臺灣博碩士論文加值系統

流感病毒(Influenza virus)主要藉由飛沫傳播感染宿主呼吸道。患者通常會有咳嗽、發燒、倦怠、四肢無力及鼻炎，嚴重者可能引起呼吸衰竭甚至死亡。
　　西元2013年春季在中國大陸東南省份爆發的H7N9新型禽流感病毒，截至2013年六月為止共造成了131例的感染及36例的死亡。H7N9感染所造成的高死亡率使得H7N9的致病能力及其是否會發生人傳人感染成為重要的研究課題。台灣在四月下旬確診一名重症住院病患感染H7N9，我們從採集的檢體中分離病毒核酸並進行質體構築，同時比較GISAID流感數據庫中所有不同宿主來源的H7N9參考株序列，釐清是否存在特異性胺基酸位點取代出現於跨宿主感染，與潛在抗藥性位點的有無。H7N9的質體與參考株經過序列分析，PB2的第627位點有屬於禽鳥病毒特異性胺基酸位點穀胺酸(glutamic acid)相異於人類的精胺酸(arginine)；神經胺酸酶則在第289位點發現抗藥型的離胺酸(lysine) 存在。此外，質體株的神經胺酸酶在第219骨架性位點由異亮胺酸(isoleucine)突變為精胺酸。
　　我們以構築之質體進行反轉錄系統製造重組病毒，重組病毒在經過培養與放大後，在MDCK細胞中觀察到細胞病變的情形，確認重組病毒的確成功製作。

Influenza virus is mainly spread by droplet to infect the respiratory tract and lungs. Patients usually have cough, fever, fatigue, weakness and rhinitis, while severe cases can cause respiratory failure and even death.
　　Novel H7N9 avian-origin influenza outbreak occurred in the coastal provinces of China in AD 2013 spring. As of June, raised great concern about a total of 131 cases of infection and 36 cases of death were reported. High mortality rates of H7N9 infection make the pathogenicity of H7N9 and the potential of human to human transmission. A severe case of H7N9 infection was diagnosed in Taiwan in late April. Viral nucleic acid was extracted from collected samples and subjected to PCR and sequencing. The H7N9 sequences from GISIAD influenza database were used to compare to specific amino acid site substitutions appear in the viral population from cross-hosts and the presence of potential drug resistance. Total H7N9 plasmids and reference strains were analyzed to release some information. At the 627 amino acid site of PB2 protein, there are glutamic acid from avian-origin virus different in the arginine from human-origin. The 289 amino acid site of neuraminidase presents lysine for resistance strain and arginine for wild type strain. In addition, as a neuraminidase skeleton site, the 219 site from plasmid is mutated from isoleucine to arginie. And the correlation between point mutation and resistance needs to be further evaluated.
　We constructed the plasmid for reverse genetic system manufacturing recombinant viruses. Cytopathic effects caused by recombinant viruses observed after culture and amplification. Confirmed the recombinant virus is indeed successful production.

口試委員會審定書 ii
致謝 iii
中文摘要 iv
Abstract v
目錄 vi
圖目錄 x
表目錄 xi
第一章 緒論 1
1-1 前言 1
1-2 A型流感病毒構造及病毒蛋白質 1
1-2.1 聚合酶蛋白質(Polymerase) 2
1-2.2 血球凝集素(Hemagglutinin；HA) 3
1-2.3 核蛋白質(Nucleoprotein；NP) 3
1-2.4 神經胺酸酶(Neuraminidase，NA) 4
1-2.5 基質蛋白質(Matrix；M) 5
1-2.6 非結構蛋白質(Non-structural protein；NS) 5
1-3 流感病毒的抗原變異 5
1-4 重要的世界性大流行 6
1-5 禽流感病毒簡介 7
1-5.1 流感病毒演化概況 7
1-5.2 禽流感病毒流行概況 7
1-5.3 H7N9疾病概況 8
1-6 H7N9基因來源及特徵 9
第二章 實驗動機與目的 10
2-1 實驗動機起源 10
2-2 本次實驗目的 11
第三章 實驗方法與材料 12
3-1 實驗材料 12
3-1.1 商業試劑套組 12
