臺灣博碩士論文加值系統

近年來陸續有高病原性H5N1血清亞型之家禽流行性感冒病毒感染人類的病例，由2003年至2007年6月12日止，已造成190人不幸死亡，經由分析後推斷病毒是由已感染的禽鳥類直接傳染給人類。家禽流行性感冒病毒除了造成嚴重之經濟損失外，更有人畜共通疾病之隱憂。臺灣於1972年第一次爆發家禽流行性感冒病毒H6N1後，1998年開始監控水禽和家禽疫情，於家禽血清監控中發現蛋雞有50％而土雞有30％呈現H6N1血清陽性。雖然臺灣目前是高病原性家禽流行性感冒病毒非疫區，但是低病原性的H6N1仍然會造成養禽業的經濟損失，而且也由於長期於蛋雞和肉雞循環感染，因此也增加了抗原變異的可能。為求有效的監控此病毒，本研究利用H6N1血清亞型的純化病毒免疫BALB/c小鼠，以E.coli原核表現的H6 Hemagglutinin（HA）蛋白來篩選抗H6血清亞型HA的單源抗體。單源抗體經純化後，分析其特異性、敏感度以及中和能力，在特異性除了可以辨識H6 HA表現蛋白以及病毒尿囊液，其他1-14型血清亞型之HA 表現蛋白以及其他RNA病毒均無法辨識，證明具有良好的特異性。而以dot-enzyme-linked immunosorbent assay來評估單源抗體7G9的敏感度，對於表現的HA蛋白敏感度為0.24ng，對於病毒尿囊液的敏感度為0.5HAU，敏感度較HA試驗為敏感。利用中和試驗來檢驗單源抗體的中和能力，結果此單源抗體不具有效的中和能力，推測其辨識點並非在病毒的抗原決定位上。因此本實驗所製備的單源抗體可以用來開發診斷試劑，用於臨床樣本的抗原、抗體檢測，有利於家禽流行性感冒的監控。

In recent years, a highly pathogenic avian influenza virus H5N1 caused disease in human; from 2003 to 2007 June 12 led 190 people die unfortunately. Nucleotide sequence analysis showed that this virus was transmitted directly from infected avian species to humans. Avian influenza viruses not only cause serious economic losses, there is a worry of the disease becoming zoonoses. The first H6N1 avian influenza viruses outbreak in Taiwan was reported in 1972. Since 1998 began surveillance in waterfowl and poultry, and a recent serologic surveillance in poultry showed that about 50％ of the layers and 30％ of the native broilers had antibodies against H6N1 viruses. Although no highly pathogenic avian influenza virus has been isolated in Taiwan, low pathogenic H6N1 AIV was still made economic loss. H6N1 viruses have long period of circulation time in layers and boilers may increase chances to antigenic drift. In order to surveillance this virus, the study used purified H6N1 virus to immune BALB/c mice and screened by hemagglutinin（HA） protein of H6 expression from E. coli. After purification, we analyzed specificity, sensitivity and neutralizing efficiency. The specificity result is that the monoclonal antibodies （MAb）could recognized the HA protein expression from E. coli and homogeneous H6N1 virus. In sensitivity, we evaluated by dot-enzyme-linked immunosorbent assay. The monoclonal antibodies can detect 0.24 ng HA protein expression from E. coli and 0.5HAU virus. It is more sensitivity than HA test. Using neutralization test to evaluate the MAb’s neutralizing efficiency, and the result is that MAb don’t have effective neutralize efficiency. May the MAb’s epitop can not recognize the antigenic site of H6N1 virus. In this study, the MAb we produced can be used to develop new detection tool to examine antigen or antibody of clinical samples for AIV surveillance.

