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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 闕玲玲(Ling-Ling Chueh) | |
| dc.contributor.author | Ying-Ting Wang | en |
| dc.contributor.author | 王映庭 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:46:23Z | - |
| dc.date.available | 2025-12-29 | |
| dc.date.copyright | 2015-03-13 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-02-02 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55076 | - |
| dc.description.abstract | 貓冠狀病毒 (Feline coronavirus, FCoV) 之感染普遍存在於貓隻族群中。FCoV感 染貓隻多不會出現臨床症狀。然而在約< 5%的血清陽性貓隻會造成一高度致死性、免疫媒介性疾病,稱為貓傳染性腹膜炎 (Feline infectious peritonitis, FIP)。目前並未有有效的疫苗及治療。由於貓隻於FIP發病後 100%死亡,一旦確診後,臨床上通常建議安樂死。直至今日,仍有許多關於病毒及宿主基因差異性於發病所中扮演之角色的問題尚待釐清。本研究深入分析FCoV之傳播、流行病學及病毒演化,並找尋與FIP相關之宿主基因多型性以期運用於未來疾病之防控。
造成FIP之病毒 (FIPV) 目前被認為散發性地由感染貓隻體內突變而來且無法有效率的於貓隻間傳播。本研究室近期於台灣的私人動物收容所中觀察到FIP之爆發, 於所有 46 隻貓中,13 隻貓出現典型 FIP之臨床症狀。進一步分析發病動物體內病毒之S及 3c基因,證明其皆感染帶有相同之重組點之第二型 FCoV,本研究並確立 FIPV 於貓隻間水平傳播之可能性,因此 FIP 發病動物可能對同一環境中之貓隻造成潛在之危害。由於第二型 FCoV 被認為與 FIP 及疾病之爆發有相關性。為了鑑別兩血清型之病毒感染並進行流行病學之調查,透 過桿狀病毒表現FCoV型別特異性棘蛋白,用以進一步分析過往八年間兩血清型 FCoV 於台灣貓隻感染之情形。 結果發現第一型 FCoV 為主要感染之血清型 。此外,比較以此血清分型方法與基因分型結果,發現兩者具有高度之相關性。此結果亦支持目前對於FCoV演化及FIP傳播之認知。基於FIP致病機制屬於免疫媒介特性,宿主基因多型性被認為影響了FIP的發生與否。為找尋與FIP相關之宿主因子,分析貓隻腫瘤壞死因子 (Tumor necrosis factor alpha, TNF-α)、FCoV 受體C-type lectin DC-SIGN (CD209) 及五個於柏曼貓被報告與 FIP相關之單核苷酸多型性 (Single nucleotide polymorphism, SNP) 之基因多型性,以了解宿主背景因子與FCoV感染後發病與否之相關性。其中,於TNF-α促進子、fCD209之外顯子 (extracellular domain) 與三個位於fCD209之內插子之 SNPs發現與FIP發病相關。相對於此,5個先前被報告與柏曼貓發病相關之SNPs,於此研究則未檢測出與疾病相關。由於宿主對於疾病之感受性/抵抗性為多因子性,且許多不同之宿主基因可能同時影響了FIP的發生與否,於本研究中所找 尋之5個基因多型性差異點可以被運用於改善抗FIP動物之篩選,以期減少未來貓隻死亡。 | zh_TW |
| dc.description.abstract | Feline coronavirus (FCoV) is an ubiquitous viral pathogen in the cat populations. The infection of FCoV is usually asymptomatic. However, less than 5% of the seropositive cats develop a highly lethal immune-mediated disease, feline infectious peritonitis (FIP). Until now, neither effective treatment nor protective vaccine is available. Euthanasia is suggested once the disease was diagnosed, as all FIP cats eventually died from this disease. To date, several questions remain unsolved regarding roles of the virus and the host genetic factors contributing to the development of FIP. The virus that causes FIP (FIPV) is believed to occur sporadically and spread infrequently from cat to cat. However, an FIP outbreak from a private animal shelter that causes 13 out of 46 cats died from FIP was confirmed in our lab recently. Sequence analysis of the S and 3c gene showed that the FIPVs of the outbreak were from the same origin and the results indicate that horizontal transmission of FIPV is possible and that FIP cats can pose a potential risk to other cats living in the same environment. Serotype II FCoV was suggested to be significantly correlated with FIP and an outbreak caused by this type of FCoV has been confirmed. A Baculovirus-expressed type-specific spike proteins based assay was used fro the seroprevalence study of two types of FCoV in the past eight years in Taiwan was firstly carried out. Type I FCoV was found to be predominant compared to type II virus. Results derived from serotyping and genotyping support our current understanding of the evolution of disease-related FCoV and the transmission of FIP. With an immune-mediated disease entity, host genetic variant was suggested to influence the occurrence of FIP. The association between the single nucleotide polymorphisms (SNPs) of tumor necrosis factor-α (fTNFA), DC-SIGN (CD209), and the five FIP-associated SNPs identified from Birman cats, and the outcome of FCoV infection was determined. Among the 57 SNPs identified, five of them, including one in the promoter region of fTNFA, one in the coding region of extracellular domain of DC-SIGN, and three in the introns of CD209 were associated with the outcome of FCoV infections. None of the five Birman FIP cat-associated SNPs showed significant differences between our FIP and non-FIP groups. As disease resistance is multifactorial, the five genetic traits identified in this study should facilitate in the future breeding of the disease-resistant animal to reduce the occurrence of cats succumbing to FIP. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T03:46:23Z (GMT). No. of bitstreams: 1 ntu-104-D00629005-1.pdf: 16251049 bytes, checksum: 2998c8c25d23d86ef6265eae8f88113b (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | Contents
