病毒檢測之新策略與抗流感藥物之研究

高病原性H5N1禽流感和H1N1豬流感病毒的出現發出了一個很嚴重的警告，主要是因為既有的流感疫苗對於這些病毒都是沒有效的。目前神經氨酸苷酶抑制劑仍是我們最有效的治療方法。但不幸地，自從2009年季節性流感突然地幾乎全部都對克流感產生了抗藥性。在這些被高病原性H5N1禽流感或H1N1豬流感病毒感染的病人中也發現了對克流感具有抗藥性的突變株。然而一般人最擔心的，豬流感病毒在未來是否也會全面都變成具有抗藥性的突變株。因此，其它不同於神經氨酸苷酶抑制劑之預防性的和治療性的化合物都是需要的。另外，研發抗流感葯物之生物分析，現有葯物的改良與修飾，利用高通量篩選來找出新抗流感的抑制劑，研發對克流感敏感度和流感病毒種類的診斷工具和針對流感進行新疫苗的研發都是非常重要的。朝著這些目標，我們研發出一個以細胞為基礎的神經氨酸苷酶分析，其最主要是測量釋放出的紅血球凝集素偽病毒顆粒來進行分析。紅血球凝集素偽病毒顆粒的釋放可以使我們得知神經氨酸苷酶抑制劑的效力。同時針對一個修飾的神經氨酸苷酶抑制劑（扎納米爾叶啉包含了四個扎納米爾和一個叶啉）進行機制的探討，扎納米爾叶啉在流感病毒的感染實驗或是在紅血球凝集素偽病毒顆粒的釋放分析中都得到比單一個扎納米爾有更好的抗病毒效果。我們利用高通量篩選系統找出新的抗流感化合物並證實其作用模式是針對流感病毒核醣核酸聚合酶為標的進行抑制作用。我們也研發了一個新的分析方法其最主要是利用競爭的結合來評估病毒之抗藥性，並且結合醣晶片的應用，建立一個快速又簡單的測試方法去分析流感病毒對克流感的抗藥性和其紅血球凝集素的種類。最後，我們利用單醣流感紅血球凝集素包覆類病毒顆粒以產生更具有效力的流感疫苗。

關鍵字

病毒檢測；抗流感藥物；假病毒顆粒

並列摘要

The appearance of the highly pathogenic H5N1 avian flu and the pandemic H1N1 swine flu cause serious alarms because the existing influenza vaccine is insufficient to prevent the spread of these viruses. The neuraminidase inhibitors are the most effective therapeutic measures we have at this time. Unfortunately, the seasonal flu since 2009 were almost all resistant to oseltamivir. The oseltamivir resistant mutant was also found in patients infected with the highly pathogenic H5N1 avian flu and H1N1 swine flu. The general concern is that whether future pandemic influenza virus will be oseltamivir resistant or not. Therefore, other prophylactics and therapeutic compounds are necessary for preventing influenza infection. The bioassays to study anti-influenza leads, the compound modifications for more efficacious neuraminidase inhibitors, the screenings to identify new anti-influenza inhibitors, the diagnostic tools for oseltamivir resistant viruses and new vaccine development for pandemic influenza are important. Toward these objectives, we developed new cell-base neuraminidase inhibition assay by measuring the releases of HA pseudotype particle (HApp). HApp release assay allows to measure the potencies of the neuraminidase inhibitors under physiological conditions. We studied the mechanism of a potent neuraminidase inhibitor, zanamivir porphyrin, that contains four zanamivirs linked to porphyrin. Zanamivir porphyrin is more efficacious than zanamivir in influenza infectivity and HApp release assays. We also elucidated the mode of action of a new anti-influenza compound identified by HTS. The anti-influenza, compound 3061, targeted influenza NP and affected the activity of RdRP (RNA dependent RNA polymerase). A novel RABC assay (Resistant Assessment by Binding Competition) was developed to determine the oseltamivir susceptibility of influenza neuraminidase. It is also combined with glycan array (influenza HA-binding specificity) to allow a quick and simple test for oseltamivir susceptibility and influenza HA typing. Finally, we also prepared mg HA-VLP (monoglycosylated hemagglutinin virus like particle) that is more effective than fg HA-VLP in preventing influenza infection.

並列關鍵字

virus detection ； anti-influenza drug ； pseudotype virus

參考文獻

1. Wagner, R., Matrosovich, M. & Klenk, H.D. Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol 12, 159-166 (2002).

2. Skehel, J.J. & Wiley, D.C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annual review of biochemistry 69, 531-569 (2000).

3. Steinhauer, D.A. Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258, 1-20 (1999).

4. Lakadamyali, M., Rust, M.J., Babcock, H.P. & Zhuang, X. Visualizing infection of individual influenza viruses. Proc Natl Acad Sci U S A 100, 9280-9285 (2003).

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病毒檢測之新策略與抗流感藥物之研究

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