目次
中文摘要…………………… Ⅰ
英文摘要…………………… Ⅱ
目次………………………… Ⅲ
表次………………………… Ⅴ
圖次………………………… Ⅵ
第一章 緒言……………… 1
第二章 文獻探討………… 2
2-1 歷史背景………………… 2
2-2 家禽流行性感冒病毒性狀……………… 4
2-2.1 病毒特性……………………………… 4
2-2.2 病毒結構與功能……………………… 5
2-3.3 病毒的複製…………………………… 8
2-3.4 病毒物理化學特性…………………… 9
2-3.5 病毒抗原變異性……………………… 9
2-3 宿主的範圍……………………………… 9
2-4 病毒實驗室診斷………………………… 10
2-4.1 偵測抗原……………………………… 10
2-5.2 偵測抗體……………………………… 12
2-5 單源抗體………………………………… 13
2-5.1 單源抗體之歷史背景………………… 13
2-5.2 融合瘤細胞篩選之原理……………… 14
2-5.3 單源抗體的特性……………………… 14
第三章 材料與方法………………………… 16
3-1 病毒的增殖與純化……………………… 16
3-1.1 病毒來源……………………………… 16
3-1.2 實驗動物……………………………… 16
3-1.3 病毒增殖……………………………… 16
3-1.4 病毒濃縮與純化……………………… 16
3-1.5 病毒力價與濃度測定………………… 17
3-2 重組蛋白之表現與確認…………………. 17
3-2.1 重組質體之萃取……………………… 17
3-2.2 重組質體之轉型作用(transformation) 18
3-2.3 重組質體之確認……………………… 18
3-2.4 重組質體之表現…………………… 19
3-2.5 表現蛋白之純化…………………… 19
3-2.6 重組蛋白電泳分析與確認………… 20
3-3 單源抗體之製備………………………… 21
3-3.1 實驗動物…………………………… 21
3-3.2 免疫計畫…………………………… 21
3-3.3 骨髓瘤細胞培養……………………… 21
3-3.4 細胞融合……………………………… 21
3-3.5 陽性融合瘤細胞之篩選……………… 22
3-3.6 融合瘤細胞之確認………………… 23
3-3.7 融合瘤細胞單株化………………… 23
3-3.8 腹水之生產…………………………… 23
3-3.9 單源抗體之純化……………………… 24
3-4 單源抗體之分析……………… 24
3-4.1 單源抗體亞型分析…………………… 24
3-4.2 單源抗體特異性之分析……………… 25
3-4.3 單源抗體敏感性之分析……………… 25
3-4.4 單源抗體中和能力之分析…………… 26
第四章 結果…………………… 27
4-1 病毒增殖、純化及力價測定…………… 27
4-1.1 病毒增殖……………………………… 27
4-1.2 病毒純化、濃度以及力價測定……… 27
4-2 重組蛋白之表現與確認 27
4-2.1 重組蛋白之表現與確認 27
4-3 單源抗體之製備………………………… 28
4-3.1 融合瘤細胞製備之結果 28
4-3.2 單源抗體之生產. 28
4-4 單源抗體特性之分析. 28
4-4.1 單源抗體亞型分析…………………………… 28
4-4.2 單源抗體特異性之分析……………………… 28
4-4.3 單源抗體敏感性之分析……………………… 29
4-4.4 單源抗體中和能力之分析…………………… 30
第五章 討論………………………………………… 46
參考文獻……………………………………………… 51

吳靜如。2001。應用大腸桿菌表現家禽流行性感冒病毒血球凝集素以區別病毒血清亞型。碩士論文。國立中興大學獸醫微生物學研究所。台灣台中市。中華民國。
邱文鴻。2002。建立以競爭性酵素連結免疫吸附分析法快速診斷H5亞型高病原性家禽流行性感冒之技術。碩士論文。國立中興大學獸醫學研究所。台灣台中市。中華民國。
Adiar BM, Todd D, Mckillop ER, McMulty MS. Detection of influenza A type-specific antibodies in chicken and turkey sera by enzyme-linked immunosorbent assay. Avian Pathol 18: 455-463, 1989.
Alexander DJ. A review of avian influenza in different bird species. Veterinary microbiology 74: 3-13, 2000.
Alexander DJ. Avian influenza. In: Manual of diagnostic tests and vaccines for terrestrial animals, 5th ed. Office International des Epizooties, Paris, France. 2004.
Bai GR, Sakoda Y, Mweene AS, Fujii N, Minakawa H, Kida H. Improvement of a rapid diagnosis kit to detect either influenza A or B virus infections. J Vet Med Sci 68: 35-40, 2006.
Bai GR, Sakoda Y, Mweene AS, Kishida N, Yamada T, Minakawa H, Kida H. Evaluation of the ESPLINE® Influenza A&B-N Kit for the diagnosis of avian and swine influenza. Microbiol Immunol 49: 1063-7, 2005.
