博碩士論文 972404009 詳細資訊




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姓名 錢葉盛(Yeh Sheng Chien)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 台灣地區未知血清型腸病毒株的基因體學分析
(Genomic analysis of serologically untypable human enteroviruses in Taiwan)
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摘要(中) 腸病毒(EV)是正鏈單股RNA病毒,分類上屬於小核糖核酸病毒科的腸病毒屬。所有腸道病毒都具有相似的基因體結構(7.2-8.5kb)。殼蛋白在稱為P1(前驅殼蛋白1)的部分中在ssRNA的5′末端編碼,P1基因可進一步區分成VP1-4基因,其中VP1基因與病毒抗原性有密切相關,非結構蛋白編碼在基因體的其餘部分,稱為P2和P3。結構蛋白基因的差異反映了腸病毒親緣關係及可能流行病學特徵。腸病毒屬包括15種(species),其中腸病毒12種(EV A-L)及鼻病毒3種(Rhinovirus A-C),會在人群造成流行的腸病毒有4種(EV A-D),此四種腸病毒共有116個血清型(EV-A有25個血清型、EV-B有63個血清型,EV-C有23种血清型、EV-D有5個血清型),因腸病毒很容易造成基因重組(gene recombination)進而演化成新血清型,所以研究腸病毒分子演化應進行基因體定序來進行分析,不過每年台灣腸病毒臨床分離株高達千株以上,需選擇有特殊意義的病毒株進行基因體定序,根據林口長庚醫院病毒室資料,每年約有10%的腸病毒分離株無法以單株抗體鑑定血清型,這些病毒很可能是基因重組所造成,值得進一步研究分析。

第二章中,我們成功建立了一個次世代定序(NGS)平台的分析方法,對無法以血清學分離型別的腸道病毒分離株,進行基因體定序。在130個無法以血清學分型的病毒株中,先使用新型RT-PCR (CODEHOP)方法擴增VP1基因來鑑定血清型,將其中的121株(93%)分類為29個血清型別。進一步我們從這些病毒樣本中,選擇了52個樣本進行次世代定序 ,並從其中51個樣本獲得了59個基因組序列,包括8個含有兩個病毒基因組的樣本。此外,我們還檢測到具有潛在遺傳重組病毒株的23種基因體變異株(與NCBI上現有的基因組序列進行比對核苷酸相似度<90%),其中 9株確認為重組病毒,其餘14株為具有未知重組來源的病毒株,此成果剛被Journal of Biomedical Science(2019年6月IMPACT FACTOR為5.2)接受發表。

第三章中,2014年EV-D68在北美造成大流行,引起全球對此病毒的重視,我們在2008年的台灣患有威爾森氏症的小兒科病人檢體中,檢測到EV-D68,將該病毒株進行次世代定序與基因體分析,進一步與從公共資料庫所獲得的EV-D68基因進行比對,將其分類為基因體型別1-B。此株病毒是台灣完成基因體定序最早的EV-D68病毒株,在演化上與2009年在越南流行的主要腸病毒D68型病毒有關,此成果已發表在Journal of the Formosan Medical Association : Feb;118(2):641-646, 2019(2019年6月IMPACT FACTOR為2.8)。

綜觀而言,我們成功地整合了CODEHOP基因檢測方法和次世代定序技術,對無法以血清學分離型別的腸道病毒分離株進行基因組分析,這不僅可以改善腸病毒檢測的方法,還可以提供完整的腸病毒基因組序列做進一步的腸病毒演化研究。
摘要(英) Enteroviruses (EV) are single-stranded, positive-sense RNA viruses in the Enterovirus genus of the Picornaviridae family. All enteroviruses have a similar genomic organization (7.2–8.5kb). The capsid proteins are coded on the 5’end of the ssRNA in a section called P1 (precursor 1). The nonstructural proteins are coded on the remaining sections of the genome, which are called P2 and P3. Changes in the structural protein genes of different enterovirus species reflect phylogenetic relationships. EV cause various clinical manifestations, including cutaneous, visceral, and neurological diseases. The Enterovirus genus consists of 12 species, including Enterovirus A (EV-A, 25 serotypes), Enterovirus B (EV-B, 63 serotypes), Enterovirus C (EV-C, 23 serotypes), Enterovirus D (EV-D, 5 serotypes), Enterovirus E (EV-E, 4 serotypes), Enterovirus F (EV-F, 6 serotypes), Enterovirus G (EV-G, 11 serotypes), Enterovirus H (EV-H, 1 serotype), Enterovirus J (EV-J, 6 serotypes), Rhinovirus A (80 serotypes), Rhinovirus B (32 serotypes), and Rhinovirus C (55 serotypes)

