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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張淑媛(Sui-Yuan Chang) | |
dc.contributor.author | Tai-Ling Chao | en |
dc.contributor.author | 趙苔伶 | zh_TW |
dc.date.accessioned | 2021-06-08T03:58:01Z | - |
dc.date.copyright | 2018-09-04 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22015 | - |
dc.description.abstract | 流感病毒為一種可造成動物與人類感染之RNA病毒,因複製時其聚合酶無校對能力、使病毒具有快速演化之特性,可迅速適應並感染不同宿主,而造成疾病。流感病毒主要感染人類上呼吸道系統,在流感重症患者會導致呼吸道系統衰竭、肺炎甚至死亡。因此,研究流感病毒在人體內的快速演化與病理學的特性,對於減緩或是預防流感病毒感染引發之疾病十分重要。本研究主要分為兩大部分,第一部分為分析H7N9流感病毒於人體內快速演化之特性,第二部分探討H1N1流感病毒對於小鼠肺部前驅細胞之病理特性。H7N9流感病毒首次於西元2013年在中國爆發流行,為來自三個地區禽鳥類病毒所重組之禽流感病毒,因其感染人類下呼吸道後會快速發展為重症而受到關注。於本研究中,我們鑑定第一起台灣境外移入病例之H7N9病毒特性,且藉由分析病毒胺基酸改變來探討H7N9病毒於人體內快速演化之模式。首先,H7N9臨床病毒株自2013年4月20、22與25日之痰液檢體與25日之喉頭拭子檢體內分離出,接著利用族群定序法(Population Sequencing)定序八段病毒全長基因序列,同時亦利用第三世代基因定序(Third Generation Sequencing)分析病毒膜蛋白質基因:血球凝集素(HA)、神經胺酸脢(NA)與基質(M)特殊胺基酸位點與比例改變。於連續時間點之痰液檢體內可觀察到病毒基因動態變化(Dynamic variation),並於HA基因上發現三個胺基酸位點:P224L、L235P與K330E可能具有影響病毒HA與不同唾液酸結合之能力;於NA基因上,觀察到野生型R289 (wild type)與抗藥性突變點K289共存之現象,與另外兩個對克流感(Tamiflu)具抗藥性之位點V115與R219,以及位於神經胺酸酶莖部(NA stalk)一突變點T68N之存在。分析具抗藥性之神經胺酸脢蛋白質對於三種抗病毒藥物之感受性,發現K289抗藥性位點對於克流感、瑞樂沙(Relenza)與Peramivir皆呈現高度抗性,但R219與V115對於瑞樂沙與Peramivir僅具有輕度抗性。此外,分析臨床病毒株對於藥物之感受性時發現,痰液內之病毒株含有較高比例的野生病毒株。T68N突變點雖然會造成神經胺酸脢損失一醣基化位點(Glycosylation site),但可加強重組病毒複製能力,且不影響對於三種抗病毒藥物之感受性。至於在不同檢體內病毒株之M基因,並未偵測到任一特殊胺基酸位點改變。我們的研究調查了H7N9病毒於該病人體內,因應環境變化所導致之快速演化與產生抗藥性之情況,可提供未來於臨床治療H7N9用藥之參考。
第二部分主要探討H1N1流感病毒對於小鼠肺部前驅細胞之病理特性。流行性A型流感病毒(Pandemic Influenza A Viruses)主要感染下呼吸道內第一型與第二型肺泡細胞,誘導細胞激素(Cytokines)與趨化因子(Chemokines)分泌,使肺部組織被免疫細胞浸潤、進而引發急性呼吸道衰竭症狀或肺炎。近期研究發現在肺泡與小支氣管交界處存在一種具有幹細胞能力,可分化成下游第一型與第二型肺泡細胞並修復受損肺泡區域之肺前驅細胞,此細胞同時也被證實可被流感病毒感染。然而、目前對於該細胞被流感病毒感染後所表現之特性,以及因應感染所分泌之細胞激素仍不清楚,因此本研究旨在建立該肺前驅細胞之細胞株(mPSCsOct4+ E3L clone),並藉此細胞株探討肺前驅細胞被病毒感染後所表現之特性與病理性。於本研究中,原生肺前驅細胞與其細胞株可被多種H1N1流感病毒所感染,其中包括實驗室病毒株PR8、季節性H1N1流感病毒與2009年H1N1新型流感病毒。然而因病毒vRNA複製能力衰減與病毒核蛋白質(Nucleoprotein; NP)堆積於細胞質內,使病毒不易於肺前驅細胞株內複製,而無法於上清液內偵測到高病毒量。此外,偵測細胞所釋放細胞激素與趨化因子表現,觀察到介白素6 (Interleukin 6; IL6)與干擾素γ (Interferon γ; IFN-γ)於感染後12小時、干擾素β (IFN-β)於感染後24小時有顯著性增加,指出肺前驅細胞之功能可能作為偵測病毒感染之傳感器(Sensor)。我們的實驗結果顯示肺前驅細胞可被流感病毒感染、並可藉由釋放促炎性細胞激素(proinflammatory cytokines)因應與調控流感病毒感染。 | zh_TW |
dc.description.abstract | Influenza virus is an RNA virus that can cause infection in animals and humans. The lack of proofreading ability of viral polymerase facilitates rapid evolution of viral genomes, which subsequently improve virus fitness, transmission and pathogenicity in different hosts. Influenza viruses mainly infect the human respiratory tract system, and can cause respiratory failure, pneumonia and even death in patients with severe infection. Therefore, characterization of the rapid virus evolution in the hosts and its pathological characteristics are very important for treatment and prophylaxis of influenza virus infection. There are two major parts in this study. The first part is to investigate the H7N9 influenza A virus evolution in an infected patient. The second part is to characterize the pathology and replication of H1N1 influenza A virus in mouse pulmonary stem/progenitor cells. Since the first outbreak of a novel avian-origin reassortant H7N9 virus in China in 2013, H7N9 infection has raised great concern due to the rapidly progressive lower respiratory tract infections in infected individuals. In this study, the first imported H7N9 case was identified and characterized. The dynamic variations of signature amino acids in patient’s specimens were investigated. First, H7N9 influenza viruses were isolated from the sputum collected at 20th, 22nd, and 25th of April and throat swab at 25th of April, from the first imported case in Taiwan. The