臺灣博碩士論文加值系統

病毒的研究需要有體外增殖系統的建立，病毒種類的鑑定則需要有形態及分生資料才能克竟全功。本研究旨在鑑定家蠶 (Bombyx mori) 的病毒病之主要病原體，因此先從黑角舞蛾細胞株NTU-LY1株系找到一株高病毒 (NPV) 接受的細胞株系LY16，再利用LY16增殖令家蠶發病之某病源，以光學及電顯觀察確定引起細胞病變的病毒形態及致病現象，證實胞質內有病毒形成的內涵體及病毒形成團。利用感染的LY16培養液回飼和回注射家蠶幼蟲，感染者皆呈典型的軟化症病徵，解剖病蠶及中腸電顯切片觀察亦得到相同結果。純化的病毒粒子經負染處理及電顯觀察，病毒粒子為28 nm大小的20面體病毒，無論細胞病變或電顯病理觀察，病原體極似透翅毒蛾軟化病病毒 (PnV, Perina nuda virus) 。利用RT-PCR增幅未感染的LY16細胞之mRNA，證實LY16細胞原來是PnV之持續感染細胞株系。因此本研究的結論是 (1) LY16是PnV持續感染 (persistent infection) 的細胞株系；(2) PnV 亦可以感染黑角舞蛾細胞株及家蠶；(3) 最起初罹病之家蠶疑是在實驗室飼養時被PnV感染，抑或是罹病家蠶的病原體未能在LY16細胞內增殖。

The viral studies need to establish in vitro viral propagation, and the morphological and molecular data are necessary for the identification and classification of an unknown virus. The aim of this study was undertaken to identify the viral pathogen from moribund silkworm larvae. For in vitro propagation, we selected a high NPV susceptible cell strain, LY16, from NTU-LY1 cells. The tissue fluid extracted from the moribund silkworm larvae was used as an inoculum to infect LY16 cells. The infected LY16 cells were examined by light and electron microscopes. The viral inclusion bodies and virogenic stroma were found in the cytoplasm of the cells. The gross anatomies of the disease silkworm larvae fed or injected with the media of the infected LY16 cells showed a typical symptom of flacheries disease. The columnar cells of the midgut of the disease silkworm were main target cells of the virus and also the muscle cells surrounded the midgut were seriously infected. The purified viral particles are icosahedral in shape and 28 nm in size by negative stain. Based on cytopathic effect and electropathogenic observations, this virus is highly similar to PnV (Perina nuda virus). By RT-PCR amplification, we found that PnV exists in uninfected LY16 cells, therefore LY16 cells are persistent infection with PnV. In conclusions, (1) LY16 cells are PnV-persistent infection cells; (2) PnV can infect both LY cells and silkworm larvae; (3) and the origin pathogen of silkworm may either PnV contaminated from lab during rearing or viral pathogens of silkworm origin which can’t propagate in LY16 cells.

