豬抗冠狀病毒血清及抗病毒藥物對貓冠狀病毒生長抑制效應之體外評估

Abstract

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.