Chapter Two indicated that we successfully established a NGS platform to conduct genome sequencing for the serologically-untypable enterovirus isolates. Among 130 serologically-untypable isolates, 121(93%) of them were classified into 29 serotypes using CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer)-based RT-PCR to amplify VP1 genes (VP1-CODEHOP). We further selected 52 samples for NGS and identified 59 genome sequences from 51 samples, including 8 samples containing two virus genomes. We also detected 23 genome variants (nucleotide identity <90% comparing with genome sequences in public domain) with potential genetic recombination, including 9 inter-serotype recombinants and 14 strains with unknown sources of recombination.

Chapter Three indicated that we retrospectively detected EV-D68 from a child with Wilson’s disease in 2008 in Taiwan. After comparing this EV-D68/Taiwan/2008 strain with EV-D68 genomes obtained from the public domain, it was classified as genome type 1-B; it is phylogenetically related to the predominant EV-D68 viruses that circulated in 2009 in Vietnam.

In conclusion, we successfully integrated VP1-CODEHOP and NGS techniques to conduct genomic analysis of serologically untypable enteroviruses, which could not only improve enterovirus surveillance but also provide genome sequences for evolution research.
關鍵字(中) ★ 腸病毒
★ 次世代基因體定序
關鍵字(英) ★ enterovirus
★ Next generation sequencing
論文目次 Chinese Abstract ……………………………………………………………… i
English Abstract ……………………………………………………………… iii
Acknowledgment ……………………………………………………………… v
Declaration ……………………………………………………………… vi
List of Figures ……………………………………………………………… ix
List of Tables ……………………………………………………………… xi
Abbreviation ……………………………………………………………… xii

