尋找新型流感病毒抑制劑及研究其藥物機轉

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Title:	尋找新型流感病毒抑制劑及研究其藥物機轉
Other Titles:	Screening Novel Influenza Virus Inhibitors and Anti-Flu Mechanism of These Drugs
Authors:	林昭仁 LIN, CHAO-JEN
Contributors:	黃光裕;萬磊 Hwang, Gung-Yuh;Wan, Lei 生命科學系
Keywords:	流感病毒;抗藥性;虎杖;白藜蘆醇;β 干擾素 Influenza virus;influenza-specific luciferase reporter assay;IFN-β;TLR9;resveratrol
Date:	2015
Issue Date:	2015-12-17T06:05:03Z (UTC)
Abstract:	流感病毒（Influenza virus）的基因，具有多樣性的抗原漂移（antigenic drift）和抗原轉變（antigenic shift）的能力，導致高致病性且具高傳染性的流感病毒暴露在人類社會，造成大流行。每年流感病毒的疫情，導致全球300萬到500萬個嚴重病例，並造成其中50萬人死亡。由於疫情的迫切性，市面上有四種美國食品藥品管理局批准的抗流感病毒藥物：amantadine、rimantadine、oseltamivir 和 zanamivir。於2005-2006年流感季節之後，發現A型流感病毒已對 amantadine 產生了九成以上的抗藥性。因此，只有神經胺酸酶抑製劑（oseltamivir 和 zanamivir）對目前的流感病毒仍然有效。2009年4月，H1N1流感大流行在世界各地蔓延，全球 oseltamivir 產生抗藥性的病例越來越多，再次顯示抗病毒藥物的大量使用讓抗藥性的病毒株快速增加。由於這些原因，研發對抗流感病毒藥物是目前迫切的課題。快速並準確檢測流感病毒的方式，是成功發展抗病毒藥物和疫苗研製的關鍵。傳統使用病毒斑分析(plaque assay)和細胞50%生長抑制所需的藥物濃度(GI50)，仍是研究新型流感病毒藥物最常使用的方式，然而這種方式相當費時費力。在這項研究中，我們使用偵測流感病毒的冷光報告系統(influenza-specific luciferase reporter assay)，快速且客觀地定量病毒、病毒中和試驗、藥物篩選試驗和抗病毒基因的影響，以證明病毒誘導的冷光細胞株之效用。本研究測試了300種中國傳統治療呼吸道疾病的藥物，利用受到流感病毒感染的A549細胞株，加入中藥後，利用流感特異性冷光報告系統分析。在這些測試的傳統中藥中，我們發現虎杖 (Polygonum cuspidatum) 和它的活性化合物白藜蘆醇 (resveratrol) 和大黃素 (emodin) 可以減少A549細胞中流感病毒的複製。受到抑制的病毒，包括2009到2011年從台灣分離的臨床流感病毒檢體和實驗室的標準A型流感病毒株A/WSN/33 (H1N1)。虎杖、白藜蘆醇和大黃素也都會抑制血球凝集素和神經胺酸酶的表現，而且會經由TLR9增加ß干擾素的表現。此外，當受到流感病毒感染的A549細胞加上抗β干擾素抗體和TLR9抑制劑之後，β干擾素的產生或白藜蘆醇的抗病毒活性也跟著降低。顯示β干擾素和白藜蘆醇對於抑制H1N1流感病毒有很好的協同作用。這個潛在的抗病毒機轉，包括直接抑制病毒的複製和刺激宿主對抗病毒的免疫反應。此一現象在單一抗病毒分子還未被發表過。總結是，我們的研究發現使用虎杖、白藜蘆醇或大黃素抑制流感病毒的複製是經由TLR9誘導β干擾素的產生。 Influenza virus cause 3-5 million cases of severe illness and up to 500000 deaths worldwide per year. Influenza viruses are highly diverse with antigenic drift and shift, which could lead to emergence of viruses that have been exposed to human population, leading to pandemic outbreaks. Because of the importance of influenza virus infection, there have been major efforts in the development of effective antivirals for the last several decadesThere are now four U.S. FDA-approved drugs on the market; amantadine, rimantadine, oseltamivir, and zanamivirin the 2005/2006 flu season, 92.3% of influenza A viruses analyzed from 26 states in the U.S. contained point mutations that conferred amantadine resistance. Therefore, only the NA inhibitors remain effective against the currently circulating viruses. In 2009, a H1N1 pandemic occurred in April and spread across the world. More and more oseltamivir-resistant viruses strains spread quickly worldwide, indicating again that antiviral resistance arises quickly with drug use. For these reasons, identification of new anti-viral compounds from natural products is important for development of therapeutic agents against newly appeared influenza virus. Accurate, specific, and rapid detection and quantification of influenza virus are critical for successful development of diagnostic tests, antiviral drugs and vaccines. Assays that are based on virus-induced cytopathic effects such as the plaque assay study and the 50% inhibition of cell growth (GI50) are still commonly used to quantify influenza virus, but such assays are time-consuming and labor- intensive. In this study, we developed a detection system for influenza A virus using a luciferase reporter gene system and demonstrate the utility of these cell lines in objective, rapid, and quantitative assays for virus quantification, virus neutralization assays, drug screening assays and cytokines response after treating influenza A virus. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9–induced IFN-β production.
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