台灣貓冠狀病毒感染之病毒分離、演化及抗病毒藥物篩選

Abstract

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).