- 學位論文
貓傳染性腹膜炎病毒之分離與基因分析
Isolation and Genetic Analysis of Feline Infectious Peritonitis Virus
摘要
中文摘要 貓冠狀病毒 (Feline coronavirus, FCoV) 廣泛存在於家貓及野生貓科動物，感 染雖大多僅出現輕微下痢或無臨床症狀，然有部分會發展為致死性之貓傳染性腹 膜炎 (Feline infectious peritonitis, FIP)。依血清型 FCoV 可被區分為第一與第二型， 兩型 FCoV 之感染皆可能發展成 FIP。為尋找有效抗病毒藥物與 FIP 致病機制探討 之需，本實驗自 2010 年至 2012 年間收集了 11 隻臨床上證實為 FIP 發病貓之新鮮 胸水及腹水，取其內之細胞與貓全胚胎細胞共同培養方式，其中一隻 10 月齡母貓 之腹水於繼代至第五代時，出現冠狀病毒感染所引起特異性細胞病變效應之多核 巨細胞，以抗 FCoV N protein 抗體對感染細胞進行免液螢光染色，結果可在多核 巨細胞質內發現強烈螢光訊號。此病毒經三次純化後，經核酸檢測確定為第二型 FCoV 並命名為 NTU204。以 Simplot 與 Bootscan 兩軟體分析 NTU204 之演化由來， 發現此病毒可能於基因體 13350nt 與 24800 nt 附近，透過第一型 FCoV 與犬或豬之 冠狀病毒經兩次重組而來，重組區域分別位於 RdRp 與 S 基因上。由於世界各地 FCoV 之感染，大多以第一型為主，相較於第二型此型病毒不易適應體外細胞培養 系統，因此分離工作相對困難。為提升第一型 FCoV 分離機率，本研究利用真核 表現載體將已被證實具提升 FCoV 感染能力之輔受器 fDC-SIGN 轉染至貓腎細胞， 將第二型 FCoV 病毒接種於此細胞時，發現在 1MOI 下表現 fDC-SIGN 之細胞確 實出現較高的病毒力價。證實此輔受器確實具提升病毒複製效應後，利用此細胞 嘗試進行第一型病毒之分離，則未能順利分離出來。
並列摘要
ABSTRACT Feline coronavirus (FCoV) is widely distributed in domestic cats and wild felids. Most of the infections are asymptomatic or causing only mild diarrhea, however, some might result in fatal feline infectious peritonitis (FIP). FCoVs are divided into two distinct serotypes I and II that both can cause FIP. In order to isolate viruses for the study of antiviral agents and pathogenesis of FIP, fresh body effusion from a total of 11 FIP cats were collected from 2010 to 2012. Through co-cultivation of the cells derived from ascites of a 10-month-old cat with feline Fcwf-4 cell line to the fifth passages, a coronavirus specific cytopathic effect was observed and confirmed by immunofluorescent staining. After three times purification, sequencing and genotyping a local FCoV-II was confirmed (NTU204). Simplot and Bootscan analysis of NTU204 revealed two crossover events took place between FCoV-I and canine or pocine coronavirus. One of the possible recombination site is located in RdRp gene (around genome 13350 nt and the other is in S gene (around genome 24800 nt). FCoV-I is the more prevalent serotype currently in circulation; however, due to difficulties associated with in vitro cultivation only limited data can be found regarding its infection. In order to increase the isolation rate of FCoV-I, a known FCoV co-receptor (Feline dendritic cell-specific ICAM-3-grabbing nonintegrin, fDC-SIGN) was expressed in the susceptible feline cell line. When stable transfected cells expressing the expected fDC-SIGN were subjected to the infection of a Taiwanese FCoV-II isolate, viral titer was higher than the control cells at 1MOI. After confirming the co-receptor function of fDC-SIGN in the enhancement of viral replication, these stable clone cells were used to isolate FCoV-I. However, none of the isolate was able to be isolated.
參考文獻
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