臺灣博碩士論文加值系統

A型流感病毒除了可能於每年當中造成數次的季節性流感之外，每隔數十年便有機會產生全球性大規模流行的高感染性流感，例如2009年的H1N1新型流感便是屬於一種具高破壞性的流感病毒。因此，如何減緩流感病毒所帶來的傷害便是現今被高度關注的一項議題。由於A型流感病毒具有高度的病毒變異性，且可透過抗原微變以及抗原移型等方式進行突變，這些因素使得A型流感病毒難以進行預測以及分析。但是在過往的研究中指出，藉由分析一些可以區分物種的重要特徵位置(Signature)對於流感病毒的毒性分析以及疫苗製作上有著相當大的助益。
在我們的論文當中，我們首先從NCBI流感病毒資料庫中獲取所需的病毒資料，並且依不同的年代區段將資料進行分割，接著使用ARI的計算方法對各個區段中的資料進行重要特徵位置的搜尋，最後根據這些選出的重要特徵位置所對應的ARI值之變化情形對其進行分群。而在我們的分群結果中可以明顯看出各個群當中的ARI值折線圖是相當密合的，並且在部分的分群結果中透過相關文獻的佐證之下，我們發現這些位置能夠影響流感病毒的毒性或著具有類似的功能性。

Infuenza A viruses(IAV) caused epidemic several times a year, and sometimes a highly pathogenic IAV will cause a global outbreak of infection, for example the 2009 influenza A(H1N1) pandemic is one of the most destructive global pandemics in human history. Therefore, how to mitigating the severity of a new influenza pandemic is now a top global public health priority. In fact, the unique host range and two mechanisms for variation in viruses, antigenic drift and antigenic shift, makes the IAV is hard to predict. Identification of the host-associated genomic signatures is a cost-effective approach to analysis amount of viral sequences data for mitigating the illness and deaths. In this thesis, we first download the IAV sequences data from NCBI, and then we divide the virus data into several chronological groups. In the chronologically experiment, after identified the chronological signatures, we purpose a cluter alalysis of chronological signatures based on the ARI values over time. In our cluster results, the chronological signatures for each cluster shows highly correlated on the ARI values trend over time, and some signature sites, which in the same cluster, shows likely in the same functional domain or correlated with the pathogenic of IAV.

摘要 i
Abstract ii
致謝 iii
表目錄 vi
圖目錄 viii
一、緒論 1
1.1研究背景與動機 1
1.2研究目標 1
1.3論文架構 2
二、背景及相關文獻研究 3
2.1 流感病毒簡介 3
2.2 重要特徵討論 5
2.3 ARI (Adjusted Rand Index) 6
2.4 Elbow criterion 8
2.5 Gap statistic 9
三、資料蒐集以及實驗方法: 11
3.1 資料蒐集 11
3.1.1流感病毒資料蒐集 11
3.1.2流感病毒亞型選擇 11
3.1.3病毒蛋白質選擇 12
3.2 資料前處理 12
3.2.1多序列排比(Multiple alignment) 12
3.2.1 Over-Sampling 13
3.2.2 依年代區分資料 13
3.3 分析方法之選擇 14
3.4 重要特徵位置之分群處理 16
3.4.1 Elbow criterion分群方法 16
3.4.2 Gap statistic分群方法 16
3.4.3 Elbow criterion與Gap statistic分群方法的選擇與比較 16
3.5 實驗流程圖 18
四、實驗結果與討論: 18
4.1總年代實驗結果 20
4.1.1 1902年以來至2014年十一月底， A型流感病毒蛋白質 Avian， Human物種間 Top-20重要特徵位置: 20
4.1.2 1902年以來至2014年十一月底A型流感病毒蛋白質 Swine Human， 物種間Top-20重要特徵位置: 35
4.2劃分年代實驗結果 45
4.2.1 根據流感大爆發年代區分八個年代區間， Avian， Human A型流感病毒蛋白質Top-20物種間重要特徵位置: 45
4.2.2 根據流感大爆發年代區分八個年代區間， Swine， Human之 A型流感病毒蛋白質Top-20物種間重要特徵位置: 58
4.3劃分年代區段之下重要特徵位置分群結果 72
4.3.1根據流感大爆發年代區分八個年代區間， Avian， Human A型流感病毒蛋白質所對應的ARI值對重要特徵位置進行分群 75
4.3.2根據流感大爆發年代區分八個年代區間， Swine， Human A型流感病毒蛋白質所對應的ARI值對重要特徵位置進行分群 89
五、結論 103
參考文獻: 105

[1] Behrens G， Kamps BS， Hoffmann C， Preiser W (2006) Influenza Report 2006. Flying Publisher.
