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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李君男 | |
| dc.contributor.author | Yi-Pei Lin | en |
| dc.contributor.author | 林宜霈 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:06:13Z | - |
| dc.date.available | 2013-09-13 | |
| dc.date.copyright | 2010-09-13 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-14 | |
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Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med 354:11-22. 148. Ruiz, M. C., A. Charpilienne, and S. R. Alonso-Torre. 1994. Rotavirus interaction with isolated membrane vesicles. J Virol 68:4009-4016. 149. Saif, L., and B. Jiang. 1994. Nongroup A rotavirus of human and animal, p. 339-371. In R. Raming (ed.), Rotaviruses, vol. 185. Springrer-Verlag, Berlin. 150. Sandino, A. M., M. Jashes, G. Faundez, and E. Spencer. 1986. Role of the inner protein capsid on in vitro human rotavirus transcription. J Virol 60:797-802. 151. Sanguinetti, C. J., E. Dias Neto, and A. J. Simpson. 1994. Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17:914-21. 152. Santos, N., and Y. Hoshino. 2005. Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev. Med. Virol 15:29-56. 153. Santos, N., R. C. Lima, C. F. Pereira, and V. Gouvea. 1998. Detection of rotavirus types G8 and G10 among Brazilian children with diarrhea. J Clin Microbiol 36:2727-9. 154. Santos, N., E. M. Volotao, C. C. Soares, M. C. Albuquerque, F. M. da Silva, T. R. de Carvalho, C. F. Pereira, V. Chizhikov, and Y. Hoshino. 2001. Rotavirus strains bearing genotype G9 or P[9] recovered from Brazilian children with diarrhea from 1997 to 1999. J Clin Microbiol 39:1157-60. 155. Sato, K., Y. Inaba, T. Shinozaki, R. Fujii, and M. Matumot | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47565 | - |
| dc.description.abstract | 輪狀病毒是全球造成嬰幼兒嚴重腹瀉、脫水甚至死亡的最重要致病原之一。輪狀病毒的變異性高，原因之一為病毒的RNA聚合脢缺乏矯正錯誤的功能而產生點突變；另一個重要因素與輪狀病毒之基因體呈片段狀有關，當兩種病毒同時感染一個細胞時，會產生基因重組 (reassortment) 之現象。依據病毒顆粒最外層的VP7和VP4兩種殼蛋白質，可將輪狀病毒分為22種G基因型和31種P基因型，其中G1P[8]、G2 P[4]、G3 P[8]與G4 P[8]是四種常見的輪狀病毒。從90年代中期開始，許多國家陸續分離出G9輪狀病毒，該病毒株已成為全球第五個主要流行的病毒株。臺大醫院在2000年首度發現四個G9輪狀病毒陽性檢體，之後便持續檢測出G9病毒株，到2002年，更成為該年主要流行的病毒，之後雖然G9病毒所佔比例有下降，但2000-2005年輪狀病毒陽性檢體中，G9病毒株所佔的數量仍是最多，G9病毒確實已在台灣地區流行。
對本地的G9病毒進行分析，大多數的病毒株屬於G9P[8]、VP6第二基因群、長型電泳型，只有五個病毒株屬於G9P[4]、VP6第一基因群、短型電泳型。針對VP7、VP4、VP6、NSP4、VP2及VP3基因進行序列比對及種系分析，發現本地G9輪狀病毒的VP7基因皆屬於分支III，且與最近國外流行的G9病毒在核酸與胺基酸序列相似性極高 (>98.6%)，在其他的基因片段，本地G9病毒株歸類在多個分支顯示變異性較大，與國內其他G血清型病毒株較相似。推測本地的G9病毒株很可能是經由國外進來的G9病毒與國內其他G血清型病毒株發生基因重組而產生。對本地代表性G9病毒株01TW591進行全基因體種系分析，其基因組合為G9-P[8]-I1-R1- C1-M1-A1-N1-T1-E1-H1 (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4- NSP5)，屬於Wa基因群的病毒，與2000年以後出現的病毒株關係較接近。 G9病毒已普遍存在於世界各地，到底有多少人曾經感染過G9病毒並不清楚，偵測血清型特異的抗體，將有助於了解個體是否感染過G9病毒。本研究利用中和試驗偵測G9輪狀病毒流行前、後血清中的抗體變化情形，比較1999年與2009年的孩童，發現G9病毒流行後，G9中和抗體有增加的情形，然而不論是1999年或2009年，個體針對G1、G3、G9病毒的中和抗體效價有相關性，很可能是因為有相同的VP4抗原造成交叉反應的現象。因此利用重組桿狀病毒只表現VP7蛋白質以去除干擾，並建立酶連試驗法來偵測血清型特異的抗體；但結果與中和試驗比較，兩者無顯著相關性。 本地流行的G9輪狀病毒株之基因變異性大且複雜，持續監控病毒的變化及是否有新興病毒的產生，並了解病毒株在族群中的感染情形及體內抗體呈現之狀況，將有助於未來控制輪狀病毒的流行。 | zh_TW |
