Virus isolation, evolution and screening of the effective antiviral drugs of feline coronavirus infection in Taiwan

貓傳染性腹膜炎 (feline infectious peritonitis; FIP) 為造成幼貓死亡的重要傳染病之一，其致病原為貓冠狀病毒 (feline coronavirus; FCoV)，除了造成FIP，此病亦能造成不顯性感染或輕微腸炎。FCoV廣泛分布於全世界貓族群中，但只有5-12%的血清陽性貓隻最後會演變成FIP。截至目前為止，臨床上並無單一血清學或分子診斷可以區分低毒力或有毒力病毒，亦無有效的疫苗可以預防此病之發生，及無有效治療方法可用。因此，本論文為針對台灣地區貓冠狀病毒感染之研究， 主要分成六部分，一 、貓冠狀病毒感染之調查；二、ORF7b 基因之分析；三、不同基因型別病毒之調查及與FIP發病間相關性之探討；四、FIP自然感染病例之病毒分離；五、台灣貓冠狀病毒分離株全基因之定序與病毒演化之分析；六、有效抑制病毒複製藥物之篩選。2000年至2009年間，有1703個FIP懷疑病例由台大動物醫院送檢至本實驗室進行FCoV檢測，結果發現檢出率為38.2% (651/1703)，其中有66個病例透過組織病理學檢查確診為FIP，此些FIP發病貓年紀以小於6月齡為主 (50.7%；34/67)，FIP發病貓隻檢體中存在較高量的病毒。針對來自有FIP病史的不同多貓家庭84隻健康貓進行FCoV檢測，結果發現有67.9% (57/84) 健康貓隻為FCoV陽性。由此部份研究結果顯示台灣地區FCoV感染相當普遍，特別發生於曾出現過FIP病例之多貓環境中之貓隻 (第一部份)。ORF 7b的缺失 (deletion)，曾被懷疑與病毒毒力降低有關，然而我們的研究結果發現ORF 7b的缺失不只出現在低毒力/腸道型病毒，也可存在有毒力/FIP病毒上 (第二部份)。FCoV以血清中和試驗可被區分成二個血清型，在此研究中我們發現，雖然第一型 FCoV的感染率非常高，與FIP發病較具顯著相關性的病毒卻為第二型 (P=0.0046)。比對五年間的代表株病毒的S gene序列，結果發現第一型 FCoV的變異性 (6.2-11.7%) 較第二型 (0.6-3.2%) 高(第三部份)。第四部份、此研究由臨床上一隻4.5 月齡小貓自然發生之FIP 病例，自其胸水與貓全胚胎細胞株進行共同培養 (co-cultivation)，成功地分離到本土株貓冠狀病毒，這是一個生長特性異於現存FCoV 的新病毒分離株，為台灣的首度報告，其命名為FCoV/NTU156/P/’07 (NTU156)。我們進一步進行台灣第二型分離株NTU156全長基因定序及另一個自然感染第二型病毒株部份基因，以軟體分析其重組點，發現NTU156的重組點位於RNA-dependent RNA polymerase (RdRp) 及M基因，另一第二型病毒重組點亦位於M基因，與國外代表株相比較，代表株的重組點位於RDRP (更上游900 nts) 及ORF3-E基因，由此結果發現每一第二型FCoV可能皆來自於獨自的重組事件。進一步分析重組熱點 (hot spot) 之序列發現可能為U(C/U/G/A)U(U/A)A (第五部份)。最後我們使用本土貓冠狀病毒株感染貓全胚胎細胞進行抗病毒藥物之篩選，當Galanthus nivalis agglutinin (GNA) 及nelfinavir合併給與時，則具加乘作用地完全抑制病毒複製，此研究結果指出，合併使用GNA及nelfinavir可做為未來預防及早期治療FIP之用 (第六部份)。Feline infectious peritonitis (FIP) is one of the major infectious causes of mortality in kitten. The causative agent, feline coronavirus (FCoV) is also associated with mild or subclinical enteric infections. Despite the high prevalence of FCoVs in feline populations around the world, only 5-12% of seropositive cats develop FIP. Up to present, there is not a single virological or serolsogical assay that can distinguish virulent from avirulent virus, moreover no effective vaccine or therapy available for FIP. The objectives of this study were to investigate the prevalence of FCoV infection in Taiwan (part I), to further clarify the correlation between FIP, ORF 7b (part II) and different types of FCoV S gene (part II), to isolate the first local FCoV isolate (part IV), to explore the evolutionary insights of type II (part V) and to screen the effective compound for their antiviral activity against a local FCoV strain in fcwf-4 cells. During 2000 to 2009, a total of 1703 cases with a history of FIP-suspected cats from National Taiwan University Animal Hospital were subjected to this study (Part I). FCoV RNA was detected from 651 (38.2%) cats. Among the 67 cases with definite diagnosis of FIP, the highest incidence was found in kittens under 6-months old (50.7%, 34/67). Significantly highest viral load in the effusive fluids of cats from confirmed FIP cases. Eighty-four clinical healthy cats co-hatitated with the FIP cats from various multi-cat households were subjected to further examination. About 67.9% (57/84) of the clinically healthy cats were FCoV positive. These data suggested that the prevalence of FCoV infection is widely in Taiwan and especially in multi-cat population. The open reading frame (ORF) 7b of FCoV has been speculated to play a determining role in virulence as deletions were found to be associated with avirulent viruses. Our part II study revealed that deletions in the ORF7b gene are not constrained to low pathogenicity/enteric biotypes but also associated with pathogenicity/FIP biotypes of FCoV. On the basis of in vitro neutralization tests, FCoVs can be divided into two serotypes. Our results showed that irrespective of the predominance of type I FCoV infection in Taiwan, type II FCoV demonstrated a higher correlation with FIP. Analysis of partial S gene sequences of the local type I and II FCoVs strains revealed that type I viruses were more genetically divergent (6.2-11.7%) than type II viruses (0.6-3.2%) within the 5-year study period (part III). By co-cultivation of pleural effusion from a 4.5-month-old FIP kitten with feline fcwf-4 cells, a local FCoV was isolated. This novel FCoV isolate with distinct growth characteristics is denominated as FCoV/NTU156/P/’07 (NTU156), the first FCoV strain in Taiwan (part IV). We further explore the evolutionary insights of local FCoVs, a complete genome of one local type II FCoV NTU156 and partial genome sequence from one type II virus NTU26-2 were determined. The genome of FCoV NTU156 was found to be 28,897 nucleotides in length, excluding the 3’ polyadenylated tail. Analysis of the sequence identified conserved open reading frames and revealed an overall genome organization similar to known FCoVs. Bearing an in frame deletion of 442 nucleotides in the ORF3c, the genome size of NTU156 was found to be the smallest among subgroup 1a (FCoV Black, FCoV C1Je, and FCoV 