[2] Chen GW， Shih SR (2009) Genomic Signatures of Influenza A Pandemic (H1N1) 2009 Virus. Emerging Infectious Diseases 15(2): 1897-1903.
[3] Miotto O， Heiny AT， Albrecht R， García-Sastre A， Tan TW， et al. (2010) Complete-Proteome Mapping of Human Influenza A Adaptive Mutations: Implications for Human Transmissibility of Zoonotic Strains. PLoS ONE 5(2): e9025.
[4] Smith GJ， Vijaykrishna D， Bahl J， Lycett SJ， Worobey M， et al. (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459: 1122-1125.
[5] Hubert L， Arabie P (1985) Comparing partitions. J Classif 2: 193-218.
[6] Rand WM (1971) Objective criteria for the evaluation of clustering methods. J Am. Stat. Assoc. 66(336): 846-850.
[7] Thorndike RL (1953) Who belongs in the family？. Psychometrika 18(4): 267-276.
[8] Tibshirani R (2001) Estimating the number of clusters in a data set via the gap statistic. Journal of the Royal Statistical Society: Series B. 63(2): 411-423.
[9] Capual， Alexander DJ (2002) Avian influenza and human health. Acta Trop 83(1): 1-6.
[10] Larkin MA， Blackshields G， Brown NP， Chenna R， McGettigan PA， et al. (2007) ClustalW and ClustalX version 2. Bioinformatics 23(21): 2947–8.
[11] Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32(5): 1792–1797.
[12] Katoh K， Misawa K， Kuma K， Miyata T. (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucl.Acids Res. 30(14): 3059-3066.
[13] Pan C， Cheung B， Tan S， Li C， Li L， et al. (2010) Genomic Signature and Mutation Trend Analysis of Pandemic (H1N1) 2009 Influenza A Virus. PLoS ONE 5(3): e9549.
[14] Milligan GW， Cooper MC (1986) A study of the comparability of external criteria for hierarchical cluster analysis. Multivariate Behavioral Research 21: 441-458.
[15] Punj G， Stewart DW (1983) Cluster Analysis in Marketing Research: Review and Suggestions for Application. Journal of Marketing Research 20(2):134-148.
[16]Milligan GW， Sokol LM (1980) A two-stage clustering algorithm with robust recovery characteristics. Educational and Psychological Measurement 40(3), 755-759.
[17] Norusis MJ (1994). SPSS Professional Statistics 6.1. (SPSS Inc., Illinois, U.S.A.).
[18] Ward J (1963) Hierarchical Grouping to Optimize an Objective
Function. Journal of the American Statistical Association 58(301):236-44.
[19] Thaa B， Herrmann A， Veit M (2009) The polybasic region is not essential for membrane binding of the matrix protein M1 of influenza virus. Virology 383(1): 150–155.
[20] Baudin F， Petit I， Weissenhorn W， Ruigrok RW (2001) In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology 281(1): 102-8.
[21] Akarsu H， Burmeister WP， Petosa C， Petit I， Műller CW， et al. (2003) Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). EMBO J 22(18): 4646–4655.
[22] Furuse Y， Suzuki A， Kamigaki T， Oshitani H (2009) Evolution of the M gene of the influenza A virus in different host species: large-scale sequence analysis. Virol J 6:67.
[23] Liu W， Li H, Chen YH (2003) N-terminus of M2 protein could induce antibodies with inhibitory activity against influenza virus replication. FEMS Immunol Med Microbiol 35(2):141–146.
[24] Xu L， Bao L， Zhou J， Wang D， Deng W， et al. (2011) Genomic polymorphism of the pandemic A (H1N1) influenza viruses correlates with viral replication, virulence, and pathogenicity in vitro and in vivo. PLoS One 6: e20698.
