表現抗登革熱病毒微小核糖核酸基因轉殖蚊抑制病毒複製之研究

登革熱是由埃及斑蚊與白線斑蚊所傳播的蟲媒病毒，盛行於熱帶與亞熱帶地區，且具有四種不同的血清型。直到現今，並無有效的疫苗與治療方法可以對抗登革熱病毒的威脅，而常規使用的殺蟲劑則極有可能導致病媒蚊產生抗藥性問題。因此，迫切需要發展新的策略控制病媒蚊傳播登革熱病毒。利用核糖核酸干擾技術(RNA interference; RNAi)在過去已被證實可以有效降低病媒蚊傳播登革熱病毒，而在最近的研究中指出:藉由表達與登革熱病毒基因體序列互補的雙股核糖核酸(double stranded RNAs; dsRNAs)可以阻止病毒的複製。在本篇研究中，利用mariner (Mos1) transposase and genomic integration system，我們製作一系列抗登革熱病毒複製的基因轉殖蚊(transgenic mosquitoes)，藉由埃及斑蚊特定表現的羧肽酶A 啟動子(CPA promoter)、泛素啟動子(Pub promoter)、內肽酶啟動子(AAEL001703 promoter)驅動生產默化登革熱病毒核糖核酸的微小核糖核酸(microRNA)，以阻斷登革熱病毒複製的路徑。利用即時定量聚合酶反應與南方墨點法分析，我們確認轉殖的表現質體(plasmid)進入埃及斑蚊的基因體內，且微小核糖核酸有顯著的表現。藉由餵食混合登革熱病毒的人工血液，CPA>8miR AAEL001703>4miR 與Pub>8miR 在感染病毒後七天，轉殖蚊體內的病毒量與野生型斑蚊相比，有明顯的下降。對CPA>8miR 的基因轉殖蚊品系在感染病毒後七天，重複餵食不含病毒之人工血液，測得對阻斷病毒複製有較高效率。對基因轉殖蚊收集唾液並檢測病毒量，發現CPA>8miR 品系對於野生型斑蚊與Pub>8miR 品系相比有明顯抑制病毒傳播的效力。綜合上述，表現對抗四型登革熱病毒的微小核糖核酸，將之組裝藉由顯微注射送入埃及斑蚊體內製作成轉殖基因蚊品系，我們提供了新的控制登革熱傳播防治策略。

關鍵字

埃及斑蚊；登革熱；微小核糖核酸；基因轉殖蚊；病毒試驗；重複血食

並列摘要

Dengue is an arthropod-borne viral disease prevalent in tropical and subtropical regions, caused by the four dengue virus serotypes (DENV 1–4), which are transmitted by Aedes mosquitoes. Until now, effective vaccine and positive treatment are not available, and the use of insecticides also leads to resistance in mosquito vectors. Therefore, the alternative strategies for mosquito vector control are needed. Genetic manipulation via RNA interference (RNAi) in the mosquito can permanently reduce vector competence and subsequent transmission of DENV to the human. Recent studies have found that expression of virus-specific double stranded RNAs is a potential way to block the DENV replication and vector competence to virus. In this study, using the mariner (Mos1) transposase and genomic integration system, we generated serial transgenic mosquito lines which express anti-viral microRNAs under the control of A. aegypti poly-ubiquitin (Pub), serine-type enodpeptidase (AAEL001703) and carboxypeptidase A (CPA) promoters. We verified the integration of the transgene in the mosquitoes by qPCR and Southern blot. By using virus challenge with viremic bloodmeal, the viral titer and infection rate was significantly suppressed in the transgenic mosquitoes of CPA>8-miRNA, Pub>8-miRNA and AAEL001703>4-miRNA at day-7 post bloodmeal, compared to those of wild type mosquitoes. Repeat bloodmeal on CPA>8-miRNA provided effective antiviral efficiency at day-14 post infection. Transmission in vitro of virus from CPA>8-miRNA line was significantly diminished when compared to Pub>8-miRNA and control mosquitoes at day-14 post bloodmeal. Our study provides an evidence for DENV control strategy which suppress viral replication via genetic manipulation of miRNA in Aedes aegypti.

並列關鍵字

DENV ； Aedes aegypti ； anti-viral miRNA ； transgenic mosquitoes ； virus challenge ； repeat bloodmeal

參考文獻

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表現抗登革熱病毒微小核糖核酸基因轉殖蚊抑制病毒複製之研究

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