Beare AS, Webster RG. Replication of avian influenza viruses in humans. Arch Virol. 119: 37-42, 1991.
Beard CW. Demonstration of type-specific influenza antibody in mammalian and avian sera by immunodiffusion. Bull WHO 42: 779-785, 1970.
Branden C, Tooza J. Bata structures. In: Introduction to protein structure, 2nd ed. Garland Publishing, New York. P.67-88, 1999.
Brown EG. Influenza virus genetics. Biomed Pharmacother 54: 196-209, 2000.
Capua I, Terregino C, Cattoli G, Mutinelli F, Rodriguez JF. Development of a DIVA (differentiating infected from vaccinated animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza. Avian Pathol 32: 47-55, 2003.
Cattoli G, Drago A, Maniero S, Toffan A, Bertoli E, Fassina S, Terregino C, Robbi C, Vicenzoni G, Capua I. Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds. Avian pathol 33: 432-7, 2004.
Chin PS, Hoffmann E, Webby R, Webster RG, Guan Y, Peiris M, Shortridge KF. Molecular evolution of H6 influenza viruses from poultry in Southeastern China: prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry. J Virol 76: 507-516, 2002.
Claas ECJ. Pandemic influenza is a zoonosis, as it requires introduction of avian-like gene segments in human population. Vet Microbiol 74: 133-139, 2000.
Claas ECJ, Osterhaus ADME, Beek R, De Jong JC, Rimmelzwaan GF, Senne DA, Krauss S, Shortridge KF, Webster RG. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351:472-477, 1998.
De St Groth DR, Scheidegger D. Production of monoclonal antibodies: strategy and tactics. J Immuno Meth 35:1-21, 1980.
Downie JC, Laver WG. Isolation of a type A influenza virus from an Australian pelagic bird. Virology 51: 259-269, 1973.
Downie JC, Webster RG, Schild GC, Dowdle WR, Laver WG. Characterization and ecology of a type A influenza virus isolated from a shearwater. Bull WHO 49: 559-566, 1973.
Dunn JJ, Gordon C, Kelley C, Carroll KC. Comparison of the Denka-Seiken INFLU A．B-Quick and BD Directigen Flu A+B Kits with direct fluorescent-antibody staining and shell vial culture methods for rapid detection of influenza viruses. J Clin Microbiol 41: 2180-3, 2003.
Easterday BC, Hinshaw VS, Halvorson DA. Influenza. In: Calnek BW, Barnes HJ, Bear CW, McDougald LR, Saif YM (ed.), Disease of poultry. Iowa State University Press, Ames, 583-605, 1997.
Ellis TM, Bousfield RB, Bissett LA, Dyrting KC, Luk GSM, Tsim ST, Sturm-Ramirez K, Webster RG, Guan Y, Peiris JSM. Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002. Avian pathol 33: 492-505, 2004.
Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, Rimmelzwaan GF, Olsen B, and Osterhaus AD. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79: 2814–2822. 2005.
García-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, Durbin JE, Palese P, Muster T. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252: 324-330, 1998.
Gómez-Puertas P, Albo C, Pérez-Pastrana E, Vivo A, Portela A. Influenza virus matrix protein is the major driving force in virus budding. J Virol 74: 11538-11547, 2000.
González S, Ortín J. Characterization of influenza virus PB1 protein binding to viral RNA: two separate regions of the protein contribute to the interaction domain. J Virol 73: 631-637, 1999.
Guan Y, Shortridge KF, Krauss S, Webster RG. Molecular characterization of H9N2 influenza viruses: were they the donors of the “internal” genes of H5N1 viruses in Hong Kong? Proc Natl Acad Sci 96: 9363-9367, 1999.
Harlow E, David L. Antibodies: a laboratory manual. Cold spring harbor labatory. New York, USA.1988.
Harvery R, Martin CR, Zambon M, Barclay WS. Restrictions to the adaptation of influenza A virus H5 hemagglutinin to the human host. Journal of virology. 78: 502-507, 2004.