[25] Leymarie O， Jouvion G， Hervé P， Chevalier C， Lorin V， et al. (2013) Kinetic characterization of PB1-F2-mediated immunopathology during highly pathogenic avian H5N1 influenza virus infection. PLoS One 8: e57894.
[26] Crowe SR， Miller SC， Brown DM， Adams PS， Dutton RW， et al. (2006) Uneven distribution of MHC class II epitopes within the influenza virus. Vaccine 24: 457–467.
[27] Mehle A， Doudna JA (2009) Adaptive strategies of the influenza virus polymerase for replication in humans. Proc Natl Acad Sci U S A. 106(50): 21312-6.
[28] Mukaigawa J， Nayak DP (1991) Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2. J Virol 65(1): 245–253.
[29] Poole E， Elton D， Medcalf L， Digard P (2004) Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites. Virology 321(1): 120–133.
[30] Honda A， Mizumoto K， Ishihama A (1999) Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA polymerase. Genes Cells 4(8): 475–485.
[31] Fechter P， Mingay L， Sharps J， Chambers A， Fodor E， et al. (2003) Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J Biol Chem 278(22): 20381–20388.
[32] Ichihashi T， Yoshida R， Sugimoto C， Takada A， Kajino K (2011) Cross-Protective Peptide Vaccine against Influenza A Viruses Developed in HLA-A*2402 Human Immunity Model. PLoS One 6(9): e24626.
[33] Ohtsu Y， Honda Y， Sakata Y， Kato H， Toyoda T (2001) Fine mapping of the subunit binding sites of influenza virus RNA polymerase. International Congress Series 1219: 463-469
[34] Nath ST， Nayak DP (1990) Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza virus (H1 N1). Mol Cell Biol. 10(8): 4139–4145.
[35] Jung TE， Brownlee GG (2006) A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase. J Gen Virol 87: 679–688.
[36] Gonzalez S， Ortin J (1999) Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates. EMBO J 18(13): 3767–3775.
[37] Yamada H， Chounan R， Higashi Y， Kurihara N， Kido H (2004) Mitochondrial targeting sequence of the influenza A virus PB1-F2 protein and its function in mitochondria. FEBS Lett 578: 1–6.
[38] Chen W， Calvo PA， Malide D， Gibbs J， Schubert U， et al. (2001) A novel influenza A virus mitochondrial protein that induces cell death. Nature Medicine 7: 1306–1312.
[39] Biswas SK， Boutz PL， Nayak DP (1998) Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins. J Virol 72(7): 5493–5501.
[40] Ozawa M， Fujii K， Muramoto Y， Yamada S， Yamayoshi S， et al. (2007) Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication. J Virol 81(1): 30–41.
[41] Gianfrani C， Oseroff C， Sidney J， Chesnut RW， Sette A (2000) Human memory CTL response specific for influenza A virus is broad and multispecific. Hum Immunol 61: 438–452.
[42] Deng Y， Yewdell JW， Eisenlohr LC， Bennink JR (1997) MHC affinity， peptide liberation， T cell repertoire， and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL. J Immunol 158: 1507-1515.
[43] Nieto A， de la Luna S， Barcena J， Portela A， Ortín J (1994) Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit. J Gen Virol 75: 29–36.
[44] Sanz-Ezquerro JJ， Zűrcher T， de la Luna S， Ortín J， Nieto A (1996) The aminoterminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis. J Virol 70(3): 1905–1911.
[45] He X， Zhou J， Bartlam M， Zhang R， Ma J， et al. (2008) Crystal structure of the polymerase PA(C)-PB1(N) complex from an avian influenza H5N1 virus. Nature 454(7208): 1123–1126.
[46] Hale BG， Randall RE， Ortín J， Jackson D (2008) The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89(10): 2359-76.
[47] Qian XY， Chien CY， Lu Y， Montelione GT， Krug RM (1995) An aminoterminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure. RNA 1(9): 948–956.
[48] Bornholdt ZA， Prasad BV (2008) X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus. Nature 456(7224): 985-8.
[49] Hale BG， Barclay WS， Randall RE， Russell RJ. (2008) Structure of an avian influenza A virus NS1 protein effector domain. Virology 378(1): 1-5.
[50] Heikkinen LS， Kazlauskas A， Melén K， Wagner R， Ziegler T， et al. (2008) Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling. J Biol Chem. 283(9): 5719-27.
