臺灣博碩士論文加值系統

登革病毒是最重要的蚊媒病毒之一，每年威脅超過三分之一的全球人口。到目前為止仍沒有有效的臨床疫苗或藥物可對抗登革病毒，因此控制登革病毒傳染媒介變成降低登革病毒疫情的主要公共衛生策略。然而登革病毒在蚊子裡的傳播方式目前仍不清楚。在本篇的研究裡我們發現，高細胞密度與細胞間的接觸可有效提高登革二型病毒於蚊子細胞間的傳染效率。利用 methylcellulose 和登革病毒中和血清限制與抑制登革病毒細胞外傳播，登革病毒仍可有效的在蚊子細胞間傳遞。登革病毒感染蚊子細胞後會大量表現tetraspanin C189 蛋白質，抑制 C189蛋白質表現會降低登革病毒細胞間傳遞的效率，但不會影響細胞外傳遞。利用共軛焦顯微鏡及細胞胞器分離實驗發現，登革病毒在感染晚期會被包裹在由C189蛋白質組成的液泡 (C189-VCs) 裡。登革病毒被包裹在 C189-VCs 後會沿著含有大量 f-actin的細胞觸手移動，並在與細胞接觸後一起被運送至鄰近細胞裡。我們的研究發現登革病毒在蚊子細胞裡的另一種傳染途徑，並發現C189蛋白質在其中扮演重要的角色。我們的研究提供了新的策略去控制登革病毒在蚊子間的傳遞。

Dengue virus (DV) is the most important mosquito-borne virus that threatens over one third of the world’s population. Since there are no clinical vaccines or antiviral drugs for DVs, the control of DV vectors becomes the major public health policy to reduce dengue epidemics. However, the knowledge of DV transmission in mosquitoes is still poorly understood. In this study, we found dengue virus type II (DV2) transmission efficiency was promoted under the high cell-density condition and cell contact significantly enhanced DV2 spread among mosquito cells. The high efficient cell-to-cell transmission of DV2 was presented in mosquito cells while cell-free transmission was limited or blocked by methylcellulose and anti-DV2 serum. Knockdown of C189, a tetraspanin protein upregulated in response to DV infection, was shown to inhibit cell-to-cell transmission but did not affect cell-free transmission of DV2. Confocal microscopy images and organelle-separation analysis pointed out DV2 particles were incorporated into C189-bound vesicles (C189-VCs) during late infection stage. After incorporation of DV2 into C189-VCs, DV-C189-VCs complexes were transported into the actin-enriched filopodia and delivered to neighboring cells upon cell-cell contact. Our study discovered an alternative transmission pathway of DVs between mosquito cells and demonstrated that the tetraspanin C189 is involved in this pathway. These findings provided insights into the development of DV vector control strategies.

指導教授推薦書
口試委員會審定書
ACKNOWLEDGMENT……………………..………….……………iii
中文摘要………………………………..……….…………………….…v
ABSTRATE……………………….…………………………………….vii
TABLE OF CONTENTS…………………………..…..………………...ix
LIST OF TABLES………………..………………….…………….…….xii
LIST OF FIGURES……………..……………………….……….……..xiii
INTRODUCTION…………………..………………………….………..1
DENGUE VIRUS…………………….………………………………….1
The virion and genome structure of dengue viruses……...………………..1
The function of viral proteins……………………….………………....….2
Structural proteins………………………….……………………..…2
Non-structural proteins…………………….…………………..…….3
The replication cycle of dengue viruses……………..…………………….3
Intercellular transmission of dengue viruses…………...………………….4
TETRASPANIN……………………………………………………..…..5
STUDY PURPOSE………………………….……………………….…..7
MATERIALS AND METHODS……………….………………….……8
Cells, viruses and antibodies……………………..………………………..8
Construction and transfection of expression vectors in C6/36 cells…….…8
Stably knockdown system in C6/36 cells………………..…………….…..9
Extracting RNA and quantitating gene expression level……..……..……10
Immunofluorescence (IF) assay……………………….…………...……10
Flow cytometry analysis………………………...…..……………….…..11
Immunoprecipitation (IP) and western blot…………….………………..11
Methylcellulose-containing-medium (MCM) overlay assay………....….12
Titration of viral titers……………………..……………………….…….12
DV transmission assay………………….………………………….……13
Separation of organelles……………….…………………………….…..16
RESULTS………………………….………………………………..…..17
Cell contact increased DV2 transmission efficiency in mosquito cells…..17
DV2 cell-to-cell transmission in mosquito cells was neutralizing-antibody resistant………………………………………………………………….18
C189 was involved in cell-to-cell transmission rather than cell-free transmission of DVs in mosquito cells……………...……….……….…..20
DV2 particles were incorporated into C189-bound vesicles (C189-VCs)........................................................................................................22
DV-containing C189-bound vesicles (DV-C189-VCs) were transported into f-actin enriched filopodia at late infection stage of DV2……………23
DV-C189-VCs were transported along the filopodia into the recipient cells…………………………………………………………………24
DISCUSSION…………………………………………………...…..26
REFERENCES………………..……………………………..…….30
TABLES………………………………………………………..……37
FIGURES…………………………..…………………………………..40


LIST OF TABLES
Table 1. A list of primer sequences used in this study…………..…….….37
Table 2. Parameters and annotations of primers in this study……..…..…38
Table 3. A list of antibodies used in this study………………………..….39


LIST OF FIGURES
Figure 1. Cell-density influenced DV2 transmission efficiency in mosquito
cells…………………………………………………………………...40
Figure 2. Cell contact promoted DV2 transmission in mosquito cells…..41
Figure 3. DV2 efficiently disseminated among mosquito cells when cellfree
transmission has been limited………………………..…..…………42
Figure 4. Blocking DV cell-free transmission by neutralizing
antiserum……………………………………..…………………………43
Figure 5. Neutralizing-antibody resistant cell-to-cell transmission of DV2
in mosquito cells…………………………………………………...……45
Figure 6. The efficiencies of DV cell-free transmission and cell-to-cell
transmission in mosquito cells………………………………….……..47
Figure 7. Construction of stable knockdown vector for mosquito cells....49
Figure 8. C189 was not involved in cell-free transmission of DVs in
mosquito cells……………………………………………………...……51
Figure 9. C189 was involved in cell-to-cell transmission of DVs in
mosquito cells………………………………………………..………...53
Figure 10. DV2 were incorporated into C189-bound vesicles (C189-
VCs)………………………………………………………………..…55
Figure 11. DV-C189-VC complexes were transported into filopodia at late
infection stage of DVs………………………………………………….57
Figure 12. DV-C189-VC complexes were transported along f-actin in
filopodia………………………………………………………….....59
Figure 13. The movement of DV-C189-VC-like structures in live C6/36
cells….……………………………………………………………....60
Figure 14. DV-C189-VC complexes were transported along the filopodia into the recipient cells………………………………………….……..61

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