79-1146). Bootscan analysis of NTU156 revealed two crossover events took place between type I FCoV and type II CCoV. One of the possible recombination site was located in RNA-dependent RNA polymerase (RdRp) gene and the other in M gene. Analysis of sequence around possible recombination spot of another local type II virus NTU26-2 identified a possible recombination site located in M gene. Comparing the finding from our local viruses with the prototype FCoV 79-1146 strain, of which one possible recombination was located in RdRp (further upstream 900 nucleotides) and the other in ORF 3-E gene, neither of them were identical to our local strains. These data indicated that each type II FCoV strain might has arisen from individual recombination event. Multiple alignments further narrowed the potential hot spot of RNA recombination to a pentanucleotide U(C/U/G/A)U(U/A)A (part V). In our search for agents that may prove clinically effective against FCoV infection, a local FCoV strain was isolated and subsequently used in the screening tests. Our results show that combined use of Galanthus nivalis agglutinin and nelfinavir to FCoV-infected cells, a synergistic antiviral effect with complete blockage of viral multiplication was observed. These results suggest that the combined use of GNA and nelfinavir has therapeutic potential in the prophylaxis and treatment of cats with early-diagnosed FIP (part VI).目錄文摘要.........................................................................................................................IX文摘要.........................................................................................................................XI一章、緒言...................................................................................................................1二章、文獻探討...........................................................................................................2 2.1 貓傳染性腹膜炎之發現歷史..................................................................................2 2.2 病毒分類..................................................................................................................2 2.3 基因體特性..............................................................................................................3 2.4 流行病學與病毒傳播..............................................................................................5 2.5 血清型別..................................................................................................................5 2.6 免疫反應與致病機制..............................................................................................7 2.7 病毒分離..................................................................................................................9 2.8 臨床症狀..................................................................................................................9 2.9 臨床診斷................................................................................................................10 2.10 治療與預防..........................................................................................................11三章、研究背景..............................................................................................................13 第一節 貓冠狀病毒感染之調查.................................................................................13 第二節 ORF7b與病毒致病相關性之分析.................................................................13 第三節 不同基因型別病毒流行病學調查及與FIP關聯性之探討...........................14 第四節 自FIP自然感染病例中分離與鑑定台灣貓冠狀病毒...................................14 第五節 台灣貓冠狀病毒分離株全基因定序及病毒演化分析.................................14 第六節 有效抑制病毒複製藥物之篩選...................................................................................15四章、材料與方法.........................................................................................................17 第一節 貓冠狀病毒感染之調查.................................................................................17 4.1.1 臨床檢體之採集.............................................................................................17 4.1.2 檢體製備及反轉錄反應.................................................................................17 4.1.3 貓冠狀病毒之篩選.........................................................................................18 4.1.4 PCR產物電泳之分析......................................................................................18 第二節 ORF7b與病毒致病相關性之分析.................................................................18 4.2.1 臨床檢體之採集及貓冠狀病毒之篩選.........................................................18 4.2.2 檢體之製備及反轉錄反應.............................................................................19 4.2.3 ORF7b gene之半巢式聚合酵素鍊鎖反應.....................................................19 4.2.4 PCR產物定序及序列之分析..........................................................................20 第三節 不同基因型別病毒流行病學調查及與FIP關聯性之探討...........................20 4.3.1 動物及臨床檢體之採集.................................................................................20 4.3.2 