| dc.description.abstract | Rotavirus infection is the most important cause of severe, dehydrating gastroenteritis among children worldwide. Rotaviruses have been characterized with high genetic variation. The viruses evolve through point mutation and genetic reassortment. According to the genetic and antigenic diversity of the two outer capsid proteins VP7 and VP4, rotaviruses are classified into G- and P-types. Globally, different surveys indicate that G1P[8], G2P[4], G3P[8] and G4P[8] are the most common G and P combinations encountered in human infections. Since the mid-1990s, novel G9 rotaviruses have been detected in many countries, suggesting that G9 is the fifth globally important serotype. In National Taiwan University Hospital, G9 rotavirus first appeared in 2000 with 4 cases, and was continuously detected until now.
Most of the G9 strains belonged to G9 P[8], VP6 genogroup II, and long electropherotype, except five belonged to G9P[4], VP6 genogroup I, and short electropherotype. Phylogenetic analysis of the VP7, VP4, VP6, NSP4, VP2, and VP3 genes of representative local G9 strains showed that the VP7 genes shared a high degree of identity (>98.6%) to overseas G9 rotaviruses detected recently. The other genes were more variable and more closely related to those of local rotaviruses of other G types, suggested that local G9 rotaviruses possibly had evolved through reassortment between overseas G9 strains and local circulating rotaviruses of other G types. Based on a full-genome classification system, a representative G9 strain, 01TW591, was shown to possess the typical Wa-like genotype constellation: G9-P[8]-I1-R1-C1-M1-A1-N1-T1- E1-H1 (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5). G9 rotaviruses have been widely spread around the world, how much is the percentage of people infected with G9 virus is still unknown. To determine serotype-specific antibody, and then understand if the individuals infected with G9 rotavirus before, is importanat. Antibody titer from serum samples collected before and after G9 rotavirus epidemic was determined by neutralization assay. Comparing the antibody titer between the sera collected from the similar age populations in 1999 and 2009, G9 antibody titer was higher in the sera collected after G9 rotavirus epidemic. There were good correlations among G1, G3, and G9 antibody titers, which was possibly due to the cross-reactivity induced by VP4 epitopes. To reduce the interference of VP4 protein, an ELISA method using baculovirus expressed VP7 protein was used to determine serotype-specific antibody. The results were compared with those of neutralization