[51] Shin YK， Liu Q， Tikoo SK， Babiuk LA， Zhou Y (2007) Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K. J Gen Virol 88: 13–18.
[52] Li Y， Yamakita Y， Krug RM (1998) Regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus NS1 protein. Proc Natl Acad Sci U S A 95: 4864–4869.
[53] Aragón T， de la Luna S， Novoa I， Carrasco L， Ortín J， et al. (2000) Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein， a translational activator of influenza virus. Mol Cell Biol 20(17): 6259–6268.
[54] Kochs G， García-Sastre A， Martínez-Sobrido L (2007) Multiple anti-interferon actions of the influenza A virus NS1 protein. J Virol 81(13): 7011–7021.
[55] Chen Z， Li Y， Krug RM (1999) Influenza A virus NS1 protein targets poly(A)- binding protein II of the cellular 3’-end processing machinery. EMBO J 18(8): 2273–2283.
[56] Obenauer JC， Denson J， Mehta PK， et al. (2006) Large-scale sequence analysis of avian influenza isolates. Science 311(5767): 1576–1580.
[57] Greenspan D， Palese P， Krystal M (1988) Two nuclear location signals in the influenza virus NS1 nonstructural protein. J Virol 62(8): 3020–3026.
[58] Babon JA， Cruz J， Orphin L， Pazoles P， Co MD， et al. (2009) Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4(+) T-cell responses to internal proteins， hemagglutinins， and neuraminidases. Hum Immunol. 70(9): 711-21.
[59] Assarsson E， Bui HH， Sidney J， Zhang Q， Glenn J， et al. (2008) Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans. J Virol 82(24): 12241–12251.
[60] Iwatsuki-Horimoto K， Horimoto T， Fujii Y， Kawaoka Y (2004) Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence. J Virol 78(18): 10149–10155.
[61] Shimizu T， Takizawa N， Watanabe K， Nagata K， Kobayashi N. (2010) Crucial role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear export of vRNP. FEBS Lett. 585(1): 41-6.
[62] Ye Z， Robinson D， Wagner RR (1995) Nucleus-Targeting Domain of the Matrix Protein (M1 )of Influenza Virus. J Virol 69(3): 1964–1970.
[63] Lamb RA， Zebedee SL， Richardson CD (1985) Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell 40(3): 627–633.
[64] Tian C， Gao PF， Pinto LH， Lamb RA， Cross TA. (2003) Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers. Protein Science 12: 2597–2605.
[65] Hull JD， Gilmore R， Lamb RA. (1988) Integration of a small integral membrane protein， M2， of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus. J Cell Biol. 106(5): 1489-98.
[66] Chen BJ， Leser GP， Jackson D， Lamb RA. (2008) The influenza virus M2 protein cytoplasmic tail interacts with the M1 protein and influences virus assembly at the site of virus budding. J Virol 82(20): 10059-70.
[67] Lee YH， Ha do LA， Simmons C， de Jong MD， Chau NV， et al. (2008) Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest 118(10): 3478–3490.
[68] Ozawa M， Basnet S， Burley LM， Neumann G， Hatta M， et al. (2011) Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses. J. Virol. 85(9): 4596-4601.
[69] Billharz R1， Zeng H， Proll SC， Korth MJ， Lederer S， et al. (2009) The NS1 protein of the 1918 pandemic influenza virus blocks host interferon and lipid metabolism pathways. J. Virol. 83(20): 10557-10570.
[70] Weeks-Gorospe JN， Hurtig HR， Iverson AR， Schuneman MJ， Webby RJ， et al. (2012) Naturally occurring swine influenza A virus PB1-F2 phenotypes that contribute to superinfection with Gram-positive respiratory pathogens. J. Virol. 86(17): 9035-9043.
[71] Zhao Z， Yi C， Zhao L, Wang S, Zhou L， et al. (2014) PB2-588I enhances 2009 H1N1 pandemic influenza virus virulence by increasing viral replication and exacerbating PB2 inhibition of beta interferon expression. J. Virol. 88(4):2260-2267.
[72] Bussey KA， Bousse TL， Desmet EA， Kim B， Takimoto T (2010) PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. J. Virol. 84(9):4395-4406.
