傳染性華氏囊炎（infectious bursal disease virus; IBDV）之VP2 基因高度變異區（hvVP2）之氨基酸變異常可導致疫苗免疫之失效，為探究IBDV-hvVP2基因之分子生物學演化，將本實驗室於2003～2005年間由台灣分離得之7株高致死率IBDV株進行其核苷酸之擴增和序列定序，以進行其親緣性的分析。結果7株IBDV- hvVP2 基因之核苷酸序列之相似度在97.4~100％間，而氨基酸序列之相似度在98.6~100％間，顯見台灣分離之7株IBDV之hvVP2之氨基酸變異度不大，且於其激發中和抗體之兩親水區內並無變異情形，目前在台灣之正確使用IBD活毒疫苗尚可提供雞隻良好之保護。

60Co所產生之γ射線可殺滅病毒，但γ射線是否會對IBDV之抗原性產生影響，尚不得而知。為瞭解γ射線照射對IBDV之免疫原性影響，將以IBDV¬ V263-TW02株製成之不活化抗原分為兩組分別進行0或10 kGy之γ射線照射後製成疫苗中抗原含量為8、4、2、1、0.5％ 之鋁膠疫苗後免疫於三週齡無特定病原雞（SPF）(0.5mL/隻)，結果發現以0.5％之抗原含量作為兩組比較之標準時，10 kGyγ射線照射後之IBDV抗原所製成之鋁膠疫苗較未經γ射線照射IBDV抗原多提供40%之免疫保護力，γ射線或可提升IBDV抗原之免疫原性。

以本實驗室先前已構築完成之桿狀病毒表現系統所表現之IBDV¬V263-TW02株之VP2全長次單位蛋白（V263/TW02-VP2-A3）製成鋁膠疫苗，並以V263/TW02株之不活化IBDV抗原製成之鋁膠疫苗分別免疫一組（每隻1 或0.5mL之含1% 細胞）三週齡SPF雞隻，結果發現以0.5％之抗原含量作為兩組比較之標準時，以V263/TW02-VP2-A3製成之次單位疫苗較V263/TW02株之傳統不活化疫苗多提供40%之免疫保護力，且於免疫後兩週之抗體力價顯較傳統不活化疫苗為佳。IBDV-VP2-A3或可製成具良好免疫保護力之次單位疫苗。

The amino acid residue changes in VP2 hypervariable region (hvVP2) of infectious bursal disease virus (IBDV) might cause some fails in IBDV vaccination. In order to determination of the molecular characterization of gene evolution we used 7 of highly virulent Taiwanese isolates obtained during 2002 to 2005 for amplification of their hvVP2 by reverse transcription-polymerase chin reaction (RT-PCR) then sequencing for phylogenetic analysis. The similarity on IBDV-hvVP2 nucleotide was 97.4~100％ and the deduced aa residues was 98.6~100％. The aa residue changes on 7 strains of IBDV-hvVP2 was mild. None aa residue change was found in the 2 major hydrophilic regions of neutralizing antibody binding sites. Correct live IBDV vaccination program in Taiwan provided a good clinical protectivity in chickens .

60Co γ-ray irradiation has long been applied in virus inactivation. The immunogenicity of viral protein affected by γ-ray still has not been well clarified. In order to know the immunogenicity of formalin-inactivated IBDV affected by γ-ray irradiation, the aluminum hydroxide (Al(OH)3) vaccine prepared from the 0 or 10 kGyγ-ray irradiated IBDV antigen (IBDV¬ V263-TW02 strain) at 8, 4, 2, 1 or 0.5% cellular content was individually used for immunized a group of 3-week-old specific pathogen free (SPF) chicken (0.5mL/bird). At 0.5% cellular content level, vaccine made up of the 10 kGyγ-ray irraditated IBDV antigen was found providing less 40% mortality in the group of chickens than the 0 kGyγ-ray irraditated IBDV did. γ-ray irraditation might provide some improvement on the immunogenicity of IBDV antigen.

The full length of V263/TW02 strain of IBDV VP2 subunit protein （V263/TW02-VP2-A3）expressed by Baculovirus expression system constructed earlier by this laboratory and a IBDV inactivated antigen prepared from V263/TW02 strain were used to prepare the Al(OH)3 type vaccine for immunizing a group of SPF chickens with 1 or 0.5 mL (1% cells) per bird. At 0.5 mL dosage level V263/TW02-VP2-A3 subunit vaccine was found with 40% better protectivity and higher antibody response at 14 days post vaccination than the V263/TW02 inactivated antigen did. V263/TW02- VP2-A3 provides some potential for making IBDV subunit vaccine.