Hoffmann E, Stech J, Leneva I, Krauss S, Scholtissek C, Chin PS, Peiris M, Shortridge KF, Webster RG. Characterization of the influenza A virus gene pool in avian species in Southern China: was H6N1 a derivative or a precursor of H5N1? J Virol 74: 6309-6315, 2000.
Horimoto T, Kawaoka Y. Direct reverse transcriptase PCR to determine virulence potential of influenza A viruses in birds. J Clin Microbiol 33: 748-751, 1995.
Huang H, Dan H, Zhou Y, Yu Z, Fan H, Tong T, Jin M, Chen H. Different neutralization efficiency of neutralizing monoclonal antibodies against avian influenza H5N1 virus to virus strains from different host. Mol Immunol 44: 1052-1055, 2007.
Hulse-Post DJ, Sturm-Ramirez KM, Humberd J, Seiler P, Govorkova EA, Krauss S, Scholtissek C, Puthavathana P, Buranathai C, Nguyen TD, Long HT, Naipospos TSP, Chen H, Ellis TM, Guan Y, Peiris JSM, Webster RG. Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia. Proc Natl Acad Sci 102: 10682-10687, 2005
Ito T, Couceiro JN, Kelm S, Baum LG, Krauss S, Castrucci MR, Donatelli I, Kida H, Paulson JC, Webster RG, Kawaoka Y. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 72: 7367-7373, 1998.
Ito T, Kawaoka Y. Host-range barrier of influenza A viruses. Vet Microbiol 74: 71-75, 2000.
Kingsbury DW. Virology. Orthomyxoviridae and their replication. Raven Press. New York.1990.
Kishida N, Sakoda Y, Isoda N, Matsuda K, Eto M, Sunaga Y, Umemura T, Kida H. Pathogenicity of H5 influenza viruses for ducks. Arch Virol. 150: 1383-1392, 2005.
Klenk HD, Rott R, Orlich M, Blödorn J. Activation of influenza A viruses by trypsin treatment. Virology 68: 426-439, 1975.
Kodiballi S, Sivanandan V, Nagaraja KV, Goyal SM, Halvorson DA. Antigen-capture enzyme immunoassay for detection of avian influenza virus in turkeys. Am J Vet Res 54: 1385-1390, 1993.
Kuszewski K, Brydak L. The epidemiology and history of influenza. Biomed & Pharmcother. 54: 188-95, 2000.
Lamb RA, Krug RM. Orthomyxoviridae: The virus and their replication. In: B.N. Fields, et al. Fundamental virology, 3rd ed. Lippincott-Raven Publisher, Philadelphia, 605-639, 1996.
Lee HC, Hsu CN, Kuo TF, Wang CH. Molecular epidemiology of avian influenza virus H6N1 in Taiwan from 2000 to 2003. Taiwan Vet J 31: 230-239, 2005.
Lee MS, Chang PC, Shien JH, Cheng MC, Chen CL, Shieh HK. Genetic and pathogenic characterization of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005. Avian Dis 50: 561-571, 2006.
Lee MS, Chang PC, Shien JH, Cheng MC, Shieh HK. Identification and subtyping of avian influenza viruses by reverse transcription-polymerase chain reaction. J Virol Meth 97: 13-22, 2001.
Lee MS, Chang PC, Shien JH, Lee LH, Shieh HK. Identification and differentiation of avian influenza and Newcastle disease viruses by multiplex reverse transcription-polymerase chain reaction. J Chin Soc Vet Sci 25: 191-198, 1999.
Lin YP, Shaw M, Gregory V, Cameron K, Lim W, Klimov A, Subbarao K, Guan Y, Krauss S, Shortridge K, Webster R, Cox N, Hay A. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc Natl Acad Sci 97: 9654-9658, 2000.
Lu H. A longitudinal study of a novel dot-enzyme-linked immunosorbent assay for detection of avian influenza virus. Avian Dis 47: 361-9, 2003.
Lu YS, Sugimura T, Shien HK, Lee YL, Jong MH. Isolation and identification of an influenza A virus in ducks in Taiwan. J Chin Soc Vet Sci 11:23-34, 1985.
Luna S, Fortes P, Beloso A, Ortin J. Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J Virol 69: 2427–2433, 1995.
Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol 78: 12665-12667, 2004.
Munch M, Nielsen LP, Handberg KJ, JØrgrnsen PH. Detection and subtyping（H5 and H7）of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA. Arch Virol 146: 87-97, 2001.
