臺灣博碩士論文加值系統

抗寄生蟲藥物氯硝柳胺被證實能抑制由蚊蟲媒介茲卡病毒的感染。本研究中，我們利用體外細胞及體內動物模式探討氯硝柳胺對抗登革病毒感染的效果。氯硝柳胺可有效地阻擋登革病毒感染人類肺腺癌細胞 (A549)、小鼠神經母細胞瘤細胞 (Neuro-2a) 以及幼倉鼠腎臟纖維母細胞 (BHK-21)。初步結果顯示氯硝柳胺不影響病毒感染進入細胞、宿主細胞因應感染生成第一型干擾素反應以及不直接影響病毒轉錄體的轉譯作用。氯硝柳胺已知可抑制mTOR、STAT3以及NF-κB訊息路徑，但利用各自的選擇性抑制劑卻無法降低登革病毒感染，故而推測氯硝柳胺應藉由其他方式抑制登革病毒感染。如同vacuolar ATPase的抑制劑巴佛洛霉素A1，氯硝柳胺以及其他的質子載體 (例如 CCCP以及FCCP) 皆能有效地抑制細胞內吞小體的酸化並減少病毒核糖核酸複製以達到降低登革病毒感染之作用。利用腦內合併腹腔注射登革病毒感染出生七天大的ICR-哺乳期小鼠，結果顯示單一劑量的氯硝柳胺能部份降低小鼠腦內的病毒量、病毒性腦炎以及小鼠死亡率。本研究結果證實氯硝柳胺透過阻擋細胞內吞小體的酸化作用進而降低登革病毒的感染。

Antiparasitic niclosamide has been demonstrated to inhibit the arthropod-borne Zika virus. Here we investigated the antiviral ability of niclosamide against dengue virus (DENV) serotype 2 infection in vitro and in vivo. Administration of niclosamide effectively retarded DENV-induced infection in vitro in human lung adenocarcinoma cells (A549), mouse neuroblastoma cells (Neuro-2a), and baby hamster kidney fibroblasts (BHK-21). Treatment of niclosamide did not retard the endocytosis of DENV or the antiviral type I interferon response. Furthermore, niclosamide did not cause a direct effect on viral replicon-based expression. Niclosamide has been reported to competitively inhibit the mTOR (mammalian target of rapamycin), STAT3 (signal transducer and activator of transcription 3), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathways; however, selective inhibitors of those pathways did not reduce DENV infection. Similar to the vacuolar-type H+-ATPase inhibitor bafilomycin A1, both niclosamide and other protonophores such as CCCP (carbonyl cyanide m-chlorophenyl hydrazone), and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) effectively reduced endosomal acidification and viral dsRNA replication. Co-administration of single dose of niclosamide partly decreased viral replication, viral encephalitis, and mortality in DENV-infected ICR suckling mice. These results demonstrate that niclosamide diminishes DENV infection by hindering endosomal acidification.

Abstract in English 1
Abstract in Chinese 2
Acknowledgement 3
Abbreviations 5
Content 8
I. Introduction 10
I-1. Epidemiology of dengue virus (DENV) 10
I-2. Characteristics of DENV 10
I-3. Life cycle of DENV 14
I-4. Clinical symptoms of DENV infection 15
I-5. Anti-DENV drugs 15
I-6. Niclosamide as a potent anti-DENV drug 17
II. Objectives and Specific Aims 19
III. Materials and Methods 20
III-1. Cells, virus strains, antibodies and reagents 20
III-2. Animals 21
III-3. DENV infection 21
III-4. Plaque assay 21
III-5. Cytotoxicity 21
III-6. Western blotting 22
III-7. Reporter assay 22
III-8. Fluorescent DENV 22
III-9. Immunostaining 23
III-10. ELISA 23
III-11. Acridine orange staining 23
III-12. Statistical analysis 23
IV. Results 24
IV-1. Niclosamide treatment decreases DENV infection in vitro 24
IV-2. Niclosamide inhibits DENV infection at early stage 24
IV-3. Blocking DENV infection by niclosamide does not affect the endocytosis of DENV 24
IV-4. Anti-DENV effect caused by niclosamide is independent of type I IFN response 25
IV-5. No effects on viral genome replication in niclosamide-treated DENV replicon cells 25
IV-6. Niclosamide initiated antiviral effects independent of inhibiting the mTOR, STAT3, and NK-kB signaling pathways 26
IV-7. Similar to other protonophores, niclosamide causes endosomal deacidification to suppress dsRNA replication and virus release 26
IV-8. Niclosamide treatment abolishes DENV infection in vivo and DENV-induced acute viral encephalitis-like disease progression 27
V. Discussion 29
VI. Conclusion 33
References 34
Appendix 53
A. Materials 53
A-1 Chemicals 53
A-2 Antibodies 55
A-3 Kits 56
A-4 Consumables 56
B. Methods 57
B-1 Cell culture 57
B-2 Western blot 59
B-3 Virus quantification 62

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