test, but no significant correlation was observed. Local G9 rotaviruses had various genetic compositions and possibly had evolved through reassortment. Therefore, it is worthwhile to continuously monitor genetic relatedness among circulating strains and understand the antibody changes in rotavirus infection, which would be helpful to control the infections in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:06:13Z (GMT). No. of bitstreams: 1 ntu-99-D93424003-1.pdf: 22417561 bytes, checksum: f8bed9e1074164447f8b3297c80bc388 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 中文摘要 iii 英文摘要 v 第一章 緒論 1 1.1 病毒結構 2 1.2 病毒基因體 2 1.3 基因之功能 3 1.3.1 核心蛋白質–VP1、VP2、VP3 3 1.3.2 內層蛋白質殼–VP6 4 1.3.3 外層棘狀蛋白質–VP4 5 1.3.4 外鞘蛋白質–VP7 5 1.3.5 非結構性蛋白質–NSP4 6 1.3.6 其他非結構性蛋白質 7 1.4 病毒之複製 7 1.5 細胞培養 7 1.6 實驗室診斷 8 1.7 免疫調控 8 1.8 院內感染 9 1.9 流行病學 10 1.10 疫苗發展 11 1.11 研究目的 15 第二章 材料與方法 16 2.1 輪狀病毒檢體與輪狀病毒株 16 2.2 血清檢體 16 2.3 萃取及純化輪狀病毒RNA 16 2.4 RNA凝膠電泳分析 17 2.4.1 鑄膠 17 2.4.2 電泳 18 2.4.3 銀染色法 18 2.5 RNA-RNA雜交試驗 18 2.5.1 電泳與電泳轉印 18 2.5.2 製備探針 19 2.5.3 雜交反應 20 2.5.4 Stringency wash 20 2.5.5 偵測 20 2.6 基因增幅 21 2.6.1 VP2, VP3, VP4、VP6、VP7及NSP4基因RT-PCR 21 2.6.2 反轉錄反應 22 2.6.3 11段基因全長序列之聚合酶連鎖反應 22 2.7 聚合酶連鎖反應產物純化 23 2.8 TA Cloning 23 2.9 轉型反應與菌落篩選 24 2.10 小量質體製備 25 2.11 限制酶切割反應 25 2.12 基因定序及分析 25 2.12.1 核酸定序反應 25 2.12.2 核酸定序反應產物純化 26 2.12.3 上機 26 2.12.4 基因序列之分析比較 27 2.13 MA104細胞之培養 27 2.14 輪狀病毒之培養 27 2.14.1 從糞便稀釋液中分離病毒 27 2.14.2 繼代培養 28 2.14.3 大量培養 28 2.15 病毒定量 29 2.16 中和試驗 29 2.16.1 螢光焦點減少中和試驗 (focus reduction neutralization, FRN) 29 2.16.2 NELISA (ELSIA-based antigen reduction neutralization assay) 30 2.17 酵素連結免疫分析法 (ELISA) 31 第三章 結果 32 3.1 G9輪狀病毒之流行 32 3.2 G9病毒株之基因型與電泳型 32 3.3 G9病毒株之基因分析 33 3.3.1 VP7基因序列分析 34 3.3.2 VP4基因序列分析 36 3.3.3 VP6基因序列分析 37 3.3.4 NSP4基因序列分析 39 3.3.5 VP2基因序列分析 40 3.3.6 VP3基因序列分析 41 3.4 本地G9與G3輪狀病毒株基因相關性分析 42 3.5 G9輪狀病毒株基因體種系分析 43 3.6 中和試驗偵測血清型特異抗體 46 3.7 酵素連結免疫分析法偵測G9特異的抗體 47 第四章 討論 49 參考文獻 56 圖 72 表 98 附錄 119 | |
| dc.language.iso | zh-TW | |
| dc.subject | 血清型特異抗體 | zh_TW |
| dc.subject | G9輪狀病毒 | zh_TW |
| dc.subject | 種系分析 | zh_TW |
| dc.subject | 基因變異性 | zh_TW |
| dc.subject | phylogenetic analysis | en |
| dc.subject | serotype-specific antibody | en |
| dc.subject | genetic variation | en |
| dc.subject | G9 rotavirus | en |
| dc.title | G9輪狀病毒基因變異與血清抗體之分析 | zh_TW |
| dc.title | Analysis of the Genetic Variation and Serotype-specific Antibody of G9 Rotavirus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 高全良,張美惠,黃立民,金傳春,施信如,張淑媛,吳宗遠 | |
| dc.subject.keyword | G9輪狀病毒,種系分析,基因變異性,血清型特異抗體, | zh_TW |
| dc.subject.keyword | G9 rotavirus,phylogenetic analysis,genetic variation,serotype-specific antibody, | en |
| dc.relation.page | 124 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-16 | |
| 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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