檢體之製備及反轉錄反應.............................................................................20 4.3.3 S gene之聚合酵素鍊鎖反應..........................................................................21 4.3.4 PCR產物定序及序列之分析..........................................................................21 4.3.5 統計分析.........................................................................................................22 第四節 自FIP自然感染病例中分離與鑑定台灣貓冠狀病毒...................................22 4.4.1 細胞培養.........................................................................................................22 4.4.2 病毒分離.........................................................................................................22 4.4.3 冠狀病毒核酸之檢測.....................................................................................23 4.4.4 冠狀病毒抗原之檢測.....................................................................................24 4.4.5 病毒純化、定量及生長曲線.........................................................................24 第五節 台灣貓冠狀病毒分離株全基因定序及病毒演化分析.................................25 4.5.1 病毒株.............................................................................................................25 4.5.2 反轉錄與聚合酵素鍊鎖反應.........................................................................26 4.5.3 5’端及3’端cDNA端點快速增幅技術............................................................26 4.5.4 PCR產物定序..................................................................................................26 4.5.5 序列分析.........................................................................................................27 第六節 有效抑制病毒複製藥物之篩選...................................................................................27 4.6.1 試驗藥物.........................................................................................................27 4.6.2 細胞與病毒.....................................................................................................27 4.6.3 細胞存活試驗.................................................................................................27 4.6.4 抗病毒藥物之篩選.........................................................................................28 4.6.5 50%有效濃度試驗..........................................................................................28 4.6.6 藥物濃度依賴性試驗.....................................................................................28 4.6.7 有效藥物合併使用試驗.................................................................................29 4.6.8 統計分析.........................................................................................................29五章、結果.................................................................................................................30 第一節 貓冠狀病毒感染之調查.................................................................................30 5.1.1 貓隻之分類.....................................................................................................30 5.1.2 疑似FIP發病貓之貓冠狀病毒盛行率及不同臨床檢體之檢出率...............30 5.1.3 FIP發病貓隻與同居健康貓隻排毒之檢測...................................................30 5.1.4 健康貓及流浪貓之檢測.................................................................................31 第二節 ORF7b與病毒致病相關性之分析.................................................................31 5.2.1 貓冠狀病毒感染貓隻.....................................................................................31 5.2.2 ORF7b 序列之分析........................................................................................32 5.2.3 ORF7b 序列之比對........................................................................................32 第三節 不同基因型別病毒流行病學調查及與FIP關聯性之探討...........................33 5.3.1 不同型別貓冠狀病毒之盛行率.....................................................................33 5.3.2 不同型別貓冠狀病毒與FIP發病之關聯性...................................................33 5.3.3 不同型別貓冠狀病毒之基因比對及演化之分析.........................................34 第四節 自FIP自然感染病例中分離與鑑定台灣貓冠狀病毒...................................34 5.4.1 貓傳染性腹膜炎之確診及病毒分離.............................................................34 5.4.2 貓冠狀病毒分離株之病原確認.....................................................................35 5.4.3 One-step growth curve....................................................................................35 第五節 台灣貓冠狀病毒分離株全基因定序及病毒演化分析.................................36 5.5.1 貓冠狀病毒株NTU156全基因之分析...........................................................36 5.5.2 5’端及3’端非轉譯區及開放讀碼區..............................................................36 5.5.3 Subgroup 1 CoVs基因比對及親緣樹狀圖之分析........................................36 5.5.4 S基因分析與APN binding site之比較...........................................................37 5.5.5 貓冠狀病毒株NTU156重組點之分析...........................................................38 5.5.6 其他第二型 FCoV重組區域之比對及重組熱點之分析..............................38 第六節 有效抑制病毒複製藥物之篩選...................................................................................39 5.6.1 細胞存活試驗.................................................................................................39 5.6.2 