Murphy BR, Webster RG. Virology. Orthomyxoviruses. Raven Press. New York. 1990.
Nakagawa Y, Kimura N, Toyoda T, Mizumoto K, Ishihama A, Oda K, Nakada S. The RNA polymerase PB2 subunit is not required for replication of the influenza virus genome but is involved in capped mRNA synthesis. J Virol 67: 728-733, 1995.
Nakagawa Y, Oda K, Nakada S. The PB1 subunit alone can catalyze cRNA synthesis and the PA subunit in addition to the PB1 subunit is required for viral RNA synthesis in replication of the influenza virus genome. J Virol 70: 6390-6394, 1996.
Neumann G, Zobel A, HobomＧ. RNA polymerase I-mediated expression of influenza viral RNA molecules. Virology 202: 477-479, 1994.
Nobusawa E, Aoyama T, Kato H, Suzuki Y, Tateno Y, Nakajima K. Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology 182: 465-485, 1991.
O''Neill RE, Talon J, Palese P. The influenza virus NEP(NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17: 288-298, 1998.
Peiris M, Yuen KY, Leung CW, Chan KH, Ip PLS, Lai RWM, Orr WK, Shortridge KF. Human infection with influenza H9N2. Lancet 354: 916-917, 1999.
Perdue ML, Swayne DE. Public health risk from avian influenza viruses. Avian Dis 49: 317-327, 2005.
Pereira HG, Tumova B, Law VG. Avian influenza A viruses. Bull WHO 32: 855-860, 1965.
Petric M, Comanor L, Petti CA. Role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics. J Infect Dis 194（Suppl 2）: S98-110, 2006.
Pinto LH, Holsinger LJ, Lamb RA. Influenza virus M2 protein has ion channel activity. Cell 69: 517-528, 1992.
Portela A, Digard P. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83: 723-734, 2002.
Potter CW. A history of influenza. J Appl Microbiol 91: 572-579, 2001.
Potter CW, Oxford JS. Determinants of immunity to influenza virus infection in man. Br Med Bull 35: 69-75, 1979.
Qui Y, Krug RM. The influenza virus NS1 protein is a poly(A)-binding-protein that inhibits nuclear export of mRNAs containing poly(A). J Virol 68:2425-2432, 1994.
Reina J, Fernandez-Baca V, Blanco I, Munar M. Comparison of Madin-Darby canine kidney cells（MDCK）with a green monkey continuous cell line（Vero）and human lung embryonated cells（MRC-5）in the isolation of influenza A virus from nasopharyngeal aspirates by shell viral culture. J Clin Microbiol 35: 1900-1901, 1997.
Ruigork RWH, Hewat EA, Wade RH. Low pH deforms the influenza virus envelope. J Gen Virol 73:995-998, 1992.
Saito T, Lim W, Suzuki T, Suzuki Y, Kida H, Nishimura SI, Tashiro M. Characterization of a human H9N2 influenza virus isolated in Hong Kong. Vaccine 20: 125-133, 2002.
Sala G, Cordioli P, Moreno-Martin A, Tollis M, Brocchi E, Piccirillo A, Lavazza A. ELISA test for the detection of influenza H7 antibodies in avian sera. Avian Dis 47: 1057-59, 2003.
Scholtissek C, Rohade W, Hoyningen V, Rott R. On the origin of the human influenza virus subtypes H2N2 and H3N2. Virology 87: 13-20, 1978.
Schultz-Cherry S, Dybdahl-Sissoko N, McGregor M, Hinshaw VS. Mink lung epithelial cells: unique cell line that supports influenza A and B virus replication. J Clin Microbiol 36:3718-3720, 1998.
Schweiger B, Zadow I, Heckler R, Timm H, Pauli G. Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. J Clin Microbiol 38: 1552-1558, 2000.
Shafer AL, Katz JB, Eernisse KA. Development and validation of competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera. Avian Dis 42: 28-34, 1998.
Shieh HK, Huang WJ, Shien JH, Chiu SY, Lee LF, Lu YS. Studies on avian influenza in Taiwan, R.O.C. (III) Isolaion, identification, and pathogencity tests of the virus isolated from breeding chicken. Taiwan J Vet Med Ani Husb 59:45-55, 1992.