Abstract.....................................................................................................1 Contents.....................................................................................................6 1. Literature review....................................................................................14 1.1. History of feline infectious peritonitis and feline coronavirus ..............14 1.2. Characteristics of coronaviruses.........................................................14 1.2.1. Classification of Coronaviridae........................................................ 14 1.2.2. Genome structure and non-structural proteins ............................... 15 1.2.3. Structural proteins and accessory proteins .................................... 16 1.2.4. Viral entry, uncoating and replication.............................................. 18 1.3. FCoV spike protein and host range ....................................................19 1.4. Serotypes of FCoVs ...........................................................................20 1.5. Genetic determinant for the pathogenicity of FCoV..............................21 1.6. FCoV and related diseases.................................................................23 1.6.1. Host and transmission .................................................................... 23 1.6.2. Immunopathogenesis of FIP............................................................ 24 1.6.3. Immune responses of FIP animals .................................................. 25 1.6.4. Immune evasion ............................................................................. 27 1.7. Clinical signs and diagnosis ..............................................................28 1.7.1. Clinical signs of FCoV enteritis ....................................................... 28 1.7.2. Clinical features of FIP .................................................................... 28 1.7.3. Diagnosis of FIP ............................................................................. 29 1.8. Treatment and prevention of FIP .........................................................30 1.8.1. Treatment of FIP............................................................................... 30 1.8.2. Prevention of FIP.............................................................................. 32 2. Introduction .......................................................................................... 34 2.1. An outbreak of FIP ..............................................................................34 2.2. Seroprevalence of two types of FCoV .................................................35 2.3. FIP-associated host genetic polymorphisms........................................36 3. Materials and methods.......................................................................... 38 3.1. An outbreak of FIP ..............................................................................38 3.1.1.Animals and specimen collection ..................................................... 38 3.1.2. Sample preparation and reverse transcription ................................. 38 3.1.3. Typing of FCoVs using nested PCR.................................................. 39 3.1.4. Amplification, sequencing and analysis of 3a and 3c gene from the type II FCoV................................................................................................ 40 3.1.5. Phylogenetic analysis and recombination site analysis of a type II FCoV ......................................................................................................... 40 3.2. Seroprevalence of two types of FCoV ..................................................41 3.2.1. Viruses and cells ...................................................................................................................... 41 3.2.2. Detection and genotyping of FCoV.............................................. 41 3.2.3. Clinical samples ............................................................................. 41 3.2.4. Detection of anti-FCoV antibody...................................................... 42 3.2.5. Amplification, cloning and expression of type-specific S genes ...... 43 3.2.6. Confirmation of recombinant proteins ............................................. 43 3.2.7. Serotyping ...................................................................................... 44 3.2.8. Neutralization test (NT) ................................................................... 44 3.3. FIP-associated host genetic polymorphisms.......................................45 3.3.1 Animals and specimens ................................................................... 45 3.3.2 Identification of SNPs in target sequences ...................................... 46 3.3.3 Association analysis ........................................................................ 47 4. Results................................................................................................. 48 4.1. An outbreak of FIP .............................................................................48 4.1.1. Confirmation of an FIP outbreak in the cat shelter............................ 48 4.1.2. Type II FIPVs were consistently detected in the FIP-succumbed cats.......................................................................................................... 48 4.1.3. A type II FIPV of the same origin was detected from FIP-succumbed cats.......................................................................................................... 49 4.1.4. An identical nonsense mutation in 3c gene was found in two FIP-succumbed cats ...................................................................................... 50 4.1.5. Shedding of the type II FIPV can be detected at the terminal stage of FIP cats ................................................................................................... 