抗病毒藥物之篩選.........................................................................................39 5.6.3 藥物濃度依賴性試驗.....................................................................................39 5.6.4 有效藥物合併使用試驗.................................................................................40六章、討論.................................................................................................................41 第一節 貓冠狀病毒感染之調查.................................................................................41 第二節 ORF7b與病毒致病相關性之分析.................................................................43 第三節 不同基因型別病毒流行病學調查及與FIP關聯性之探討...........................44 第四節 自FIP自然感染病例中分離與鑑定台灣貓冠狀病毒...................................46 第五節 台灣貓冠狀病毒分離株全基因定序及病毒演化分析.................................47 第六節 有效抑制病毒複製藥物之篩選...................................................................................50七章、結論.................................................................................................................53八章、展望.................................................................................................................55九章、參考文獻.........................................................................................................57目錄1-1 各個臨床檢體數及FCoV陽性率.........................................................................761-2 疑似發病及確診為FIP貓隻各檢體陽性率之比較..............................................761-3 全世界貓冠狀病毒流行病學調查之比較..............................................................772-1 ORF7b 增幅之特異性引子..................................................................................782-2 40隻貓隻之健康狀況、基本資料及病毒命名...................................................792-3 全世界之ORF7b定序結果...................................................................................802-4 全世界ORF7b基因缺失病毒株之比較...............................................................813-1 台灣貓冠狀病毒之分型及不同型別病毒之盛行率...........................................823-2 不同型別貓冠狀病毒與FIP發病之關聯性.........................................................823-3 10個進一步分析S基因序列的病例資料.............................................................833-4 台灣地區第一型貓冠狀病毒序列之變異性.......................................................843-5 台灣地區第二型貓冠狀病毒序列之變異性.......................................................843-6 全世界不同型別貓冠狀病毒流行病學調查之比較............................................854-1 貓小病毒及貓卡里西病毒特異性引子...............................................................865-1 FCoV NTU156之開放讀碼區大小及transcription regulatory sequences...........875-2 FCoV NTU156與其他group 1 CoV基因之相似性.............................................885-2 已確認及推測之重組熱點...................................................................................896-1 抗病毒藥物對貓全胚胎細胞之毒性及對FCoV之抗病毒作用.........................90amp;#8232;圖目錄2-1 自臨床檢體分析FCoV ORF7b基因....................................................................91 2-2 ORF7b 基因序列之比對及親緣樹狀圖之分析..................................................923-1 12株第一型貓冠狀病毒株及3株犬冠狀病毒株之S gene 168 bp片段之比對分析.....................................................................................................................................933-2 11株第一型貓冠狀病毒株、2株犬冠狀病毒株及二株野生動物冠狀病毒株之S gene 168 bp片段之比對分析......................................................................................943-3 本土貓冠狀病毒株、犬冠狀病毒與文獻貓冠狀病毒株、犬冠狀病毒株及野生動物冠狀病毒株之部份S gene親緣樹狀圖之分析..................................................954-1 貓傳染性腹膜炎之確診.......................................................................................964-2 FIP發病貓隻之胸水與貓全胚胎細胞共同培養.................................................974-3 貓冠狀病毒核酸檢測...........................................................................................984-4 細胞培養上清液以nPCR或RT-nPCR排除其他貓病毒之汙染..........................994-5 貓冠狀病毒抗原之確認..............................................................................100-1014-6 NTU156在貓全胚胎細胞之生長曲線...............................................................1024-7 NTU156對貓腎細胞之敏感性...........................................................................1035-1 FCoV NTU156之ORF3基因..............................................................................1045-2 FCoV NTU156各基因與其他subgroup 1a CoVs之親緣樹狀圖...............105-1075-3 FCoV NTU156 S基因之分析......................................................................108-1105-4 FCoV NTU156全基因重組區域之分析.....................................................111-1125-5 FCoV NTU156及NUT26-2重組區域序列比對.................................................1135-6 FCoV 79-1146全基因重組區域之分析.............................................................114 圖5-7 FCoV 79-1146及79-1683重組區域序列比對.....................................................1156-1 FCoV NTU156感染貓全胚胎細胞.....................................................................1166-2 藥物抗病毒能力之評估..............................................................................117-1186-3 藥物對不同病毒濃度依賴性試驗.....................................................................119圖6-4 Nelfinavir及GNA合併使用試驗........................................................................12