傳染性腹膜炎貓隻病毒感染細胞之偵測與分析

Abstract

貓傳染性腹膜炎 (feline infectious peritonitis, FIP) 是由貓冠狀病毒 (feline coronavirus, FCoV) 所引起之免疫性致死性疾病。根據體腔滲出液之與否， FIP 可區分為滲出型與非滲出型；而經由血清學分析， FCoV 可區分為第一型及第二型。為了解滲出型與非滲出型 FIP 貓隻體內病毒感染細胞分佈情況之差異，本研究針對曾就診於台大動物醫院，並經由解剖及組織病理學確診的 FIP 貓隻，挑選其中典型的非滲出型 FIP 五例，以及滲出型 FIP 八例。將其組織切片進行免疫化學染色 (immunohistochemistry, IHC) ，結果在檢測之患貓臟器都可被觀察到 FCoV 感染的細胞。在乾式病例中腎臟 (80%) 與腦 (100%) 的 FCoV 陽性率，分別高於溼式病例之 37.5% 及 40% 。與 FCoV 相關之病灶在數量及嚴重程度上，均有乾式病例甚於溼式病例之趨勢。為了建立原位增幅技術並以此分析不同型別病毒與致病 性之關連性，本研究利用型別特異性引子於相關組織切片上進行直接式原位反轉錄聚合酶鏈鎖反應 (direct in situ RT-PCR) 。由於組織之消化過程對於反應結果影響甚鉅，首先需將其條件最佳化；針對 F96-156 該同時感染第一及第二型 FCoV 之 FIP 病例，其最佳化條件為肝臟切片以 50 μg/mL proteinase K 於 37℃ 作用 1 小時，及肺臟切片以 2 mg/mL pepsin 於室溫下作用 4 小時。以此最佳化消化條件進行後續之 direct in situ RT-PCR ，其肝臟及肺臟均可觀察到第二型 FCoV 所感染的細胞。

Feline infectious peritonitis (FIP) is an immune-mediated disease manifesting clinically as effusive or non-effusive form. The causative agents, feline coronaviruses (FCoVs), are serologically classified into type I and II. In order to understand the differences in the distribution pattern of FCoV-infected cells within the lesions between effusive and non-effusive FIP, cats presented to the Veterinary Hospital at National Taiwan University with the final diagnosis of FIP were selected. Tissue samples from five typical non-effusive cases and eight typical effusive cases were subjected to immunohistochemistry staining. FCoV-infected cells were found in nearly all kinds of the examined organ samples. The FCoV-positive rates of kidney (80%) and brain (100%) in non-effusive FIP were higher than those of effusive FIP (37.5% and 40%, respectively). As comparing to effusive cases, a tendency that the lesions associated with FCoV were more abundant and severe in non-effusive ones was noted. To correlate different type of FCoVs with the development of FIP, in situ amplification of FCoV nucleic acid in affected tissues was attempted using type-specific primers via direct in situ RT-PCR. As the process of digestion might greatly influence the test results, optimization of the assay condition was first established. For F96-156, a FIP case that infected with type I and II FCoVs, incubation with 50 μg/mL of proteinase K at 37℃ for one hour in liver sections, and 2 mg/mL of pepsin at room temperature for four hours in lung sections were found to be optimal. Under these optimized conditions, type II FCoV-infected cells were detected in both liver and lung.