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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王金和(Chin-Ho Wang) | |
| dc.contributor.author | Wei-En Hsu | en |
| dc.contributor.author | 許偉恩 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:28:58Z | - |
| dc.date.available | 2020-06-12 | |
| dc.date.copyright | 2017-06-12 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-03-29 | |
| dc.identifier.citation | 1. ATCC VIrology Guide, 2016 https://www.atcc.org/~/media/PDFs/Culture%20Guides/Virology_Guide.ashx
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59586 | - |
| dc.description.abstract | A型流感病毒為威脅人類健康的重要病原。一般情形下，A型流感病毒具有宿主專一性。如H6N1亞型會感染禽鳥。在台灣於2014年發現H6N1感染犬隻的案例。該病毒由犬隻的鼻腔拭子經胚胎蛋增殖2代所分離，命名為A/canine/Taiwan/E01/2014 (H6N1)。此犬H6N1於PB2蛋白質中擁有已知能增加病毒在哺乳類宿主複製的E627K突變。然而，發現將此病毒在胚胎蛋增殖1代後於PB2基因中見K627E的胺基酸突變。將該病毒以MDCK細胞增殖2代後恢復PB2 627K。顯示於不同系統的增殖導致了該病毒基因的突變。本研究目的為對H6N1在細胞的感染性進行分析。包含 (1)犬H6N1以胚胎蛋或MDCK細胞增殖後胺基酸的突變。(2)病毒對不同物種來源細胞的複製能力及細胞傷害性。 (3) 於台灣所分離到的H6N1在HA、PB2、PB1及PA基因中自1972年至2014年的演化趨勢。 (4)演化趨勢與病毒在細胞中複製動力學與傷害性的關係。透過病毒溶斑純化試驗，純化確認PB2的627個別為E (病毒93)和K (病毒99)的2株H6N1病毒，並重新做8段基因的定序。此外，挑選5株實驗室中於不同年份分離之家禽來源H6N1 (病毒04、11、20、37及72)，以RT-PCR增幅HA、PB2及PA基因片段，與病毒99及基因資料庫中其他台灣H6N1比對，繪製親緣樹狀圖。依據親緣樹狀圖將病毒分作2組，是為2012年前分離組 (病毒04、37及72)與2012年後分離組 (病毒11、20及99)。以MOI=0.01的病毒感染MDCK、A549及DF-1此3種不同物種來源細胞檢視病毒在細胞中的複製動力學。此外，經由MTT assay檢視感染後病毒對細胞的傷害性。結果顯示，病毒93與病毒99有3個胺基酸位點的胺基酸改變。93的HA基因有142I、PB2基因有627E，及PA基因有739E。99的HA基因有142N、PB2基因有627K，及PA具有739G。病毒99比病毒93可在MDCK細胞上產生較大的病毒溶斑。病毒99在MDCK、A549及DF-1中都有比93更高的病毒複製力價，並在感染後能對MDCK細胞造成比病毒93更顯著的傷害。親緣樹狀圖顯示，2012年後於台灣所分離的H6N1於HA、PB2及PA都有相近的親緣。在HA胺基酸中，2005年後H6N1普遍具有E190V和G228S此2會可能病毒在H6亞型增加與哺乳類受體結合能力的突變點。在PB2胺基酸中只有於2014年分離的犬H6N1和病毒99具有627K的突變。病毒99比病毒04、11、20、37及72在MDCK、A549及DF-1細胞都能更有效複製。在MDCK細胞中病毒99可造成嚴重的細胞傷害。然而，對2012年前分離的病毒與2012年後分離的病毒做組間比較，發現2012年後分離的病毒並沒有都比2012年前分離的病毒有更好的複製能力。即使組內基因具有較高的相似度，2012年後分離的病毒並沒有都比2012年前分離的病毒有更好的複製能力。而在A549及DF-1細胞中，病毒複製能力和病毒的細胞傷害性沒有絕對的相關性。結論，擁有PB2 E627K此一位點突變，可能是導致病毒99比93和其他禽來源H6N1在哺乳類細胞有更好複製效率的原因。 | zh_TW |
| dc.description.abstract | Influenza A viruses are important life threatening pathogen to human. Generally, influenza A viruses have host restriction. H6N1 subtype viruses infect avian species.