Shortridge KF. Poultry and the influenza H5N1 outbreak in Hong Kong, 1997: Abridged chronology and virus isolation. Vaccine 17: 26-29, 1999.
Shu LL, Bean WJ, Webster RG. Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. J Virol 67: 2723-2729, 1993.
Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT, Suarez DL. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 40: 3256-3260, 2002.
Spackman E, Stallknecht DE, Slemons RD, Winker K, Suarez DL, Scott M, Swayne DE. Phylogenetic analyses of type A influenza genes in natural reservoir species in North America reveals genetic variation. Virus Res: 114: 89-100, 2005.
Stallknecht DE, Kearney MT, Shane SM, Zwank PJ. Effects of pH, temperature, and salinity on persistence of avian influenza viruses in water. Avian Dis 34:412-418, 1990.
Starick E, RÖmer-oberdÖrfer A, Werner O. Type- and subtype-specific RT-PCR assays for avian influenza A viruses (AIV). J Vet Med B 47: 295–301, 2000.
Steinhauer DA, Skehel JJ. Genetics of influenza viruses. Annu Rev Genet 36: 305-32, 2002.
Sturm-Ramirez KM, Ellis T, Bousfield B, Bissett L, Dyrting K, Rehg JE, Poon L, Guan Y, Peiris M, Webster RG. Reemerging H5N1 influenza viruses in Hong Kong in 2002 are highly pathogenic to ducks. J Virol 78: 4892-4901, 2004.
Suarez DL. Evolution of avian influenza viruses. Vet Microbiol 74: 15-27, 2000.
Suarez DL, Perdue ML, Cox N, Rowe T, Bender C, Huang J, Swayne DE. Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong. J Virol 72: 6678-6688, 1998.
Suarez DL, Schultz-Cherry S. Immunology of avian influenza virus: a review. Dev Comp Immunol 24: 269-283, 2000.
Subbarao K, Katz J. Avian influenza viruses infecting humans. Cell Mol Life Sci 57: 1770-1784, 2000.
Swayne DE, Suarez DL. Highly pathogenic avian influenza. Rev Sci Tech 19: 63-482, 2000.
Synder DB, Marquardt WW, Yancey FS, Savage PK. An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus. Avian Dis 29: 136-144, 1984.
Wang CW, Wang CH. Experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity H6N1 avian influenza virus field isolate. Avian Dis 47: 1416-1422, 2003.
Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev 56: 152-179, 1992.
Wester RG, Laver WG. The origin of pandemic influenza. Bull WHO. 47: 449-452, 1972.
Webster RG and Walker EJ. Influenza: The world is teetering on the edge of a pandemic that could kill a large fraction of the human population. American Scientist 91: 122-129, 2003.
Wiley DC, Skehel JJ. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Ann. Rev. Biochem. 56: 365-94, 1987.
Wiley DC, Wilson IA, Skehel JJ. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289: 373-378, 1981.
Woolcock PR, Cardona CJ. Commercial immunoassay kits for the detection of influenza virus type A: evaluation of their use with poultry. Avian Dis 49: 477-81, 2005.
Ye Z, Liu Y, Offringa DP, MCinnis J, Levandowsk RA. Association of influenza virus matrix protein with ribonucleoproteins. J Virol 73: 7467-7473, 1999.
Zambon MC. Epidemiology and pathogenesis of influenza. J Antimicrob Chemother 44 Suppl B: 3-9, 1999.
Zebedee SL, Lamb RA. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virus. J. Virol. 62: 2762-72, 1988.
Zhou EM, Chan M, Heckert RA, Riva J, Cantin MF. Evalution of a competitive ELISA for diction of antibodies against avian influenza virus nucleoprotein. Avian Dis 42: 517-522, 1998.
Zhou NN, Senne DA, Landgraf JS, Swenson SL, Erickson G, Rossow K, Liu L, Yoon KJ, Krauss S, Webster RG. Genetic reassortment of avian, swine, and human influenza A viruses in American pig. J Virol 73: 8851-8856, 1999.
Ziegler T, Hall H, Sánchez-Fauquier A, Gamble WC, Cox NJ. Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assy. J Clin Microbiol 33: 318-21, 1995.