3-1.2 病毒反轉錄-聚合酶鏈鎖反應(Reverse Transcription-Polymerase Chain Reaction, RT-PCR) 12
3-1.3 勝任細胞(competent cell) 12
3-1.4 細菌培養基 13
3-1.5 限制酶與緩衝溶液 13
3-1.6 轉染實驗(transfection) 13
3-2 實驗方法 14
3-2.1 病毒核酸萃取 14
3-2.2 反轉錄-聚合酶鏈鎖反應(Reverse Transcription-Polymerase Chain Reaction, RT-PCR) 14
3-2.3 DNA連接 (ligation)作用 15
3-2.4 熱衝擊轉型(Heat shock transformation) 16
3-2.5 細菌質體萃取 16
3-2.6 限制酶切割 16
3-2.7 產物膠體純化 17
3-2.8 pHW2000載體製備 17
3-2.9 以轉染實驗製造H7N9病毒 18
3-2.10 病毒序列分析軟體 18
3-2.11 參考病毒株序列來源 18
第四章 實驗結果 19
4-1 H7N9參考株病毒序列分析 19
4-2 台灣與中國大陸H7N9胺基酸特異位點分析 20
4-3 H7N9核苷酸密碼子使用分析 23
4-4 構築新型禽流感病毒株H7N9全片段基因體 23
4-5 H7N9構築質體序列分析 24
4-5.1 聚合酶鹼蛋白質2( Polymerase Basic protein 2；PB2) 25
4-5.2 聚合酶鹼蛋白質1( Polymerase Basic protein 1；PB1) 25
4-5.3 聚合酶酸蛋白質(Polymerase Acidic protein；PA) 25
4-5.4 血球凝集素蛋白質(Hemagglutinin；HA) 26
4-5.5 核蛋白質(Nucleoprotein；NP) 26
4-5.6 神經胺酸酶(Neuraminidase，NA) 26
4-5.7 基質蛋白質(Matrix；M) 27
4-5.8 非結構蛋白質(Non-structural protein；NS) 27
4-6 利用構築之H7N9質體在細胞模式中重組病毒 27
第五章 討論 28
參考文獻 32
附圖 43

1.Fields, B.N., D.M. Knipe, P.M. Howley, D.E. Griffin, and Ovid Technologies Inc., Fields'' virology, 2007, Wolters kluwer/Lippincott Williams & Wilkins: Philadelphia.
2.Lu, X., Y. Shi, F. Gao, H. Xiao, M. Wang, et al., Insights into avian influenza virus pathogenicity: the hemagglutinin precursor HA0 of subtype H16 has an alpha-helix structure in its cleavage site with inefficient HA1/HA2 cleavage. J Virol, 2012. 86(23): p. 12861-70.
3.Zhu, X., H. Yang, Z. Guo, W. Yu, P.J. Carney, et al., Crystal structures of two subtype N10 neuraminidase-like proteins from bat influenza A viruses reveal a diverged putative active site. Proc Natl Acad Sci U S A, 2012. 109(46): p. 18903-8.
4.Zhu, X., W. Yu, R. McBride, Y. Li, L.M. Chen, et al., Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities. Proc Natl Acad Sci U S A, 2013. 110(4): p. 1458-63.
5.Hoffmann, E., J. Stech, Y. Guan, R.G. Webster, and D.R. Perez, Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol, 2001. 146(12): p. 2275-89.
6.Fujiyoshi, Y., N.P. Kume, K. Sakata, and S.B. Sato, Fine structure of influenza A virus observed by electron cryo-microscopy. EMBO J, 1994. 13(2): p. 318-26.
7.Chu, C.M., I.M. Dawson, and W.J. Elford, Filamentous forms associated with newly isolated influenza virus. Lancet, 1949. 1(6554): p. 602.
8.Kilbourne, E.D. and J.S. Murphy, Genetic studies of influenza viruses. I. Viral morphology and growth capacity as exchangeable genetic traits. Rapid in ovo adaptation of early passage Asian strain isolates by combination with PR8. J Exp Med, 1960. 111: p. 387-406.
9.Schroeder, C., H. Heider, E. Moncke-Buchner, and T.I. Lin, The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein. Eur Biophys J, 2005. 34(1): p. 52-66.
10.Mukaigawa, J. and D.P. Nayak, Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2. J Virol, 1991. 65(1): p. 245-53.
