特選抗病毒試劑對抗魚類傳染性胰臟壞死病毒活體外之研究

Abstract

魚類傳染性胰臟壞死病毒（infectious pancreatic necrosis virus; IPNV）是一種廣存於魚貝類的病毒，台灣水產養殖業常受此病毒之感染而遭受損失。今就IPNV病毒複製過程及現存抗病毒試劑的分子級抗病毒機制的瞭解，本實驗嚐試以四類的抗病毒試劑（1）影響polyprotein 轉譯後切割的蛋白質?抑制劑（2）常用來抑制同為雙股RNA的Rotavirus和Reoviurs之RNA甲基化抑制劑（3）影響病毒醣蛋白的醣化作用抑制劑（4）影響病毒複製的RNA聚合?抑制劑等21種抗病毒試劑，來篩選有效試劑對抗本實驗室自1987年鱒魚鰓部分離而得較一般IPNV具更強感染力的T42G病毒株，並研究其對病毒蛋白質及遺傳物質之影響，藉以瞭解藥物抑制病毒複製及病毒本身複製之分子生物學。 由病毒斑減低試驗，篩選出兩種絲胺酸（serine）蛋白質?抑制劑：N-Chlorosuccinimide, PMSF和兩種RNA甲基化抑制劑：Ribavirin和Tubercidin，其ID50（50%抑制劑量；50% inhibitory dose）分別為50,80,3和0.1μg/ml ，由細胞毒殺試驗顯示，其最小使細胞變形的毒殺劑量MTC(minimal toxic concentration）分別為60,180,200，及3μg/ml ，且其毒殺細胞程度與劑量成正比。由蛋白質及核醣核酸的抑制試驗，可知N-Chlorosuccinimide在ID50劑量時，可抑制95.8%病毒蛋白質合成?當PMSF在ID50劑量時，病毒核醣核酸之合成被抑制76%，而蛋白質合成也被抑制72.5%。Tubercidin則在ID50劑量時，可抑制約50%病毒核醣核酸之合成，卻發現增加Tubercidin之劑量時，其核醣核酸之合成又增加，其抑制機制顯然與Ribavirin不同。此四種抗病毒試劑之抑制病毒機制亦加以討論。

Infectious pancreatic necrosis virus (IPNV) is the causal agent of a contagious and high mortality disease of fishes and mollusca. It caused a severe economic loss of aquaculture in Taiwan. From the studies of the replication of IPNV and the elucidation of the molecular mechanism of existing antiviral compounds. We chose four kinds of antiviral agents containing 21 compounds for our research. Such as proteinase, methylation, glycosylation and RNA polymerase inhibitors. The T42G(IPNV), isolated from the gill of rainbow trout ,was chosen for screening the antiviral drugs. From our studies, these antiviral agents affect the synthesis of viral proteins and/or RNA. Their antiviral mechanisms and the molecular biology of the virus also can be elucidated. From the plaque reduction tests, two serine proteinase inhibitors: N-chlorosuccinimide and PNSF; and two RNA methylation inhibitors: Ribavirin and Tubercidin were screened out from 21 compounds, their ID50 value (50% inhibitory dose) are 50,80,3 and 0.1μg/ml respectively. From the cytotoxicity tests on the CHSE-214 monolayer, their MIC(minimal toxic concentration )are80,160,200,and 3μg/ml respectively. From the inhibition of viral protein and RNA synthesis experiments , N-chlorosuccinimide inhibited 95.8% viral protein synthesis at its ID50 value. PMSF inhibited 67% viral RNA and 72.5% viral protein at its ID50. Ribavirin inhibited 67% viral protein and 72.5% viral RNA at its ID50 value. Tubercidin inhibited 50% viral RNA at its ID50 value, but viral RNA synthesis increased in higher dose. Obviously, the antiviral mechanisms of Ribavirin and Tubercidin are different. The inhibition mechanisms of these four antiviral drugs were also discussed.