目錄
目錄…………………………………………………………………...……………………..i
圖目錄……………………………………………………………….......……………….....iv
表目錄………………………………………………………..………………………….…..v
壹、緒言………………………………………………..……………….…..………………1
貳、往昔研究……………………….………………………………………………………4
一、細胞株之建立及其在病毒增殖上的應用……………………………………….4
二、昆蟲軟化症之研究……………………………………………………………….4
三、昆蟲小RNA病毒之分類地位及其相關研究…….……………………………..7
四、昆蟲小RNA病毒之研究進展…………………………………..........................9
參、材料與方法…………………….…………………………………………………..…11
一、供試昆蟲……………………………………………………………...…………11
二、供試細胞株………………………………………………………..………….…11
三、供試病毒株……………………………………………………………..….……11
四、黑角舞蛾株系之特性分析…………………………………………………...…11
(一) 細胞株培養及細胞株系之選殖………………………………..……….11
(二) 各細胞株系之形態及病毒感受性之比較……………..……………….11
五、罹病家蠶之病原體鑑定……………………………………………...…………14
(一) 供試家蠶…………...……………………………………………………14
(二) 罹病家蠶…………...……………………………………………………14
(三) 罹病家蠶內病原體之體外增殖………………...………………………14
(四) 利用染色法以初步鑑定內涵體內病毒基因體屬性…….…………..…15
(五) 細胞RNA 之抽取…...……………………..………….…………….…..15
(六) 純化病毒粒子…………………...………………………………...…….16
六、家蠶之病毒感染…………………………………………………......................16
(一) 以餵食法感染病毒……………………………...………...…………....16
(二) 以注射方式感染病毒……………………………………...…...…...….17
七、病毒感染之細胞與家蠶組織之電顯觀察……………………………….…….17
(一) 感染細胞之處理…………………………………………………….….17
(二) 家蠶組織之固定及包埋……………………………….……………….18
(三) 厚切片染色…………………………………………….……………….18
(四) 電顯薄切片染色………………………………………………….…….18
八、病毒基因組之鑑定……………………………………………………….…….19
(一) 未感染病毒之LY16細胞之病毒持續性感染之鑑定…………………19
肆、結果………………………………………………………………………….…..…..20
一、 黑角舞蛾細胞株系之特性……………………………….……………………20
(一) 細胞形態…………………………………………………………………20
(二) 細胞生長曲線及倍增時間………………………………………………25
(三) 細胞對病毒的感受性……………………………………………………27
(四) 六種細胞株酯酶 (esterase) 圖譜之比較……………………….………..30
二、 病蠶病原體體外增殖…………………………………………………....……..31
(一) 感染細胞之超微構造觀察………………………………………...……..31
(二) 病毒粒子形態………………………………………….…………………43
三、 家蠶病原體之生體感染…………………………………………………….….44
(一) 家蠶幼蟲之死亡率…………………………………….…………………44
(二) 罹病家蠶之中腸超微病理之觀察……………………………………….46
四、 病毒基因組之鑑定…………………………………………………….……….56
(一) 未感染之LY16細胞之病毒持續性感染之鑑定…….…………………..56
(二) 測試未感染之LY16細胞是否感染BmCPV, IFV……….……………..56
伍、討論……………………………………………………………………………….…..57
一、LY16細胞株是其同源株系細胞中對病毒感受性最高的細胞株系………….57
二、LY16細胞株具有可辨識其身份的特殊band…………………………………58
三、原罹病家蠶之病原體為PnV…………………………………………………...58
四、本研究對瞭解家蠶病毒病PnV之貢獻…………………………………...…...59
(一) PnV對家蠶的影響…………………………………………...……………59
(二) PnV的病變現象與其複製之可能機制……………………….…………..59
陸、參考文獻…………………………………………………………………….………..63
柒、附錄…………………………………….………………………………………….….69
附錄 1、 同功異構酶之電泳與染色………………………………………….…..….69
附錄 2、 電顯樣品固定相關藥品之配方…………………………………...……….72
附錄 3、 電顯切片染色…………………………………………………….……..….72
附錄 4、 實驗所使用的相關引子名稱與序列……………………………...……….73





















圖目錄
圖 1、 黑角舞蛾1號細胞株之細胞形態…...…………………………..……..…….......21
圖 2、 黑角舞蛾各細胞株系之細胞形態觀察………….…………………….…………24
圖 3、 黑角舞蛾細胞株系 (LY16) 感染LyxyMNPV 12 d後之細胞病變……..………29
圖 4、 以含有紅色螢光基因之黑角舞蛾核多角重組病毒感染黑角舞蛾細胞株系，
可見LY16之紅色螢光表現量優於其他株...…………………….………….….30
圖 5、 六種細胞株之同功異構酯酶 (Esterase) 圖譜……………………...………….…31
圖6、 源自家蠶病蟲之過濾液感染14 d後，黑角舞蛾細胞株
LY16之細胞病變現象……………………………….…………………………..33
圖7、 感染家蠶病蟲過濾液10 d後細胞之病變……….……………...………………34
圖 8 、 感染家蠶病蟲過濾液14 d後之LY16細胞病變………...……………….…….35
圖 9、 LY16 細胞感染14 d後之病變……..…………………………………….….…..36
圖 10、 纖維內涵體之病變現象………………………………………………......……...37
圖 11、 3種含有病毒之內涵體………………………………………………….…….…38
圖 12、 感染25 d後的LY16細胞之胞質病變部份……….………………………..…..39
圖 13、 以DAPI / Evans blue測染感染家蠶病蟲過濾液第25 d後
之LY16細胞 ..…………………………………………………………….….…42
圖 14、 以負染法觀察純化之病毒粒...…………………………………………...……..43
圖 15、 經注病毒液10 μl (A和B) 及20 μl (C和D) 17 d 後之病蠶
外部及內部形..……………………………………………………….………....45
圖 16、 六張電顯圖組合的家蠶中腸細胞…………………………………….………...48
圖 17、 家蠶中腸細胞被膜包圍的內涵體病變……………………………….………...54
圖 18、 以RT-PCR方法確定感染病源……………………………………….………...57





表目錄
表 1 LY 細胞株系母株與各次選殖株含細胞種類之比例…………………….………22
表 2 LY 細胞株系母株與各次選殖株所含各細胞形態大小…………………….……23
表 3 黑角舞蛾1號細胞株所選殖出的株系培養於28 ℃,8% FBSTNM-FH
之細胞倍增時間………………………..……………………….………….……….26
表 4 LY 細胞株系 (11-18) 在感染 LyxyMNPV第 7 d之感染細胞
比率及細胞病變…………………………………………………………………...28

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