11.Li, M.L., B.C. Ramirez, and R.M. Krug, RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites. EMBO J, 1998. 17(19): p. 5844-52.
12.Kawaguchi, A., T. Naito, and K. Nagata, Involvement of influenza virus PA subunit in assembly of functional RNA polymerase complexes. J Virol, 2005. 79(2): p. 732-44.
13.Chen, W., P.A. Calvo, D. Malide, J. Gibbs, U. Schubert, et al., A novel influenza A virus mitochondrial protein that induces cell death. Nat Med, 2001. 7(12): p. 1306-12.
14.Zamarin, D., A. Garcia-Sastre, X. Xiao, R. Wang, and P. Palese, Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1. PLoS Pathog, 2005. 1(1): p. e4.
15.Jagger, B.W., H.M. Wise, J.C. Kash, K.A. Walters, N.M. Wills, et al., An overlapping protein-coding region in influenza A virus segment 3 modulates the host response. Science, 2012. 337(6091): p. 199-204.
16.Wilson, I.A., J.J. Skehel, and D.C. Wiley, Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature, 1981. 289(5796): p. 366-73.
17.Bullough, P.A., F.M. Hughson, A.C. Treharne, R.W. Ruigrok, J.J. Skehel, et al., Crystals of a fragment of influenza haemagglutinin in the low pH induced conformation. J Mol Biol, 1994. 236(4): p. 1262-5.
18.Weis, W., J.H. Brown, S. Cusack, J.C. Paulson, J.J. Skehel, et al., Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature, 1988. 333(6172): p. 426-31.
19.Xu, Q., W. Wang, X. Cheng, J. Zengel, and H. Jin, Influenza H1N1 A/Solomon Island/3/06 virus receptor binding specificity correlates with virus pathogenicity, antigenicity, and immunogenicity in ferrets. J Virol, 2010. 84(10): p. 4936-45.
20.Koerner, I., M.N. Matrosovich, O. Haller, P. Staeheli, and G. Kochs, Altered receptor specificity and fusion activity of the haemagglutinin contribute to high virulence of a mouse-adapted influenza A virus. J Gen Virol, 2012. 93(Pt 5): p. 970-9.
21.Martin-Benito, J., E. Area, J. Ortega, O. Llorca, J.M. Valpuesta, et al., Three-dimensional reconstruction of a recombinant influenza virus ribonucleoprotein particle. EMBO Rep, 2001. 2(4): p. 313-7.
22.Shu, L.L., W.J. Bean, and R.G. Webster, Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. J Virol, 1993. 67(5): p. 2723-9.
23.Kheiri, M.T., A. Jamali, M. Shenagari, H. Hashemi, F. Sabahi, et al., Influenza virosome/DNA vaccine complex as a new formulation to induce intra-subtypic protection against influenza virus challenge. Antiviral Res, 2012. 95(3): p. 229-36.
24.Palese, P., J.L. Schulman, G. Bodo, and P. Meindl, Inhibition of influenza and parainfluenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA). Virology, 1974. 59(2): p. 490-8.
25.Matrosovich, M.N., T.Y. Matrosovich, T. Gray, N.A. Roberts, and H.D. Klenk, Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol, 2004. 78(22): p. 12665-7.
26.Jefferson, T.O., V. Demicheli, C. Di Pietrantonj, M. Jones, and D. Rivetti, Neuraminidase inhibitors for preventing and treating influenza in healthy adults. Cochrane Database Syst Rev, 2006(3): p. CD001265.
27.Gaur, A.H., B. Bagga, S. Barman, R. Hayden, A. Lamptey, et al., Intravenous zanamivir for oseltamivir-resistant 2009 H1N1 influenza. N Engl J Med, 2010. 362(1): p. 88-9.
28.Brennan, B.J., B. Davies, G. Cirrincione-Dall, P.N. Morcos, A. Beryozkina, et al., Safety, tolerability, and pharmacokinetics of intravenous oseltamivir: single- and multiple-dose phase I studies with healthy volunteers. Antimicrob Agents Chemother, 2012. 56(9): p. 4729-37.
29.Okomo-Adhiambo, M., K. Sleeman, C. Lysen, H.T. Nguyen, X. Xu, et al., Neuraminidase inhibitor susceptibility surveillance of influenza viruses circulating worldwide during the 2011 Southern Hemisphere season. Influenza Other Respi Viruses, 2013.