[73] Gíria M， de Andrade HR (2014) Genetic evolution of PB1 in the zoonotic transmission of influenza A(H1) virus. Infection, Genetics and Evolution 27:234-243.
[74] Wanitchang A， Patarasirin P， Jengarn J， Jongkaewwattana A (2011) Atypical characteristics of nucleoprotein of pandemic influenza virus H1N1 and their roles in reassortment restriction. Arch. Virol. 156:1031–1040.
[75] Thippamom N， Sreta D， Kitikoon P， Thanawongnuwech R， Poovorawan Y， et al. (2010) Genetic variations of nucleoprotein gene of influenza A viruses isolated from swine in Thailand. Virol J 7:185.
[76] Lindstrom SE， Hiromoto Y， Nerome R， Omoe K， Sugita S (1998) Phylogenetic analysis of the entire genome of influenza A (H3N2) viruses from Japan: Evidence for genetic reassortment of the six internal genes. J Virol 72: 8021–8031.
[77] Surichan S， Wirachwong P， Supachaturas W， Utid K， Theerasurakarn S, et al. (2011) Development of influenza vaccine production capacity by the Government Pharmaceutical Organization of Thailand: addressing the threat of an influenza pandemic. Vaccine 29S:A29-A33.
[78] Muraki Y， Murata T， Takashita E， Matsuzaki Y， Sugawara K, et al. (2007) A mutation on influenza C virus M1 protein affects virion morphology by altering the membrane affinity of the protein. J Virol 81: 8766–8773.
[79] Jonges M， Bataille A， Enserink R， Meijer A， Fouchier RA， et al. (2011) Comparative analysis of avian influenza virus diversity in poultry and humans during a highly pathogenic avian influenza A (H7N7) virus outbreak. Journal of virology 85: 10598–10604. doi: 10.1128/jvi.05369-11.
[80] Graham M， Liang B， Van Domselaar G， Bastien N， Beaudoin C， et al. (2011) Nationwide molecular surveillance of pandemic H1N1 influenza A virus genomes: Canada, 2009. PLoS One 6: e16087.
[81] Tsurumura T， Qiu H， Yoshida T， Tsumori Y， Hatakeyama D (2013) Conformational Polymorphism of m7GTP in Crystal Structure of the PB2 Middle Domain from Human Influenza A Virus. PLoS ONE 8(11): e82020.
[82] Taubenberger JK， Reid AH， Lourens RM， Wang R， Jin G (2005) Characterization of the 1918 influenza virus polymerase genes. Nature 437: 889-893.
[83] Mushtaq M， Juan H， Jiang P， Li Y， Li T， et al. (2008) Complete genome analysis of a highly pathogenic H5N1 influenza A virus isolated from a tiger in China. Arch. Virol. 153:1569-1574.
[84] Basler CF， Reid AH， Dybing JK， Janczewski TA， Fanning TG， et al. (2001) Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. PNAS 98(5):2746-2751.
[85] Wang JF， Wei DQ， Chou KC (2009) Insights from investigating the interactions of adamantane-based drugs with the M2 proton channel from the H1N1 swine virus. Biochem Biophys Res Commun 388: 413–417.
[86] Yao Y， Mingay LJ， McCauley JW， Barclay WS (2001) Sequences in influenza A virus PB2 protein that determine productive infection for an avian influenza virus in mouse and human cell lines. J Virol 75: 5410–5415.
[87] Mänz B， Dornfeld D， Götz V， Zell R， Zimmermann P， et al. (2013) Pandemic influenza A viruses escape from restriction by human MxA through adaptive mutations in the nucleoprotein. PLoS Pathog. 9(3): e1003279.
[88] Elleman CJ， Barclay WS (2004) The M1 matrix protein controls the filamentous phenotype of influenza A virus. Virology 321:144–153.
[89] Burleigh LM， Calder LJ， Skehel JJ， Steinhauer DA (2005) Influenza A viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes. J. Virol. 79:1262–1270.
[90] Chen BJ， Takeda M， Lamb RA (2005) Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes differ in their ability to support assembly. J. Virol. 79:13673-13684.
[91] Le L， Leluk J (2011) Study on phylogenetic relationships, variability, and correlated mutations in M2 proteins of influenza virus A. PLoS One. 6: e22970.