建構黃病毒誘導基因表達系統作為新興抗病毒策略

病媒蚊是許多重大疾病的傳播媒介，包含瘧疾、登革熱以及茲卡病毒感染症等等。其中登革熱作為世界上傳播速度最快的蚊媒傳染疾病之一，全世界約有一半的人口生活在登革熱流行的區域之中。隨著全球暖化，登革熱在世界各地蔓延的情形有愈發嚴峻的趨勢。由於登革熱目前尚無安全有效的疫苗可供接種，亦無藥物可供治療，因此，控制病媒蚊族群的數量為主要的防治策略，以減緩疾病的傳播。而噴灑殺蟲劑為目前病媒蚊防制的主要方法，然而，隨著具有抗藥性病媒蚊的增加，積極研發新興抗病毒策略便成了當務之急。在本研究中，我們在埃及斑蚊細胞株(ATC10)中建立一個能夠受黃病毒誘導之基因表達系統，並且利用此系統作為新興的抗病毒方式。首先我們以第二型登革病毒的五端及三端未轉譯蛋白區域序列之負股選殖至表現載體中，並加入登革病毒的RNA聚合酶基因 (Non-structural protein 5 RNA-dependent RNA-polymerase, NS5 RdRp)。並且以綠色螢光蛋白(Green fluorescent protein, GFP)基因作為報導基因 (Reporter gene)。我們的結果顯示，當帶有病毒誘導表達系統之細胞分別被四種血清型的登革病毒以及茲卡病毒感染後，細胞內GFP的表現量隨之顯著上升，顯示此系統可被不同黃病毒科的病毒所啟動。接著我們將此系統中的GFP置換成能夠誘導蚊子細胞凋亡的兩個基因，Michelob_x (AaMx)以及IAP-antagonist Michelob_x like protein (AaIMP)，並且測試此兩種系統的抗病毒能力。實驗結果顯示，當帶有病毒誘導AaMx或AaIMP表達系統之細胞分別被四種血清型的登革病毒以及茲卡病毒感染後，細胞內的細胞凋亡酶(Caspase-3)活性有顯著的上升。我們也發現此兩種細胞分別感染四種血清型的登革病毒以及茲卡病毒後，細胞內病毒RNA以及蛋白質的表現量相較於控制組有顯著的降低。除此之外，我們亦利用Focus-forming assay (FFA)偵測帶有病毒誘導系統之細胞在感染病毒後產生具有感染力的病毒的效價。實驗結果發現，三種帶有病毒誘導系統的細胞(GFP, AaMx, and AaIMP)在分別感染四種血清型的登革病毒以及茲卡病毒後，產生具有感染力的病毒的效價相較於控制組有顯著的降低。上述的實驗結果顯示了此病毒誘導系統的抗病毒能力，除此之外，我們也初步確認帶有病毒誘導系統的蚊子對於第二型登革病毒具有顯著的抑制效果。我們的研究顯示病毒誘導系統可以成功的抑制登革病毒及茲卡病毒的複製及傳播，可提供未來建構新型抗病毒策略的參考模式。

關鍵字

埃及斑蚊；登革病毒；茲卡病毒；細胞凋亡；病毒誘導基因表現系統

並列摘要

Nearly half of the world population is now at risk for contracting dengue virus (DENV) and Zika virus (ZIKV), the two mosquito-borne flaviviruses. The current vaccination and medication of these infectious diseases are either non-satisfactory or entirely lacking. Vector control, to a greater extent than vaccines or drugs, has been responsible for shrinking the map of mosquito-borne diseases. With the rising threat from insecticide-resistant mosquitoes, there is an urgent need to develop novel alternative approaches to combat these diseases. Here, we developed a novel antiviral approach using a virus-inducible gene expression system in the mosquito cell line. The constructs containing the smallest replication units (3’UTR and 5’UTR) of dengue virus serotype-2 (DENV2) with negative-stranded DENV2 artificial genome and genes of interest were established in the Aedes aegypti cell line, allowing the expression of target genes after DENV2 infection. The GFP gene was then incorporated into this construct to verify the potency of the virus-inducible gene expression system. Our results revealed that the GFP protein was produced in the cells after dengue virus serotype 1-4 and Zika virus infection, indicating the virus-inducible feature of this system. Next, the GFP gene was replaced with two apoptosis-related genes, AaMx and AaIMP, and the antiviral activity were examined. Our results showed that the production of viral RNA and viral protein were inhibited in this virus-inducible gene expression system with incorporated AaMx or AaIMP after all tested viruses infection. In addition, the caspase-3 activity was induced in cells with AaMx or AaIMP-incorporated expressing system after all viruses infection. Interestingly, the virus-inducible gene expression system with incorporated GFP, AaMx or AaIMP failed to produce infectious virus particles, suggesting the antiviral activity of this system. Furthermore, we demonstrated that mosquito with virus-inducible gene expression system exhibited significant inhibition of infectious virus particles production after DENV2 infection. Our results revealed that the virus-inducible gene expression system is capable of inhibition in DENV and ZIKV replication and transmission, which provide new avenue for novel vector control approach.

並列關鍵字

Aedes aegypti ； dengue virus ； Zika virus ； apoptosis ； virus-inducible gene expression system

參考文獻

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建構黃病毒誘導基因表達系統作為新興抗病毒策略

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