50 4.2. Seroprevalence of two types of FCoV ................................................51 4.2.1. Prevalence of FCoV in Taiwan ........................................................ 51 4.2.2. Amplification of S region based on RBD of type I and II FCoV for type-specific protein expression....................................................................... 51 4.2.3. Establishment and confirmation of type-specific IFA....................... 52 4.2.4. Association between FCoV genotype and serotype ........................ 52 4.3. FIP-associated host genetic polymorphisms.......................................53 4.3.1. Polymorphism at fTNFA - 421 was found to be significantly associated with resistance to FIP ............................................................................... 53 4.3.2. Polymorphisms in the extracellular domain and introns 6 and 7 of fCD209 were found to be significantly associated with the disease outcome ................................................................................................................. 54 4.3.3. Evaluation of the association between the FIP-associated SNPs reported in Birman cats and disease susceptibility in a cat population with higher genetic variability .......................................................................... 56 4.3.4. Association study .......................................................................... 56 4.3.5. The number of the FIP-associated SNPs harbored is correlated to the disease outcome...................................................................................... 56 5. Discussion ........................................................................................... 58 5.1. An outbreak of feline infectious peritonitis: molecular evidence of horizontal transmission of a type II FCoV....................................................58 8 5.2. An eight-year epidemiologic study based on baculovirus-expressed type- specific spike proteins for the differentiation of type I and II FCoV.....60 5.3. Polymorphisms in the feline TNFA and CD209 genes are associated with the outcome of FCoV infection...................................................................63 6. Summary .............................................................................................. 67 7.Prospect..................................................................................................70 8. References ...........................................................................................101 Tables Table 1. Information for all FIP-suspected and confirmed cats from the cat shelter...72 Table 2. Detection and typing of FCoV in fecal samples from healthy cats in the same shelter.................................................................73 Table 3. The characteristics of 3c genes of FCoV recovered from various specimens of FIP cats. ..............................................................................74 Table 4. Shedding and serotypes of feline coronavirus detected in FIP cats in the cat shelter............................................................................................75 Table 5. Prevalence of FCoV serotypes I and II in cats with different disease statuses in Taiwan. ....................................................................................76 Table 6. Genotype, serotype and NT titer against FCoV from FIP cats........77 Table 7. Correlation between serotype and genotype in FCoV antibody-positive animals without FIP. .....................................................................78 Table 8. Primers used for SNP identification of fTNFA and fCD209 gene....79 Table 9. Primers used for SNP identification of five suspected FIP-associated SNPs in Birman cats .................................................................................80 Table 10. Frequencies of the fTNFA genotypes and alleles and associations with the outcome of FCoV infection ...........................................................81 Table 11. Frequencies of the fCD209 genotypes and alleles and associations with the outcome of FCoV infection ...........................................................82 Table 12. Frequencies of the genotypes and alleles of the proposed FIP-associated SNPs in Birman cats and associations with FIP .......................89 Table 13. FIP-associated genetic polymorphisms identified in the fIFNG, fTNFA, and fCD209 gene and their associations with FIP ..........................90 Table 14. Association of the number of the disease-associated SNPs harbored, including fIFNG + 428, fTNFA - 421, and fCD209 + 1900, + 2276, + 2392, and + 2713, and the outcome of FIP ................................................91 Figures Figure 1. Recombination of FIPV from cats 1, 7, 9, 10, 11, 12 and 13 on the S gene. The alignment of the 3′ end of the S gene to the downstream genes of FCoV isolated from seven FIP cats with type I FCoV and CCoV. The light and dark shaded areas encompass higher similarity to CCoV and type I FCoV, respectively. The putative recombination event occurred at nucleotide 4250 based on the comparison with FCoV NTU2 and is indicated with an arrow. The sequences