A canine H6N1influenza virus was first noted in Taiwan in 2014. The virus on nasal swab was isolated from 2 passages in chicken embryo eggs and designated as A/canine/Taiwan/E01/2014 (H6N1). PB2 protein of the virus possessed E627K mutation, which was already known to enhance virus replication in mammalian hosts. However, the amino acid had mutated to PB2 627E after a passage in chicken embryo eggs. The virus recovered PB2 E627K after being cultivated in MDCK (Madin-Darby canine kidney) cell for 2 passages. It showed that passaging in different systems may lead to gene mutation. The aims of this study were cellular infectivity analysis of the H6N1 influenza viruses: (1) The amino acid mutations of the dog virus after passaging in chicken embryo eggs and MDCK cells. (2) Viral replication efficiency and injury of cells from different hosts. (3) The evolutionary trend of HA, PB2, PB1 and PA genes in H6N1 in Taiwan from 1972 to 2014. (4) The relationship between evolutionary trend, viral replication efficiency and injury of cells from different hosts. Plaque purification was used for purifying both the canine H6N1 viruses which possessed PB2 627E (virus 93) and PB2 627K (virus 99). Virus 93 and virus 99 were sequenced. In addition, five H6N1 viruses (virus 04, 11, 20, 37 and 72) were selected. HA, PB2, PB1 and PA segments of the viruses were amplified by RT-PCR. Phylogenetic tree was built up in order to analyze evolutionary trend. Viruses were grouped into the viruses isolated before 2012 (included 04, 37 and 72) and the viruses isolated after 2012 (included 11, 20 and 99) based on phylogenetic tree. Using MOI=0.01 viruses to infect MDCK, A549 and DF-1 cells to investigate viral replication efficiency. Moreover, MTT assay was used for detecting cell injury. Results showed that virus 93 had three amino acids different from virus 99. Virus 93 possessed 142I in HA gene, 627E in PB2 gene and 739E in PA gene. Virus 99 possessed 142N in HA gene, 627K in PB2 gene and 739G in PA gene. Plaques generated by virus 99 were larger than virus 93. Higher virus titers were observed in MDCK, A549 and DF-1 cells infected with virus 99 than infected with virus 93. Infected with virus 99 caused distinct cytopathic effect in MDCK cells than virus 93. Phylogenetic tree showed that the HA, PB2, PB1 and PA genes of H6N1 which were isolated after 2012 shared close relationship. Most of H6N1 possessed E190V and G228S in HA gene, which may enhance the binding between viruses and receptor of mammalian hosts. Only the canine virus isolated in 2014 and virus 99 possessed E627K in PB2 gene. Virus 99 could replicate better in MDCK, A549 and DF-1 cells than virus 04, 11, 20, 37 and 72. Infected with virus 99 could couse severe cell injury in MDCK cells. However, the viruses isolated after 2012 did not show better viral replication efficiency than the viruses isolated before 2012. Although having similar amino acid compositions, not every virus isolated after 2012 could have better viral replication efficiency than the viruses isolated before 2012. The correlation between viral replication efficiency and cell injury in A549 and DF-1 cells was poor. In concusion, possessed E627K in PB2 gene may lead virus 99 to have better viral replication efficiency in mammalian hosts than virus 93 and other H6N1 viruses in this study. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:28:58Z (GMT). No. of bitstreams: 1 ntu-106-R03629018-1.pdf: 2495137 bytes, checksum: 75ff792d11ff071acb8f6bb6180a926e (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書......................................................................................................i 誌謝.............................................................................................................................ii 中文摘要...................................................................................................................iii-iv 英文摘要...................................................................................................................v-vi 目錄…………………………………………………………………………………1 圖之目錄……………………………………………………………………………3 表之目錄……………………………………………………………………………4 第1章─序言……………………………………………………………………….5 第2章─文獻回顧……………………………………………………….………….7 2.1 A型流感病毒…………………………………………….............………….7 2.1.1 病毒組成與特性…………………………………….……………….7 2.1.2 病毒生態學………………………………………….……………….9 2.2 影響A型流感病毒之毒力的要素………………………….............………9 2.2.1 HA與唾液酸 (sialic acid)受體………………………….…….…….9 2.2.2 NA蛋白質...........................................................................................10 2.2.3 M與NS蛋白質..……………………………………………….…...11 2.2.4 病毒核醣核酸蛋白質複合體 ( PB1、PB2、PA與NP) …………………………………………….……………………….11 2.3 犬A型流感病毒…………………………………............…………………13 2.3.1 非H6N1的犬流感病毒…………………………………………….13 2.3.2 H6N1犬流感病毒…………………………………………………..15 第3章─材料與方法………………………………………………………………..16 3.1 細胞實驗設計……………………………………………………………….16 3.2 病毒………………………………………………………………………….16 3.2.1 實驗中所使用病毒株……………………………………………….16 3.2.2 病毒培養 (雞胚胎蛋)……………………………………………....17 3.2.3 經TPCK (L-(tosylamido-2-phenyl) ethyl chloromethyl ketone ) 處理之胰蛋白質酶 (TPCK-trypsin)………………......................17 3.2.4 病毒培養 (Madin-Darby canine kidney, MDCK cells)……………..18 3.2.5 病毒的病毒溶斑純化 (plaque purification)………………………..18 3.3 細胞培養………………………………………..……….................………..19 3.3.1 MDCK、A549及DF-1細胞………………………………………..19 3.3.2 細胞繼代………………………………………..………………..19 3.4 病毒定量………………………………………..……………………………20 3.4.1 病毒溶斑染色試驗 (plaque assay)……………………………………20 3.4.2 50％組織培養感染劑量 (50% Tissue culture infectious dose, TCID50)………………………………………………....................21 3.5 複製動力學分析 (growth kinetic analysis)..............................................21 3.6 細胞存活率分析[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] (MTT assay) ………………………………………………....22 3.7 分子檢測………………………………………………………………...23 3.7.1 核酸萃取………………………………………...........................23 3.7.2 核酸定序………………………………………...........................23 3.7.3 序列分析………………………………………...........................24 第4章─結果…………………………………………………………….............25 4.1 病毒93與99之序列分析結果……………………….............................25 4.2 病毒93與99在MDCK細胞感染後呈現顯著不同的病毒溶斑型態………………………………………...................................................25 4.3 病毒93與99在MDCK、A549、DF-1三種不同物種來源細胞的複製動力學……………………………………….......................................26 4.4 病毒93與99在MDCK、A549、DF-1三種不同物種來源細胞的細胞傷害性……………………………………….......................................26 4.5 不同年代之犬與禽的H6N1在MDCK、A549、DF-1三種不同來源細胞於感染後第24小時的複製動力學………………….....................27 4.6 不同年代之犬與禽的H6N1在MDCK、A549、DF-1三種不同來源細胞於感染後第24小時的細胞傷害性……………………………….27 4.7 實驗中所應用病毒的序列分析結果………………………………......28 第5章─討論……………………………………………………………...........29 5.1 台灣H6N1的序列演化趨勢…………………………………...............29 5.1.1 在HA基因中的突變…………………………………...............29 5.1.2 在PB2基因中的突變…………………………………..............30 5.1.3 