full-length genome of viruses was sequenced by population sequencing, and the dynamic variation of hemagglutinin (HA), neuraminidase (NA) and matrix (M) genes was additionally determined by the third generation sequencing (TGS) PacBio RS II platform. Dynamic variation of H7N9 influenza viruses was observed in the sequential sputum specimens. Three HA substitutions: P224L, L235P and K330E were identified and might alter the binding preference to different sialic acids. Three oseltamivir-resistant mutations R289K, E115V and I219R, and a T68N mutation on the stalk of NA were detected on NA genes in our study subjects. The results of the drug susceptibility of N9 proteins to neuraminidase inhibitors indicated that the R289K was highly resistant to oseltamivir, zanamivir and peramivir, but I219R and E115V were only mildly resistant to zanamivir and peramivir. Furthermore, a higher proportion of wild type viruses were identified from the sputum specimens. The T68N mutation which can cause removal of one glycosylation site at NA can enhance the virus replication in MDCK cells, but it has no effect on the drug susceptibility. As for the M gene of the virus in all specimens, no amino acid substitution was detected. In conclusion, the rapid evolution of substitutions and resistant mutations of H7N9 influenza viruses for responding the alteration of environment were demonstrated in our study.
Pandemic influenza A viruses or avian influenza viruses mainly infect the type I and II alveolar epithelial cells in the lower respiratory tract and induce the releasing of cytokines and chemokines, which subsequently lead to infiltration of immune cells into the alveolus, and then trigger acute respiratory distress syndrome (ARDS) or pneumonia. Recently, the pulmonary stem/progenitor cells (PSCs) at the junction of alveolus and bronchiole, which could be differentiated into downstream cells to repair tissue damage caused by influenza A virus, have been shown to be the target cells of influenza virus infection. However, the characteristics of virus infection and cytokines or chemokines release from PSCs remain obscured. In this study, a mouse pulmonary stem/progenitor cells (mPSCs) with capability to differentiate into type I or type II alveolar cells was used as an in vitro cell model to characterize replication and pathogenic effects of influenza viruses in PSCs. First, mPSCs and its immortalized cell line mPSCsOct4+ were shown to be susceptible to PR8, seasonal H1N1 and 2009 pandemic H1N1 influenza viruses and could generate infectious virus particles, although with a lower virus titer, which could be attributed to the reduced vRNA replication and NP aggregation in the cytoplasm. Nevertheless, a significant increase of IL6 and IFN-γ at 12 hours and IFN-beta at 24 hours post infection in mPSCs implicate that mPSCs might function as a sensor to modulate immune responses to influenza virus infection. In summary, our results demonstrated mPSCs, as one of the target cells for influenza A virus, could modulate early proinflammatory responses to influenza virus infection. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:58:01Z (GMT). No. of bitstreams: 1 ntu-107-D01424001-1.pdf: 7427347 bytes, checksum: 6b0fcb17d419bef046ae21391d3db5cd (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 致謝 I