were obtained from FIPV found in individual samples and tissues and are shown collectively..............................................................92 Figure 2. Recombination site analysis of FIPVs from cats 1, 7, 9, 11, 12 and 13 on the S gene. Similarity plot analysis with the Kimura (two-parameter) distance model, neighbor-joining tree model and 1000 bootstrap replicates showed a recombination event, and the putative crossover site is indicated with an arrow. .........................................................................................93 Figure 3. Alignment of complete 3c genes of FIPVs from cats 1, 7, 9, 11, 12 and 13. (a) Full-length 3c genes analyzed in this study were aligned a type I FCoV, FCoV NTU2. A box represents identified premature stop codons. (b) Scheme shows the location of premature stop codons (PT) of 3c gene of various specimens from different FIP cats. ..............................................94 Figure 4. A schematic of the timeline of the FIP outbreak. Solid line indicates the period that animals lived in the shelter. Dotted line: the periods of animals leaved the shelter to another rehoming center. Inverted triangle: fever observed. Open circle: animals enterimg the shelter. X: death of FIP cat...........................................................................................................95 Figure 5. Amplification and expression of the type-specific S region of type I and II FCoV. (A) The alignment of the amino acid sequences of the putative RBD of S protein from type II FCoV and type I FCoV with the sequences from prototypic viral strains. The boxed nucleotides represent N-linked glycosylation sites. (B) Sf-9 cells were infected with two r-viruses (rFCoV-I and rFCoV-II) at different MOI values, and the cells were harvested at different times post-infection. The putative RBD of S proteins (23 kDa in size) from type I and type II FCoV were confirmed by Western blotting using an anti-histidine mAb.....................................................................................96 Figure 6. Establishment and confirmation of the type-specific IFA. Sera from confirmed type I and/or II FCoV-infected cats with FIP, with type I or II FCoV antisera or SPF cat serum as a reference, were applied to the IFA to characterize its specificity. ........................................................................97 Figure 7. A schematic of the 5' terminal of the fTNFA gene analyzed in this study. A partial fTNFA sequence of 1018 bp was sequenced in this study, including the PRR, the 5′-UTR, and part of exon 1. All the SNPs and the corresponding positions are indicated with lines. Gray box: exon 1. Black boxes: repeat regions. SNPs located in the exon were shaded..................98 Figure 8. Plasma concentration of TNF-α in cats carrying different genotypes at positions -421 in the FIP cases (closed circles) and control group (opened circles). The concentration of TNF-α was measured using antigen capture ELISA. A dotted line indicates the detection limit (80 pg/ml) of the assay....99 Figure 9. A schematic of the partial fCD209 gene analyzed in this study. The PRR, the 5′-UTR, and the ECD of fCD209 were sequenced in this study. All the SNPs and the corresponding positions were indicated with lines. Gray boxes: exons. Black boxes: repeat regions. White box: 5′ UTR. SNPs located in the exon were shaded. ........................................................................100  | |
| dc.language.iso | en | |
| dc.subject | 宿主基因多型性 | zh_TW |
| dc.subject | 腫瘤壞死因子 | zh_TW |
| dc.subject | C 型凝集素 | zh_TW |
| dc.subject | 桿狀病毒表現型別特異性棘蛋白 | zh_TW |
| dc.subject | 血清盛行率 | zh_TW |
| dc.subject | 第二型貓冠狀病毒 | zh_TW |
| dc.subject | 貓傳染性腹膜炎 | zh_TW |
| dc.subject | 貓冠狀病毒 | zh_TW |
| dc.subject | 貓傳染性腹膜炎爆發 | zh_TW |
| dc.subject | host genetic polymorphisms | en |
| dc.subject | feline infectious peritonitis | en |
| dc.subject | type II FCoV | en |
| dc.subject | outbreak of FIP | en |
| dc.subject | baculovirus expressed type specific S protein | en |
| dc.subject | serosurveillance | en |
| dc.subject | feline coronavirus | en |
| dc.subject | tumor necrosis factor-alpha | en |
| dc.subject | C-type lectin DC-SIGN | en |
| dc.title | 貓冠狀病毒流行病學之調查與貓傳染性腹膜炎相關宿主因子之探討 | zh_TW |
| dc.title | Epidemiological surveillance of feline coronavirus infection and feline infectious peritonitis associated host genetic polymorphisms | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 萬灼華(Cho-Hua Wan) | |
| dc.contributor.oralexamcommittee | 郭育良(Yue-Liang Leon Guo),謝世良(Shie-Liang Hsieh),伍安怡(Betty An-Ye, Wu-Hsieh),張惠雯(Hui-Wen Chang),陳宜寧(Yi-Ning Chen) | |
| dc.subject.keyword | 貓冠狀病毒,貓傳染性腹膜炎,第二型貓冠狀病毒,貓傳染性腹膜炎爆發,桿狀病毒表現型別特異性棘蛋白,血清盛行率,宿主基因多型性,腫瘤壞死因子,C 型凝集素, | zh_TW |
| dc.subject.keyword | feline coronavirus,feline infectious peritonitis,type II FCoV,outbreak of FIP,baculovirus expressed type specific S protein,serosurveillance,host genetic polymorphisms,tumor necrosis factor-alpha,C-type lectin DC-SIGN, | en |
| dc.relation.page | 117 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-02-03 | |
| 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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