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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 闕玲玲(Ling-Ling Chueh) | |
dc.contributor.author | Li-En Hsieh | en |
dc.contributor.author | 謝立恩 | zh_TW |
dc.date.accessioned | 2021-06-16T05:08:13Z | - |
dc.date.available | 2019-08-25 | |
dc.date.copyright | 2014-08-25 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55768 | - |
dc.description.abstract | 貓傳染性腹膜炎 (Feline infections peritonitis, FIP) 為一發生於所有家貓及野生貓科動物 (Felidae) 之免疫媒介性、高度致死性之疾病,其病原為隸屬於冠狀病毒科 (Family Coronaviridae) ,甲型冠狀病毒屬 (Genus Alphacoronavivrus) 之貓冠狀病毒 (Feline coronavirus, FCoV)。FCoV之感染廣泛分布於全世界,包括台灣。其主要造成貓隻不顯性感染,僅於部分年幼貓隻造成輕微之腸炎 (Enteritis),然而,於血清陽性 (Seropositive) 貓隻中,約有5-12%動物會發展為FIP,且一旦出現臨床症狀,多數病貓會於一個月內死亡。因此,FIP為貓隻重大疾病之一。然而,目前對於此疾病並無具保護力之疫苗或有效的治療藥物可供使用。目前對於FCoV及其所造成之疾病仍有許多問題尚待解決。
根據感染後的疾病呈像,FCoV可以進一步的被分為兩個生物型 (Biotypes):貓常腸道型冠狀病毒 (Feline enteric coronavirus, FECV),其主要造成消化道之感染且可以廣泛的於貓群中傳播;貓傳染性腹膜炎病毒 (Feline infectious peritonitis virus, FIPV),造成高死亡率之FIP。除疾病呈像上之差異外,兩型病毒於體外複製 (Replication) 之能力上亦具有其差異。FIPV相較於FECV可以有效率的感染其標的細胞 (Target cell),巨噬細胞 (Macrophage),此外亦可快速的傳播並維持其於細胞中之感染。目前雖有許多病毒毒力 (Virulence) 相關之基因被提出,例如棘 (Spike) 蛋白、膜 (Membrane) 蛋白、及附屬基因 (Accessory gene) 3c及7b,其於毒力上影響之角色仍不清楚。在本研究中,完整之附屬基因3c經選殖後,使其成功於細胞中表現,用以評估3C蛋白質對附屬基因3c缺失之病毒株複製上之影響。結果發現3C蛋白質於病毒複製之角色中為一抑制因子,此一效應或與FECV具有較低之毒力有所相關;此外,於該實驗中亦發現,此一3C蛋白質所造成之病毒複製抑制效應並非透過細胞自噬 (Autophagy pathway) 而達成。 由於FIP目前仍缺乏疫苗及治療可用,臨床上一旦診斷為FIP,多為建議進行安樂死 (Euthanasia),然而此造成了金錢、情感及道德很大之衝擊。為了找尋其他具有治療潛力之物質,第二部分之實驗評估了16種可購得之化合物,包含了核苷酸類似物 (Nucleoside analogue)、蛋白酶抑制劑 (Protease inhibitor)、反轉錄酶抑制劑 (Reverse transcriptase inhibitor) 及具有其他活性之化合物,之抗FCoV活性,其中蛋白酶抑制劑nelfinavir及甘露醣結合凝集素 (Mannose-binding lectin) Galanthus nivalis agglutinin顯示具有顯著之抗病毒效應,此外,兩藥物之合併更可看到加乘 (Synergy) 之抑制病毒複製效應。 然縱使成功找到具有治療潛能之抗病毒藥物,臨床上前來就診之貓隻多為疾病之較晚期,限制了治療藥物的成效。為了能夠更有效率的避免疾病的發生,第三部分的實驗嘗試找尋貓隻疾病相關之基因多型性 (Disease-associated genetic polymorphism)。由於在FIP動物中,許多細胞激素 (Cytokines) 之表現被發現會顯著改變,其中,γ干擾素 (Interferon-γ, IFN-γ) 在許多研究中都發現於發病貓體內會顯著的下降,此外在人類的研究中,IFN-γ之基因多型性亦被發現與許多的疾病,如病毒感染及免疫媒介性疾病有所相關。為了找尋與疾病相關之遺傳因子,分析了六年間一共63隻FIP貓及82隻無臨床症狀之FCoV感染貓隻,於貓IFN-γ之第一內插子 (Intron 1) 之序列上共找到了16個單核苷酸多型性 (Single nucleotide polymorphism, SNP),其中三個分別位於+401、+408及+428號位置之SNPs顯示與FIP之發生有顯著相關。此外+428號位置之SNP亦與FIP血漿中IFN-γ濃度有明顯之關聯性。為了進一步運用本研究所找到之疾病相關多型性於篩選具FIP抗性之貓隻,本研究亦針對三個基因多型性,利用擴增受阻突變體系聚合酶鏈反應 (Amplification refractory mutation system polymerase chain reaction) 及限制片段長度多態性 (Restriction fragment length polymorphism) 設計基因分型之技術。未來可運用此基因分型方法藉此找出FIP抗性及感受性貓隻,以降低於貓群中FIP之發生。 | zh_TW |
dc.description.abstract | Feline infectious peritonitis (FIP) is an immune mediated highly lethal disease in all domestic and non-domestic Felidae. The causative pathogen of FIP is feline coronavirus (FCoV), which belongs to the Alphacoronavirus in the family Coronaviridae. The infection of FCoV is distributed widely around the world, including Taiwan. Most of the infection is asymptomatic and only mild enteritis could be observed in some kittens. However, there are about 5-12% seropositive cats will develop FIP. Once the clinical signs appeared, diseased cats usually died within one month. Hence, FIP is one of the most important diseases in cats. As a disease with nearly 100% of mortality, no preventive vaccine and effective therapeutic agents are currently available for FIP. Several questions regarding the virus and its related disease remain to be answered.
