibodies to Lyssavirus in the serum samples from the bat populations of Taiwan, and all 121 serum samples were negative. No Lyssavirus or Lyssavirus-specific antibodies was detected in the bat samples collected in this study. Further investigation can provide more comprehensive knowledge of Lyssavirus infection status in the bat populations of Taiwan for wild animal management and public health control.