摘要 II Abstract IV Index of Figures X Index of Tables XII Chapter I. Introduction 1 1. Influenza A viruses 1 1. 1 Influenza A virus structure and morphology 1 1. 2 Influenza A virus genome 2 1. 3 Life cycle of influenza A viruses in the cell 3 1. 4 Influenza A viruses infection in lower respiratory tract 12 1. 5 Antiviral drugs and Resistance 14 1. 6 H7N9 history, features and epidemiology 17 1. 7 The disease development of the first imported H7N9 case in Taiwan 21 2. Pulmonary stem/progenitor cells 22 2. 1 The mechanism of damage repairing in the alveolus 22 2. 2 The pulmonary stem/progenitor cells 23 2. 3 Repairment of influenza virus induced alveolar damages via pulmonary epithelial stem/progenitor cells 24 2. 4 Repairment of influenza virus induced alveolar damages via pulmonary mesenchymal stromal stem/progenitor cells 27 2. 5 Preliminary investigation of mouse pulmonary stem/progenitor cells 28 Chapter II. Motivation and study aim 30 Chapter III. Materials and Methods 31 Cells 31 Virus strains and reverse genetics 32 Virus infection 33 Plaque assay and plaque reduction assay 33 Microneutralization assay (mNT assay) 34 Hemagglutinin inhibition (HI) assay 35 Binding, penetration and entry assay 35 RNA extraction 37 Reverse transcription and Polymerase chain reaction (PCR) 37 Real-time PCR 38 Immunofluorescence assay 38 Western blot analysis 40 Sucrose gradient ultracentrifugation 40 Cytokine array 41 Detection of IFNβ in the supernatant 42 Transmission electron microscopy 42 Library construction and sequencing 43 Bioinformatics analysis 44 Homology modeling for HA and NA proteins 44 Neuraminidase inhibition assay 45 Statistical analysis 46 Chapter IV. Results 47 Part I. The studies of serology and virology of the first imported human case of avian H7N9 influenza A virus infection in Taiwan. 47 1.1 Trends of H7N9 viral loads and anti-H7N9 antibody responses 47 1.2 Molecular characterization of H7N9 viruses from respiratory specimens 48 1.3 The susceptibility of S0422, T0422 and T0425 viruses to NAIs 50 1.4 Replication of S0422 and T0422 viruses in A549, DF-1 and MDCK cell lines 51 1.5 Statistics of next-generation sequencing results 52 1.6 Molecular characterization of HA gene in S0420, S0422, S0425 and T0425 viruses 53 1.7 Structural and predict functional analysis of HA 55 1.8 Molecular characterization of NA gene in S0420, S0422, S0425 and T0425 viruses 56 1.9 Drug resistance mutations in NA during antiviral treatment 57 1.10 The E115V, I219R and R289K mutations in NA partially contributed to H7N9 resist oseltamivir, zanamivir and peramivir 59 1.11 T68N mutation caused removal of one glycosylation site at NA stalk, and enhanced virus replication, but had no influence to drug susceptibility 59 Part II. Establishment of infection model of mouse pulmonary stem cell line to influenza virus 61 2. 1 Susceptibility of mPSCs to influenza virus infection 61 2. 2 Establishment and characterization of immortalized mPSCs using Oct4 over-expression. 62 2. 3 Susceptibility of mPSCsOct4+ to influenza virus infection 63 2. 4 mPSCsOct4+ E3L clone can support influenza viruses’ replication 64 2. 5 Life cycle of influenza virus in E3L clone 65 2. 6 Cytokine and chemokines released from mPSCsOct4+ after PR8 infection. 66 Chapter V. Discussion 68 Chapter VI. Figures 86 Chapter VII. Tables 112 Chapter VIII. Reference 129 Appendix 141 | |
dc.language.iso | en | |
dc.title | 研究流感病毒在體內的演化與在小鼠肺部幹細胞之感染 | zh_TW |
dc.title | Investigation of Influenza A Virus Evolution in Infected Patients and Infection in Mouse Pulmonary Stem/progenitor Cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 林泰元(Thai-Yen Ling),高全良(Chuan Liang Kao),李君男(Chun-Nan Lee),施信如(Shin Ru Shih) | |
dc.subject.keyword | 流感病毒,H7N9,小鼠肺部幹細胞, | zh_TW |
dc.subject.keyword | Influenza A virus,H7N9,mouse pulmonary stem/progenitor cells, | en |
dc.relation.page | 141 | |
dc.identifier.doi | 10.6342/NTU201802995 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2018-08-13 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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