Inhibitory effect of swine anti-coronaviral serum and antiviral agents against feline coronavirus in vitro

貓傳染性腹膜炎 (Feline infectious peritonitis, FIP) 為由貓冠狀病毒 (Feline coronaviurs, FCoV) 所引起之免疫媒介、高度致死性疾病，目前為尚無有效之治療方法。本研究之目的為探討利用豬抗冠狀病毒血清及部份上市之抗病毒藥物對清除FCoV感染及控制FIP之可能性。以表現重組豬傳染性胃腸炎病毒 (Transmissible gastroenteritis virus, TGEV) spike protein之痘病毒免疫無特定病原 (Specific pathogen free, SPF) 豬隻之血清作為抗血清。進行其對FCoV中和試驗評估，結果顯示其抗第二型FCoV中和抗體力價為1024倍，接近FIP貓的抗體力價平均值。抗病毒藥物實驗中，首先於fcwf-4細胞中以MTT試驗評估11個藥物之細胞毒性，選取細胞毒性小於5%之藥物濃度，以計算細胞病變效應病灶 (Cytopathic effect loci, CPE loci) 進行抗病毒效力之篩選，結果顯示，所有藥物於測試濃度皆無法有效抑制病毒複製。若選取此11個藥物細胞毒性小於20%之濃度分別與本實驗室先前證實具抗病毒效應之藥物nelfinavir合併使用，結果發現與單獨給予nelfinavir 6.25 μg/ml 相比，nelfinavir 6.25 μg/ml 合併不同濃度ribavirin顯現具有加乘之抗病毒效應 (Synergistic antiviral effect)，而能顯著降低上清液之病毒力價同時對細胞毒性亦未增加。於本實驗中觀察到豬抗TGEV spike protein血清及ribavirin/ nelfinavir之合併使用於體外試驗中具有抑制FCoV複製之活性，或有更進一步運用於臨床FIP預防及控制之可行性。Feline infectious peritonitis (FIP), is an immune-mediated fatal disease caused by feline coronavirus (FCoV). Currently no effective vaccine and therapy is available for the disease. This study aims to evaluate the possibility of using swine anti-coronaviral serum and other new antiviral agents as a mean for the prevention and control of FIP. Serum harvested from specific pathogen free (SPF) pigs inoculated with vaccinia virus expressing recombinant transmissible gastroenteritis virus (TGEV) spike protein were tested for its neutralization capacity against FCoV. The result showed this swine origin anti-coronaviral serum exhibited the ability to neutralize FCoV type II to a relative high neutralizing titer (1024 ×). A titer that is nearly reach the mean of those serum obtained from other naturally occurring FIP. In the evaluation of inhibitory activity of antiviral agents, the cytotoxicity of 11 commercialized antiviral agents was first tested by MTT assay on fcwf-4 cell. Based on the results of MTT assay, the highest concentration of every agents with less than 5% of cytotoxicity were determined for further antiviral screening by counting of Cytopathic effect loci (CPE loci). None of the 11 antiviral agents tested showed reduction of CPE loci. Whether these agents can inhibit replication of FCoV when combined with nelfinavir, the agent that has been proven to be effective against FCoV, was further tested. The results showed 6.25 μg/ml of nelfinavir combined with 50 μg/ml and 25 μg/ml of ribavirin exhibited significant inhibitory effect. Taken together, swine anti-TGEV spike protein serum alone and combined use of 2 antiviral agents, i.e. ribavirin and nelfinavir, showed inhibitory effect against FCoV in vitro. These agents will have potential in the future clinical application for the prevention and control of FIP.目錄 ---------------------------------------------------------------------------------------------- I次----------------------------------------------------------------------------------------------- V次 ---------------------------------------------------------------------------------------------- VI文摘要 ---------------------------------------------------------------------------------------- VII文摘要 ----------------------------------------------------------------------------------------VIII一章 序言 ------------------------------------------------------------------------------------ 1二章 文獻探討 ------------------------------------------------------------------------------ 3第一節 歷史背景 ----------------------------------------------------------------------- 3第二節 病毒特徵 ----------------------------------------------------------------------- 3 2.1.1 病毒分類 ------------------------------------------------------------ 3 2.1.2 基因體特性 --------------------------------------------------------- 4 2.1.3 貓冠狀病毒之分型 ------------------------------------------------ 6 2.2 宿主 --------------------------------------------------------------------------- 7第三節 貓冠狀病毒與疾病 ----------------------------------------------------------- 7 3.1 流行病學、致病機制及免疫反應 --------------------------------------- 7 3.1.1 流行病學 ------------------------------------------------------------ 8 3.1.2 致病機制 ------------------------------------------------------------ 8 3.1.3 免疫反應 ------------------------------------------------------------ 9 3.2 臨床症狀與診斷 ------------------------------------------------------------ 11 3.2.1 臨床症狀 ------------------------------------------------------------ 11 3.2.2 診斷 ------------------------------------------------------------------ 11 3.3 治療及預防 ------------------------------------------------------------------ 12 3.3.1 臨床上之治療 ------------------------------------------------------ 12 3.3.2 預防與控制 --------------------------------------------------------- 14三章 材料與方法 --------------------------------------------------------------------------- 16第一節 病毒確認及定量 -------------------------------------------------------------- 16 3.1.1 細胞及病毒 --------------------------------------------------------- 16 3.1.2 RNA萃取 ------------------------------------------------------------ 16 3.1.3 反轉錄反應 --------------------------------------------------------- 17 3.1.4 聚合酶鍊鎖反應 --------------------------------------------------- 17 3.1.5 電泳分析 ------------------------------------------------------------ 18 3.1.6 病毒斑試驗 --------------------------------------------------------- 18 3.1.7 間接免疫螢光染色 ------------------------------------------------ 19第二節 抗血清效力之體外評估 ----------------------------------------------------- 19 3.2.1 血清 ------------------------------------------------------------------ 19 3.2.2 血清中和試驗 ------------------------------------------------------ 19第三節 抗病毒藥物效力之體外評估 ----------------------------------------------- 20 3.3.1 抗病毒藥物 --------------------------------------------------------- 20 3.3.2 細胞活性 ------------------------------------------------------------ 20 3.3.3 抗病毒效應之篩選 ------------------------------------------------ 21 3.3.4 抗病毒藥物與nelfinavir合併給予之抗病毒效應篩選 ------ 21 3.3.5 評估抗病毒藥物與nelfinaivr合併給予之抗病毒效應 ------ 22 3.3.6 Ribavirin合併nelfinaivr之細胞活性 ---------------------------- 22 3.3.7 濃度相關性抗病毒效應 ------------------------------------------ 22 3.3.8 統計分析 ------------------------------------------------------------ 22四章 結果 ------------------------------------------------------------------------------------ 23第一節 病毒確認及定量 -------------------------------------------------------------- 23 4.1.1 病毒核酸之確認 --------------------------------------------------- 23 4.1.2 病毒力價 ------------------------------------------------------------ 23 4.1.3 間接免疫螢光染色 ------------------------------------------------ 23第二節 抗血清效力之體外評估 ----------------------------------------------------- 23 4.2.1 血清中和試驗 ------------------------------------------------------ 23第三節 抗病毒藥物效力之體外評估 ----------------------------------------------- 24 4.3.1 藥物之細胞毒性評估 --------------------------------------------- 24 4.3.2 抗病毒藥物之篩選 ------------------------------------------------ 24 4.3.3 抗病毒藥物合併nelfinaivr給予抗病毒效果之評估 --------- 24 4.3.4 Ribavirin與nelfinavir合併給予抗病毒效果之評估 ---------- 25 4.3.5 Ribavirin與nelfinavir合併給予之細胞毒性 ------------------- 25 4.3.6 濃度相關性抗病毒效應 ------------------------------------------ 25五章 討論 ----------------------------------------------------------------------------------- 27圖 ---------------------------------------------------------------------------------------------- 33表 ---------------------------------------------------------------------------------------------- 43考文獻 ---------------------------------------------------------------------------------------- 4