30.Colman, P.M., P.A. Hoyne, and M.C. Lawrence, Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase. J Virol, 1993. 67(6): p. 2972-80.
31.Ye, Z., T. Liu, D.P. Offringa, J. McInnis, and R.A. Levandowski, Association of influenza virus matrix protein with ribonucleoproteins. J Virol, 1999. 73(9): p. 7467-73.
32.Ali, A., R.T. Avalos, E. Ponimaskin, and D.P. Nayak, Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein. J Virol, 2000. 74(18): p. 8709-19.
33.Wharton, S.A., R.B. Belshe, J.J. Skehel, and A.J. Hay, Role of virion M2 protein in influenza virus uncoating: specific reduction in the rate of membrane fusion between virus and liposomes by amantadine. J Gen Virol, 1994. 75 ( Pt 4): p. 945-8.
34.Bukrinskaya, A.G., N.K. Vorkunova, G.V. Kornilayeva, R.A. Narmanbetova, and G.K. Vorkunova, Influenza virus uncoating in infected cells and effect of rimantadine. J Gen Virol, 1982. 60(Pt 1): p. 49-59.
35.Centers for Disease, C. and Prevention, High levels of adamantane resistance among influenza A (H3N2) viruses and interim guidelines for use of antiviral agents--United States, 2005-06 influenza season. MMWR Morb Mortal Wkly Rep, 2006. 55(2): p. 44-6.
36.Wainright, P.O., M.L. Perdue, M. Brugh, and C.W. Beard, Amantadine resistance among hemagglutinin subtype 5 strains of avian influenza virus. Avian Dis, 1991. 35(1): p. 31-9.
37.Schmidtke, M., R. Zell, K. Bauer, A. Krumbholz, C. Schrader, et al., Amantadine resistance among porcine H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany between 1981 and 2001. Intervirology, 2006. 49(5): p. 286-93.
38.Stasakova, J., B. Ferko, C. Kittel, S. Sereinig, J. Romanova, et al., Influenza A mutant viruses with altered NS1 protein function provoke caspase-1 activation in primary human macrophages, resulting in fast apoptosis and release of high levels of interleukins 1beta and 18. J Gen Virol, 2005. 86(Pt 1): p. 185-95.
39.Fernandez-Sesma, A., S. Marukian, B.J. Ebersole, D. Kaminski, M.S. Park, et al., Influenza virus evades innate and adaptive immunity via the NS1 protein. J Virol, 2006. 80(13): p. 6295-304.
40.O''Neill, R.E., J. Talon, and P. Palese, The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J, 1998. 17(1): p. 288-96.
41.Bizebard, T., C. Barbey-Martin, D. Fleury, B. Gigant, B. Barrere, et al., Structural studies on viral escape from antibody neutralization. Curr Top Microbiol Immunol, 2001. 260: p. 55-64.
42.Alexander, D.J. and I.H. Brown, Recent zoonoses caused by influenza A viruses. Rev Sci Tech, 2000. 19(1): p. 197-225.
43.Claas, E.C., Y. Kawaoka, J.C. de Jong, N. Masurel, and R.G. Webster, Infection of children with avian-human reassortant influenza virus from pigs in Europe. Virology, 1994. 204(1): p. 453-7.
44.Burt, D.S., K.H. Mills, J.J. Skehel, and D.B. Thomas, Diversity of the class II (I-Ak/I-Ek)-restricted T cell repertoire for influenza hemagglutinin and antigenic drift. Six nonoverlapping epitopes on the HA1 subunit are defined by synthetic peptides. J Exp Med, 1989. 170(2): p. 383-97.
45.Ghendon, Y., Introduction to pandemic influenza through history. Eur J Epidemiol, 1994. 10(4): p. 451-3.
46.Brasseur, J.W., Pandemic influenza: a brief history and primer. JAAPA, 2007. 20(1): p. 24-8.
47.in The Threat of Pandemic Influenza: Are We Ready? Workshop Summary, S.L. Knobler, et al., Editors. 2005: Washington (DC).
48.Pankhurst, R., The Great Ethiopian Influenza (Ye Hedar Beshita) Epidemic of 1918. Ethiop Med J, 1989. 27(4): p. 235-42.