1.Chapter One Literature Review………………………………………...………….… 1
1-1. Virology………………………………………………………………… 2
1-2. Epidemiology and Evolution…….……………………………………. 3
1-3. Molecular Techniques…………………………………………………. 7
1-3.1 COnsensus- DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) …………………………………………………………...
7
1-3.2 Next-Generation Sequencing (NGS)…………………………………. 7
1-4. References……………………………………………………………… 9
2.Chapter Two Genomic Analysis of Serologically Untypable Human Enteroviruses in Taiwan……………………………………………….
22
2-1. Introduction……………………………………………………………. 24
2-2. Materials and Methods………………………………………………... 26
2-2.1 Viruses………………………………………………………………….. 26
2-2.2 CODEHOP method……………………………………………………. 26
2-2.3. Virus purification and concentration………………………………… 27
2-2.4. Real-time PCR reaction and primers design………………………… 27
2-2.5. Next-generation sequencing (NGS)…………………………………… 28
2-2.6. Recombination detection and phylogenetic analysis………………… 28
2-2.7. Data access……………………………………………………………... 28
2-3. Result…………………………………………………………………… 29
2-3.1 Characterization of IFA-untypable enteroviruses using the VP1- CODEHOP method…………………………………………………….
29
2-3.2 Identification of untypable enterovirus by NGS…………………….. 29
2-3.3 Recombination detection……………………………………………… 30
2-3.4 Virus co-infection and clinical presentation…………………………. 32
2-4. Discussion………………………………………………………………. 33
2-5. Conclusion……………………………………………………………… 37
2-6. References…………………………………………………………….... 38
3.Chapter Three Genomic Analysis of Enterovirus D68, Including One Strain Isolated from a Child with Wilson’s Disease in Taiwan………….…
60
3-1. Introduction……………………………………………………………. 62
3-2. Materials and Methods……………………………………………….. 63
3-2.1 Clinical isolates collection……………………………………………... 63
3-2.2 Molecular analysis……………………………………………………... 63
3-2.3. Next-generation sequencing…………………………………………... 64
3-2.4. Phylogenetic analysis…………………………………………………... 64
3-3. Result and Discussion………………………………………………...... 65
3-4. Conclusion……………………………………………………………… 67
3-5. References……………………………………………………………… 68
Figures 1-5..……………………………………………………………………… 14
6-11..……………………………………………………………………. 43
12-13..…………………………………………………………………… 71
Tables 1-2………………………………………………………………………. 20
3-8………………………………………………………………………. 50
9…………………………………………………………………………. 74
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Chapter 2:
Guidelines for enterovirus surveillance in support of the Polio Eradication Initiative.: World Health Organization; 2015.
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Chapter 3:
1. Brown B.A., Nix W.A., Sheth M., Frace M. and Oberste M.S. Seven Strains of Enterovirus D68 Detected in the United States during the 2014 Severe Respiratory Disease Outbreak. Genome Announc. 2014; 2(6).
2. Chiang P.S., Huang M.L., Luo S.T., Lin T.Y., Tsao K.C. and Lee M.S. Comparing molecular methods for early detection and serotyping of enteroviruses in throat swabs of pediatric patients. PLoS One. 2012; 7(10):e48269.
3. Foster C.B., Friedman N., Carl J. and Piedimonte G. Enterovirus D68: a clinically important respiratory enterovirus. Cleve Clin J Med. 2015; 82(1):26-31.
4. Furuse Y., Chaimongkol N., Okamoto M., Imamura T., Saito M., Tamaki R., Saito M., Tohoku R.C.R.T., Lupisan S.P. and Oshitani H. Molecular epidemiology of enterovirus d68 from 2013 to 2014 in Philippines. J Clin Microbiol. 2015; 53(3):1015-1018.
5. Greninger A.L., Naccache S.N., Messacar K., Clayton A., Yu G., Somasekar S., Federman S., Stryke D., Anderson C., Yagi S., Messenger S., Wadford D., Xia D., Watt J.P., Van Haren K., Dominguez S.R., Glaser C., Aldrovandi G. and Chiu C.Y. A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012-14): a retrospective cohort study. Lancet Infect Dis. 2015; 15(6):671-682.
6. Guillaud O., Dumortier J., Sobesky R., Debray D., Wolf P., Vanlemmens C., Durand F., Calmus Y., Duvoux C., Dharancy S., Kamar N., Boudjema K., Bernard P.H., Pageaux G.P., Salame E., Gugenheim J., Lachaux A., Habes D., Radenne S., Hardwigsen J., Chazouilleres O., Trocello J.M., Woimant F., Ichai P., Branchereau S., Soubrane O., Castaing D., Jacquemin E., Samuel D. and Duclos-Vallee J.C. Long term results of liver transplantation for Wilson′s disease: experience in France. J Hepatol. 2014; 60(3):579-589.
7. Imamura T. and Oshitani H. Global reemergence of enterovirus D68 as an important pathogen for acute respiratory infections. Reviews in medical virology. 2015; 25(2):102-114.
8. Kyriakopoulou Z., Pliaka V., Amoutzias G.D. and Markoulatos P. Recombination among human non-polio enteroviruses: implications for epidemiology and evolution. Virus Genes. 2015; 50(2):177-188.
9. Lee M.-S., Lin T.-Y., Chiang P.-S., Li W.-C., Luo S.-T., Tsao K.-C., Liou G.-Y., Huang M.-L., Hsia S.-H., Huang Y.-C. and Chang S.-C. An investigation of epidemic enterovirus 71 infection in Taiwan, 2008: clinical, virologic, and serologic features. The Pediatric infectious disease journal. 2010; 29(11):1030-1034.
10. Messacar K., Schreiner T.L., Maloney J.A., Wallace A., Ludke J., Oberste M.S., Nix W.A., Robinson C.C., Glode M.P., Abzug M.J. and Dominguez S.R. A cluster of acute flaccid paralysis and cranial nerve dysfunction temporally associated with an outbreak of enterovirus D68 in children in Colorado, USA. Lancet. 2015; 385(9978):1662-1671.
11. Nix W.A., Oberste M.S. and Pallansch M.A. Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J Clin Microbiol. 2006; 44(8):2698-2704.
12. Oermann C.M., Schuster J.E., Conners G.P., Newland J.G., Selvarangan R. and Jackson M.A. Enterovirus d68. A focused review and clinical highlights from the 2014 U.S. Outbreak. Ann Am Thorac Soc. 2015; 12(5):775-781.
13. Renois F., Bouin A. and Andreoletti L. Enterovirus 68 in pediatric patients hospitalized for acute airway diseases. J Clin Microbiol. 2013; 51(2):640-643.
14. Tsao K.C., Huang C.G., Huang Y.L., Chen F.C., Huang P.N., Huang Y.C., Lin T.Y., Shih S.R. and Chang S.C. Epidemiologic features and virus isolation of enteroviruses in Northern Taiwan during 2000-2008. J Virol Methods. 2010; 165(2):330-332.
指導教授 李敏西 高永旭(Min Shi Lee Yung Hsi Kao) 審核日期 2019-7-25
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