目 錄
中文摘要.................................................I
英文摘要.................................................III
謝誌.....................................................V
目錄.....................................................VI
圖表目錄..................................................IX
第1章 前言..................................................1
第2章 文獻回顧..............................................4
2.1傳染性華氏囊病（IBD）...................................4
2.1.1歷史背景.............................................4
2.1.2流行病學.............................................5
2.1.3感染症狀與診斷.......................................6
2.2傳染性華氏囊病毒（IBDV）之特性...........................7
2.2.1IBDV病毒結構及理化特性................................7
2.2.2IBDV之核苷酸及其蛋白質特性............................7
2.2.3IBDV之分子流行病學...................................9
2.2.4IBDV病毒株的抗原性..................................11
2.2.5IBDV的分子診斷學....................................11
2.2.6IBDV病毒株的毒力標誌.................................12
2.3IBDV的致病機制........................................13
2.4vvIBDV之預防與控制....................................14
2.5桿狀病毒表現系統（baculovirus expression system）......15
2.6γ射線................................................16
2.6.1輻射...............................................16
2.6.2輻射作用於微生物之直接反應............................17
2.6.3輻射作用於微生物之間接反應............................17
2.6.4輻射滅菌法..........................................18
第3章 材料與方法...........................................19
3.1IBDV hvVP2基因之親緣性分析.............................19
3.1.1供親源性分析用之IBDV hvVP2基因來源....................19
3.1.2病毒之增殖..........................................19
3.1.3IBDV之純化.........................................20
3.1.4病毒RNA之抽取.......................................20
3.1.5引子之設計..........................................21
3.1.6反轉錄--聚合酶連鎖反應（RT-PCR）......................21
3.1.7PCR產物之純化、選殖與定序............................21
3.2IBDV抗原經γ射線照射後之免疫原性及以桿狀病毒表現系統表之次單位 疫苗之免疫保護力比較...............................23
3.2.1試驗雞隻...........................................23
3.2.2試驗用不活化抗原之製備...............................23
3.2.2.1不活化抗原之無菌試驗...............................23
3.2.2.2不活化抗原之確認...................................24
3.2.3次單位蛋白之製備.....................................24
3.2.4不活化抗原之γ射線照射................................24
3.2.5自製IBD不活化疫苗及次單位疫苗.........................24
3.2.6自製疫苗之評估......................................24
3.2.6安全試驗............................................24
3.2.6.2 效力試驗.........................................25
3.2.6.3 EID50測定.......................................26
3.2.6.4 ELISA抗體檢測....................................26
3.2.6.5 華氏囊／體重比值（BBR）之測定......................26
3.2.6.6 華氏囊之肉眼及組織病變分級（BGL and BGL）..........27
第4章 結果.................................................28
4.1IBDV hvVP2基因選殖定序及其親源關係研究..................28
4.1.1七株野外分離IBDV之RT-PCR產物之選殖與定序..............28
4.1.2IBDV hvVP2之親源關係分析............................28
4.1.3IBDV hvVP2 核苷酸與氨基酸序列比較分析.................29
4.2自製疫苗之評估........................................30
4.2.1V263- TW02 bursatin經γ射線照射後之免疫原性影響評估....30
4.2.2V263 bursatin不活化疫苗與次單位疫苗之免疫保護力........31
第5章 討論................................................48
5.1 IBDV hvVP2基因選殖定序及其親源關係研究..............48
5.2 自製疫苗之評估....................................50
5.2.1 V263 bursatin經γ射線照射後之免疫原性影響評估......50
5.2.2 V263 bursatin不活化疫苗與次單位疫苗之免疫保護力....52
參考文獻...................................................55
中、英文對照縮寫............................................65
附錄一 Map of yT&A cloning vector（Yeastern Biotech）.....66
附錄二 The virion and genome of Birnaviridae..............67
附錄三 D10 values of vegetative cells of some foodborne bacteria in non frozen high moisture foods................68
附錄四 Summary of previously reported D10 values..........70
附錄五 主要溶液配製........................................72
作者簡介...................................................76

參考文獻

許郁停。2003。以傳統方法和桿狀病毒表現VP2次單位傳染性華氏囊病毒（IBDV）蛋白製作IBDV不活化疫苗抗原在雞隻免疫保護力比較。碩士論文，國立屏東科技大學生物科技研究所。

蔡向榮，呂榮修。1993。1992年台灣雞傳染性華氏囊病大流行之疫情分析。中華獸醫誌，19: 249-258。

蔡明成。2002。市售傳染性華氏囊病疫苗病毒株之免疫原性及其分子生物學研究。碩士論文，國立屏東科技大學獸醫學研究所。

Abdel-Alim GA, Saif YM. Immunogenicity and antigenicity of very virulent strains of infectious bursal disease viruses. Avian Diseases 45: 92-101. 2001.

Almedia JD, Morris R. Antigenically-related viruses associated with infectious bursal disease. Journal of General Virology 20: 369-375. 1973.