49.Shortridge, K.F., N.N. Zhou, Y. Guan, P. Gao, T. Ito, et al., Characterization of avian H5N1 influenza viruses from poultry in Hong Kong. Virology, 1998. 252(2): p. 331-42.
50.Prevention, C.f.D.C.a., 2009 H1N1 flu: international situation update., C.f.D.C.a. Prevention, Editor. 2010, Atlanta, GA.
51.Webster, R.G., W.J. Bean, O.T. Gorman, T.M. Chambers, and Y. Kawaoka, Evolution and ecology of influenza A viruses. Microbiol Rev, 1992. 56(1): p. 152-79.
52.Kida, H., T. Ito, J. Yasuda, Y. Shimizu, C. Itakura, et al., Potential for transmission of avian influenza viruses to pigs. J Gen Virol, 1994. 75 ( Pt 9): p. 2183-8.
53.Alexander, D.J., Avian influenza, in OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2008, World Organisation for Animal Health.
54.Claas, E.C., A.D. Osterhaus, R. van Beek, J.C. De Jong, G.F. Rimmelzwaan, et al., Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet, 1998. 351(9101): p. 472-7.
55.Subbarao, K., A. Klimov, J. Katz, H. Regnery, W. Lim, et al., Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science, 1998. 279(5349): p. 393-6.
56.Tweed, S.A., D.M. Skowronski, S.T. David, A. Larder, M. Petric, et al., Human illness from avian influenza H7N3, British Columbia. Emerg Infect Dis, 2004. 10(12): p. 2196-9.
57.Peiris, M., K.Y. Yuen, C.W. Leung, K.H. Chan, P.L. Ip, et al., Human infection with influenza H9N2. Lancet, 1999. 354(9182): p. 916-7.
58.Butt, K.M., G.J. Smith, H. Chen, L.J. Zhang, Y.H. Leung, et al., Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J Clin Microbiol, 2005. 43(11): p. 5760-7.
59.Taubenberger, J.K., The origin and virulence of the 1918 "Spanish" influenza virus. Proc Am Philos Soc, 2006. 150(1): p. 86-112.
60.Yang, F., J. Wang, L. Jiang, J. Jin, L. Shao, et al., A fatal case caused by novel H7N9 avian influenza A virus in China. Emerging Microbes & Infections, 2013. 2(4): p. e19.
61.Gao, R., B. Cao, Y. Hu, Z. Feng, D. Wang, et al., Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med, 2013. 368(20): p. 1888-97.
62.Cowling, B., G. Freeman, J. Wong, P. Wu, Q. Liao, et al., Preliminary inferences on the age-specific seriousness of human disease caused by avian influenza A(H7N9) infections in China, March to April 2013. Euro Surveill, 2013. 18(19).
63.Liu, D., W. Shi, Y. Shi, D. Wang, H. Xiao, et al., Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet, 2013. 381(9881): p. 1926-32.
64.Liu, Q., L. Lu, Z. Sun, G.W. Chen, Y. Wen, et al., Genomic signature and protein sequence analysis of a novel influenza A (H7N9) virus that causes an outbreak in humans in China. Microbes Infect, 2013. 15(6-7): p. 432-9.
65.Tian, J., W. Qi, X. Li, J. He, P. Jiao, et al., A single E627K mutation in the PB2 protein of H9N2 avian influenza virus increases virulence by inducing higher glucocorticoids (GCs) level. PLoS One, 2012. 7(6): p. e38233.
66.Kageyama, T., S. Fujisaki, E. Takashita, H. Xu, S. Yamada, et al., Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013. Euro Surveill, 2013. 18(15): p. 20453.
67.Chang, S.Y., P.H. Lin, J.C. Tsai, C.C. Hung, and S.C. Chang, The first case of H7N9 influenza in Taiwan. Lancet, 2013. 381(9878): p. 1621.
68.Reperant, L.A., T. Kuiken, and A.D. Osterhaus, Adaptive pathways of zoonotic influenza viruses: from exposure to establishment in humans. Vaccine, 2012. 30(30): p. 4419-34.
69.Ng, A.K., W.H. Chan, S.T. Choi, M.K. Lam, K.F. Lau, et al., Influenza polymerase activity correlates with the strength of interaction between nucleoprotein and PB2 through the host-specific residue K/E627. PLoS One, 2012. 7(5): p. e36415.