According to the outcome of infection, FCoV could be further categorized into two biotypes: feline enteric coronavirus (FECV), which leads to the enteric infection and is widely distributed in cat populations especially in multicat environment, and feline infectious peritonitis virus (FIPV), which induces the highly lethal FIP. In addition, these two biotypes of FCoV showed a difference in infection kinetics in vitro and in disease manifestation as well. FIPV infects the target cell, macrophages, more efficiently, spread to other susceptible cells faster, and sustained the infection better than FECV. Despite several virulence related genes, i.e., spike, membrane, 3c, and 7b, have been proposed, the actual role of the mutation of these genes is unclear. In this study, full length of accessory gene 3c was cloned and the effect of the expression of 3C protein on the FCoV harboring a truncated accessory gene 3c is assessed. The 3C protein is confirmed to play an inhibitory role in the viral replication, which may be associated with the low virulence of FECV, and this effect is independent to the autophagy pathway. Due to the lack of vaccine and therapy, the suggested clinical management of FIP is euthanasia that causes an enormous financial, emotional and ethical impact. To search the agents with potential therapeutic effect, the anti-FCoV activity of 16 commercially available compounds belongs to four categories, i.e., nucleoside analogue, protease inhibitor, reverse transcriptse inhibitor, and compounds with other activities, were examined. Among all the compounds tested, nelfinavir, a protease inhibitor, and Galanthus nivalis agglutinin, a mannose-binding lectin, were found to exhibit a significant anti-FCoV effect. In addition, the combination of these two compounds were demonstrated to complete inhibit the viral replication, synergistically. Despite the antiviral compounds with therapeutic potential were identified, as FIP cats presented in the clinics are usually at the late stage of the disease, the effect of therapy is often very limited. In order to prevent the occurrence of disease more efficiently, the identification of the disease-associated genetic polymorphism in cats was attempted. Expression of several cytokines was shown to be significantly altered in FIP cats when compared to the FCoV-infected non-FIP cats. Among the cytokines that have been studied, the transcription level and plasma concentration of IFN-γ is consistently decreased in FIP cats. Since the polymorphisms of IFN-γ gene in human have been demonstrated to be associated with various virus infections and immune-mediated diseases. In order to identify the possible genetic factor associated with the occurrence of FIP, a survey of feline IFN-γ gene were conducted in 63 FIP cats and 82 asymptomatic cats from our teaching hospital during the past six years. Sixteen single nucleotide polymorphisms were successfully identified in the intron 1, and three of them, i.e., polymorphisms at position +401, +408, and +428, were found significantly associated with the occurrence of FIP. In addition, polymorphism at position+428 also showed a clear correlation with the plasma concentration of IFN-γ in FIP cats. Since the disease-associated polymorphism could be used as a marker for the selection of FIP-resistant cats, three genotyping methods using amplification refractory mutation system polymerase chain reaction and restriction fragment length polymorphism for fIFNG+401, +408, and +428 were established in this study. These selection markers could be utilized for the further identification of FIP-resistant and -susceptible cats, and decrease the occurrence of FIP in cats populations. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:08:13Z (GMT). No. of bitstreams: 1 ntu-103-D98629008-1.pdf: 16222357 bytes, checksum: 540a2dc75ad0f0666c3188d8e150ea0d (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 1. Literature review 17