在PA基因中的突變.....................................................................31 5.1.4 在PB1基因中的突變………………………………................31 5.1.5 各片段序列比對的結果…………………………………...........................................32 5.2 2012年前與後H6N1在細胞感染性試驗上的比較……………............32 5.3 病毒93及99在細胞特性的比較……….................................................33 圖……………………………………………………………...............................34 表……………………………………………………………...............................45 參考文獻……………………………………………………………...................60 圖之目錄 圖一 細胞操作實驗流程圖.................................................................................34 圖二 93 (原液稀釋倍數10-4)與99 (原液稀釋倍數10-4)於MDCK細胞所呈 現的病毒溶斑型態.....................................................................................35 圖三 93與99於MDCK、A549及DF-1細胞的複製動力學.........................36 圖四 以MTT assay測試93與99於MDCK、A549及DF-1細胞的細胞傷 害性.............................................................................................................37 圖五 MOI=0.01的93與99在感染後第24小時的複製動力學與細胞傷害 性................................................................................................................38 圖六 2012年前、後分離到的病毒於感染後24小時在MDCK、A549及DF-1 細胞的複製動力學....................................................................................39 圖七 以MTT assay測試2012年前與後分離到的病毒於感染後24小時在 MDCK、A549及DF-1細胞的的細胞傷害性........................................40 圖八 試驗中所有應用H6N1與其他台灣家禽H6N1於HA基因片段之親緣 樹狀圖.........................................................................................................41 圖九 試驗中所有應用H6N1與其他台灣家禽H6N1於PB2基因片段之親 緣樹狀圖.....................................................................................................42 圖十 試驗中所有應用H6N1與其他台灣家禽H6N1於PA基因片段之親緣 樹狀圖.........................................................................................................43 圖十一試驗中所有應用H6N1與其他台灣家禽H6N1於PB1基因片段之親 緣樹狀圖.....................................................................................................44 表之目錄 表一 實驗中應用的primer................................................................................45 表二 經病毒溶斑純化後93與99毒株在8段基因中核苷酸或胺基酸的 改變...........................................................................................................47 表三 針對會增加病毒感染哺乳動物趨勢的胺基酸位點，分析1972年-2014 年間分離的所有台灣H6N1毒株與實驗中應用病毒株的HA胺基酸....48 表四 針對會增加病毒感染哺乳動物趨勢的胺基酸位點，分析1972年-2014 年間分離的所有台灣H6N1毒株與實驗中應用病毒株的PB2胺基酸...51 表五 針對會增加病毒感染哺乳動物趨勢的胺基酸位點，分析1972年-2014 年間分離的所有台灣H6N1毒株與實驗中應用病毒株的PA胺基酸.....54 表六 針對會增加病毒感染哺乳動物趨勢的胺基酸位點，分析1972年-2014 年間分離的所有台灣H6N1毒株與實驗中應用病毒株的PB1胺基酸...56 表七 針對會增加病毒感染哺乳動物趨勢的胺基酸位點，分析1972年-2014 年間分離的所有台灣H6N1毒株與實驗中應用病毒株的PB1-F2胺基 酸............................................................................................................57 附錄一 實驗用試劑、耗材及廠牌及配方.......58 | |
| dc.language.iso | zh-TW | |
| dc.title | H6N1流感病毒在細胞的感染性分析 | zh_TW |
| dc.title | Cellular Infectivity Analysis of the H6N1 Influenza Viruses | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 王儷蒨(Lih-Chiann Wang) | |
| dc.contributor.oralexamcommittee | 陳慧文(Hui-Wen Chen),鄭益謙(Ivan-Chen Cheng) | |
| dc.subject.keyword | H6N1流感病毒,親緣樹狀圖,病毒複製,細胞傷害性,台灣, | zh_TW |
| dc.subject.keyword | H6N1 Influenza Virus,Phylogenetic tree,Viral Replication,Cell Injury,Taiwan, | en |
| dc.relation.page | 66 | |
| dc.identifier.doi | 10.6342/NTU201700730 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-03-30 | |
| 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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