70.Steel, J., A.C. Lowen, S. Mubareka, and P. Palese, Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog, 2009. 5(1): p. e1000252.
71.Chen, G.W., S.C. Chang, C.K. Mok, Y.L. Lo, Y.N. Kung, et al., Genomic signatures of human versus avian influenza A viruses. Emerg Infect Dis, 2006. 12(9): p. 1353-60.
72.Yamada, S., Y. Suzuki, T. Suzuki, M.Q. Le, C.A. Nidom, et al., Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature, 2006. 444(7117): p. 378-82.
73.Connor, R.J., Y. Kawaoka, R.G. Webster, and J.C. Paulson, Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology, 1994. 205(1): p. 17-23.
74.Matrosovich, M., A. Tuzikov, N. Bovin, A. Gambaryan, A. Klimov, et al., Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol, 2000. 74(18): p. 8502-12.
75.Martin, J., S.A. Wharton, Y.P. Lin, D.K. Takemoto, J.J. Skehel, et al., Studies of the binding properties of influenza hemagglutinin receptor-site mutants. Virology, 1998. 241(1): p. 101-11.
76.Hoffmann, T.W., S. Munier, T. Larcher, D. Soubieux, M. Ledevin, et al., Length variations in the NA stalk of an H7N1 influenza virus have opposite effects on viral excretion in chickens and ducks. J Virol, 2012. 86(1): p. 584-8.
77.Sun, Y., Y. Tan, K. Wei, H. Sun, Y. Shi, et al., Amino acid 316 of hemagglutinin and the neuraminidase stalk length influence virulence of H9N2 influenza virus in chickens and mice. J Virol, 2013. 87(5): p. 2963-8.
78.Carr, J., J. Ives, L. Kelly, R. Lambkin, J. Oxford, et al., Influenza virus carrying neuraminidase with reduced sensitivity to oseltamivir carboxylate has altered properties in vitro and is compromised for infectivity and replicative ability in vivo. Antiviral Res, 2002. 54(2): p. 79-88.
79.Abed, Y., B. Nehme, M. Baz, and G. Boivin, Activity of the neuraminidase inhibitor A-315675 against oseltamivir-resistant influenza neuraminidases of N1 and N2 subtypes. Antiviral Res, 2008. 77(2): p. 163-6.
80.Bouvier, N.M., A.C. Lowen, and P. Palese, Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol. J Virol, 2008. 82(20): p. 10052-8.
81.Simon, P., B.P. Holder, X. Bouhy, Y. Abed, C.A. Beauchemin, et al., The I222V neuraminidase mutation has a compensatory role in replication of an oseltamivir-resistant influenza virus A/H3N2 E119V mutant. J Clin Microbiol, 2011. 49(2): p. 715-7.
82.Wu, N.C., A.P. Young, S. Dandekar, H. Wijersuriya, L.Q. Al-Mawsawi, et al., Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening. J Virol, 2013. 87(2): p. 1193-9.
83.Shi, B., S. Xia, G.J. Yang, X.N. Zhou, and J. Liu, Inferring the potential risks of H7N9 infection by spatiotemporally characterizing bird migration and poultry distribution in eastern China. Infect Dis Poverty, 2013. 2(1): p. 8.
84.Ahn, I. and H.S. Son, Evolutionary analysis of human-origin influenza A virus (H3N2) genes associated with the codon usage patterns since 1993. Virus Genes, 2012. 44(2): p. 198-206.
85.Goni, N., A. Iriarte, V. Comas, M. Sonora, P. Moreno, et al., Pandemic influenza A virus codon usage revisited: biases, adaptation and implications for vaccine strain development. Virol J, 2012. 9: p. 263.
86.De Groot, A.S., M. Ardito, F. Terry, L. Levitz, T.M. Ross, et al., Low immunogenicity predicted for emerging avian-origin H7N9: Implication for influenza vaccine design. Hum Vaccin Immunother, 2013. 9(5).
87.Liu, C.Y. and J.H. Ai, [Virological characteristics of avian influenza A H7N9 virus]. Zhongguo Dang Dai Er Ke Za Zhi, 2013. 15(6): p. 405-8.