1.1. Discovery of feline infectious peritonitis and feline coronavirus 17 1.2. Characteristics of CoVs 17 1.2.1. Coronaviridae 17 1.2.2. Genome structure 18 1.2.3. Viral replication 19 1.2.4. Structural proteins and accessory proteins 20 1.3. FCoV spike protein and host range 22 1.4. Serotypes of FCoVs 23 1.5. Genetic determinant for the pathogenicity of FCoV 25 1.6. FCoV and related diseases 26 1.6.1. Host and transmission 26 1.6.2. Immunopathogenesis and immune responses 28 1.6.3. Immune evasion 31 1.7. Clinical signs and diagnosis 31 1.7.1. Clinical signs of coronavirus enteritis 32 1.7.2. Clinical signs of FIP 32 1.7.3. Diagnosis of FIP 33 1.8. Treatment and prevention of FIP 34 1.8.1. Treatment of FIP 34 1.8.2. Prevention of FIP 36 2. Introduction 38 2.1. Investigation of virulence factors of FCoV 38 2.2 Screening of antiviral agents against FCoV 39 2.3 Identification of host factors of FIP 40 3. Materials and methods 42 3.1 Investigation of virulence factors of FCoV 42 3.1.1. Specimens 42 3.1.2 Amplification of the 3c gene 42 3.1.3. Cells and virus 42 3.1.4. Construction of a plasmid for the expression of full-length 3C protein 43 3.1.5. Establishment and confirmation of the 3C protein-expressing cells 43 3.1.6. Effect of the expression of the 3c gene on the replication of FCoV 44 3.1.7. Involvement of autophagy in the effect of the 3C protein 44 3.1.8. Statistical analysis 45 3.2 Screening of antiviral agents against FCoV 45 3.2.1. Test compounds 45 3.2.2. Cells and virus 46 3.2.3. Cell viability 46 3.2.4. Screening of antiviral effects 47 3.2.5. Concentration-dependence of antiviral effects 47 3.2.6. Fifty percent of inhibitory concentration (IC50) 48 3.2.7. Combined use of the effective compounds in fcwf-4 cells 48 3.2.8. Statistical analysis 48 3.3. Identification of host factors in FIP 49 3.3.1. Specimens 49 3.3.2. Identification of SNPs in the partial fIFNG sequences 49 3.3.3. Linkage disequilibrium (LD) test and association analysis 50 3.3.4. Quantification of IFN-γ levels in the plasma samples of FIP cats 50 3.3.5. Genotyping of SNPs by tetra-primer amplification refractory mutation system PCR (ARMS-PCR) and restriction fragment length polymorphisms (RFLP) 50 4. Results 52 4.1 Investigation of virulence factors of FCoV 52 4.1.1. Integrity of the 3c gene of FCoV from FIP and asymptomatic cats 52 4.1.2 Alteration of viral replication in cells transiently expressing 3C protein 52 4.1.3. Expression of the 3C protein 53 4.1.4. Changes in viral replication in 3c-expressing cells. 53 4.1.5. The 3C protein-mediated inhibitory effect on FCoV replication is not mediated by autophagy 54 4.2 Screening of antiviral agents against FCoV 54 4.2.1. Cell viability 54 4.2.2. Screening the 16 test compounds for antiviral activity 55 4.2.3. Concentration-dependence of antiviral effects 55 4.2.4. Combined use of nelfinavir and GNA in fcwf-4 cells 55 4.3 Identification of host factors in FIP 56 4.3.1. Identification of hIFNG+874 homologue on feline genome 56 4.3.2. Polymorphisms in fIFNG 57 4.3.3. Association study 57 4.3.4. Production of IFN-γ in FIP cats carrying different genotypes 58 4.3.5. Genotyping for disease-related SNPs 59 5. Discussion 60 5.1 3C protein of feline coronavirus inhibits viral replication independently of the autophagy pathway 60 5.2 Synergistic antiviral effect of Galanthus nivalis agglutinin and nelfinavir against feline coronavirus 63 5.3 Identification and genotyping of feline infectious peritonitis-associated single nucleotide polymorphisms in the feline interferon-γ gene 66 6. Prospect 70 7. References 101 | |
dc.language.iso | en | |
dc.title | 貓傳染性腹膜炎病毒毒力、抗病毒藥物及宿主基因感受性之探討 | zh_TW |
dc.title | Investigation of the virulence factor, antiviral agents, and host genetic susceptibility of feline infections peritonitis | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 蘇璧伶(Bi-Ling Su),林昭男(Chao-Nan Lin),黃溫雅(Wen-Ya Huang),林大盛(Dah-Sheng Lin),陳慧文(Huei-Wen Chen) | |
dc.subject.keyword | 貓冠狀病毒,貓傳染性腹膜炎,3C蛋白,細胞自噬作用,抗病毒藥物,宿主基因因子,γ-干擾素, | zh_TW |
dc.subject.keyword | Feline coronavirus,feline infectious peritonitis,3C protein,autophagy,antiviral agents,host genetic factor,interferon-γ, | en |
dc.relation.page | 118 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-20 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
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