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研究生：	謝雲傑 Hsieh, Yun-Chieh
論文名稱：	白點症病毒感染對於蝦脂肪酸組成之影響 The cellular fatty acid profile is profoundly altered in WSSV-infected shrimp
指導教授：	王涵青 Wang, Han-Ching
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	53
中文關鍵詞：	白點症病毒、脂質生合成、長鏈脂肪酸
外文關鍵詞：	White spot syndrome virus, lipogenesis, long chain fatty acids
相關次數：	點閱：154 下載：1
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導致全球蝦類養殖產業經濟重創之蝦類白點症 (White spot disease - WSD)，其病原體為白點症病毒 (White spot syndrome virus - WSSV)。於 WSSV 基因體複製時期—感染早期 (12 hpi)，WSSV 會誘發瓦氏效應 (Warburg effect)，得以有效提升能量獲得及生合成之基礎材料。在過去癌症研究中，發現當瓦氏效應被誘發時，同時會伴隨著脂質生合成 (lipogenesis)，而在本研究中則發現，在 WSSV 感染誘發瓦氏效應之時，長鏈脂肪酸 (LCFAs) 皆顯著減少，由此可知此期進行脂質降解作用 (lipolysis)，推測 LCFAs 進入 β-oxidation，以填補檸檬酸循環 (TCA cycle)。另一方面，於 WSSV 感染晚期 (24 hpi)，WSSV 所誘發之瓦氏效應已消失，此時發現 LCFAs 則顯著增加，代表病毒於此期乃促使脂肪酸之生合成。本研究中進一步發現，於病毒感染晚期，WSSV 會透過活化 PI3K-Akt-mTOR-HIF1α 訊息傳遞路徑，進而誘發Fatty acid synthase (FAS) 基因之表現。而當利用 FAS 抑制劑 C75 抑制 FAS 活性後，病毒顆粒之形成及釋放因而受阻。總結本研究發現，WSSV 為了完成其複製週期，會策略性的活化 PI3K-Akt-mTOR 訊息傳遞路徑，於感染早期促使瓦氏效應發生，進而獲取生合成所需能量與材料；而至晚期則透過相同訊息傳遞路徑促使 lipogenesis，以利於病毒顆粒組裝與釋放。

The disease most impacting the global shrimp aquaculture industry is white spot disease or WSD. The WSD causative agent, white spot syndrome virus (WSSV), is a virus. The virus is a novel large dsDNA virus, and pathogenesis is still quite poorly understood. Recently we found that WSSV induces a metabolic rerouting known as the invertebrate Warburg effect, which boosts the availability of energy and biosynthetic building blocks in the WSSV genome replication stage (12 hpi). Here we show that unlike the lipogenesis that is seen in cancer cells that are undergoing the Warburg effect, at 12 hpi, all of the long chain fatty acids (LCFAs) were significantly decreased. This means that a non-selective lipolysis was induced by WSSV, the LCFAs were apparently diverted into β-oxidation and used to supplement the TCA cycle. Conversely, at 24 hpi, when the Warburg effect had ceased, the significant increase of most of the LCFAs implied that fatty acid synthesis was triggered at the late stage. We also found that, at 24 hpi, but not at 12 hpi, the PI3K-Akt-mTOR-HIF1α pathway induced the expression of fatty acid synthase (FAS). WSSV virion formation was impaired in the presence of the FAS inhibitor C75, although viral gene and viral genome levels were unaffected. In order to complete its replication cycle, WSSV therefore appears to use the same PI3K-Akt-mTOR signaling pathway to trigger both the Warburg effect at 12 hpi and lipogenesis at 24 hpi.

Table of contents
中文摘要(Chinese abstract)...............................Ⅰ
Abstract...............................................Ⅱ
Acknowledgements.......................................Ⅲ
Table of contents......................................Ⅳ
Table Index............................................Ⅶ
Figure Index...........................................Ⅷ
Abbreviation...........................................Ⅹ
1. Introduction........................................1
2. Materials and Methods...............................6
2-1 Experimental animals and virus inoculum............6
2-2 Preparation of WSSV-infected crayfish stomachs and 
WSSV virions for lipidomic analysis....................6
2-3  Using gas chromatography–mass spectrometry (GC/MS) 
to analyze the lipidome of WSSV-infected shrimp stomachs 
and the fatty acid profiles of WSSV-infected crayfish stomachs and WSSV virion particles.....................7
2-4 Preparation and use of the mTOR inhibitor Torin 1..8
2-5 Effect of the PI3K-Akt-mTOR-HIF1α inhibitors 
LY294002, Torin 1 and 2-ME on FAS expression...........9
2-6 Quantification of the shrimp fatty acid synthase 
(FAS) gene and WSSV VP28 gene.........................10
2-7 Quantification of the WSSV genome copy number.....10
2-8 Effect of the FAS inhibitor C75 on viral genes and 
virus copy number.....................................11
3. Results............................................13
3-1 WSSV infection promotes lipogenesis at the late 
stage of WSSV replication (24 hpi)....................13
3-2 LCFA composition in WSSV-infected shrimp stomach 
cells is altered at the    late stage of WSSV infection 
(24 hpi)..............................................13
3-3 Differences in the fatty acid compositions of WSSV-infected stomachs and  WSSV virions...................14
3-4 mTOR plays an important role in the WSSV-induced lipogenesis at the late   stage of WSSV infection 
(24 hpi)..............................................15
3-5 WSSV induces FAS expression through the PI3K-Akt-
mTOR-HIF1α signaling pathway..........................16
3-6 FAS is important for WSSV virion morphogenesis....17
4. Discussion.........................................19
5. References.........................................26
 
Table Index
Table 1 PCR primers used in this study................32

 
Figure Index
Figure 1. Relative quantities of the long chain fatty 
acids (LCFAs) of shrimp stomachs during WSSV 
infection.............................................33
Figure 2. The percentage (%) of the total fatty acids 
of shrimp stomachs during WSSV infection..............35
Figure 3. The relationship between viral copy number 
and each LCFA.........................................37
Figure 4. GC/MS analysis of the fatty acid composition 
of stomach cells from WSSV-infected P. clarkii 
(host; grey) and the WSSV virion particles themselves  (black)...............................................39
Figure 5. Fatty acid profiles of the stomachs of 
Torin 1-pretreated shrimp during WSSV infection.......41
Figure 6. WSSV-induced fatty acid synthase (FAS) 
expression is regulated by the PI3K-Akt-mTOR-HIF1α 
pathway...............................................43
Figure 7. Pretreatment with inhibitors of the PI3K-
Akt-mTOR-HIF1α pathway   reduced the WSSV genome copy 
number in shrimp pleopods.............................46
Figure 8. In vivo FAS inhibition prevented the release 
of WSSV into the hemolymph............................48
Figure 9. The abnormal decrease in plasma triglyceride 
can be rescued when FAS was inhibited by C75..........50
Figure 10. Treatment with an inhibitor of LCFA transportation into the mitochondria reduced the WSSV 
genome copy number in shrimp pleopods.................51
Figure 11. Schematic representation of the involvement 
of the PI3K-Akt-mTOR  pathway in WSSV-induced lipolysis 
and lipogenesis at the viral genome replication  stage 
(12 hpi) and the late stage (24 hpi) of the first WSSV replication cycle.....................................52

                                    

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