Augustine PC, Danforth HD, Jenkins MC. Avian Eimeria: effects of gamma irradiation on development of cross-species immunity in foreign and natural host birds. Avian Disease 37(2): 349-57. 1993.

Banda A, Villegas P, El-Attrache J, Estevez C. Molecular characterization of seven field isolates of infectious bursal disease virus obtained from commercial broiler chickens. Avian Diseases 45: 620-630. 2001.

Barnes HJ, Wheeler J, Reed D. Serological evidence of infectious bursal disease virus infection in lowa turkeys. Avian Diseases 26: 560-565. 1982.

Benton, W. J., Cover, M. S., Rosenberger, J. K., Lake, R. S. Physicolchemical properties of the infectious bursal agent ( IBA) of chicken. Avian Diseases 11: 438-445. 1967.
Brian DR. The sterways process: a new approach to inactivating viruses using gamma radiation. Biologicals 26: 125-130. 1998.

Brown F. The classification and nomenclature of viruses: summary of results of the meeting so the International Committee on Taxonomy of Viruses in Sendai, September, 1984. Intervirology 25: 141-143. 1986.

Bumstead M, Reece RL, Cook JK. Genetic differences in susceptibility of chicken lines to infection with infectious bursal disease virus. Poultry Science 72: 403-410. 1993.

Byun MW, Lee JW, Seo JH, Kim JH, Jo CR, Kim DH, Chung HW. Changes of the immune reactivities of antibodies produced against gamma-irradiated antigen. Radiation physics and chemistry 71: 125-127. 2004.

Cao YC, Yeung WS, Law M, Bi YZ, Leung FC, Lim BL. Molecular characterization of seven Chinese isolates of infectious bursal disease virus: classical, very virulent, and variant strains. Avian Diseases 42: 340-351. 1998.

Chevill NF. Studies on the pathologenesis of Gumboro disease in bursa of Fabricius, spleen, and thymus of chicken. American Journal of Pathology 51: 527-551.1967.

Cho Y, Edgar SA. Characterization of the infectious bursal disease. Poultry Science 51: 60-69. 1972.

Cloud SS, Lillehoj HS, Rosenberger JK. Immune dysfunction following infection with chicken anaemia virus and infectious bursal disease virus. I. Kinetic alterations of avian lymphocytes subpopulations. Veterinary Immunology and Immunopathology 34: 337-352.1992.

Di Fabio J, Rossini LI, Eterradossi N, Toquin D, Gardin Y. European-like pathogenic infectious bursal disease viruses in Brazil. Veterinary Record 145: 203-204. 1999.

Dong H, Jiao X, Pan Z, Yin Y, Sun L, Liu S. Immunogenicity comparision of Listeria monocytogenes inactivated by gamma-irradiation or traditional treatments. Wei Sheng Wu Xue Bao 49(2):269-73. 2009.

Dubey JP, Jenkins MC, Thayer DW, Kwok OC, Shen SK. Killing of Toxoplasma gondii oocysts by irradiation and protective immunity induced by vaccination with irradiated oocysts. Journal of Parasitology 82(5): 724-7. 1996.

Dybing JK and Jackwood DJ. Antigenic and immunogenic properties of baculovirus-expressed infectious bursal disease viral proteins. Avian Diseases 42: 80-91. 1998.

Elankumaran S, Heckert RA, Moura L. Pathogenesis and tissue distribution of a variant strain of infectious bursal disease virus in commercial broiler chickens. Avian Diseases 46: 169-176. 2002.

Eterradossi N, Picault JP, Drouin P, Guittet M, Hospitalier R, Bennejean G. Pathogenicity and preliminary antigenic characterization of six infectious bursal disease virus strains isolated in France from acute outbreaks. Journal of
Veterinary Medicine 39: 683-691. 1992.

Eterradossi N, Rivallan G, Toquin D, Guittet M. Limited antigenic variation among recent infectious bursal disease virus isolates from France. Archives of Virology 142: 2079-2087. 1997b.

Eterradossi N, Toquin D, Rivallan G, Guittet M. Modified activity of a VP2-located neutralizing epitope on various vaccine, pathogenic and hypervirulent strains of infectious bursal disease virus. Archives of Virology 142: 255-270. 1997a.

Fahey KJ, Erny K, Crooks J. A conformational immunogen on VP2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chickens. Journal of General Virology 70: 1473-1481. 1989.

Giambrone JJ, Dormitorio T, Brown T. Safety and efficacy of in ovo administration of infectious bursal disease viral vaccines. Avian Diseases 45: 144-148. 2001.

Habib M., Hussain I, Fang WH, Rajput ZI, Yang ZZ, Irshad H. Inactivation of infectious bursal disease virus by binary ethylenimine and formalin. Journal of Zhejiang University SCIENCE B 7(4): 320-323. 2006.

Hassan MK, Nielsen CK, Ward LA, Jackwood DJ, Saif YM. Antigenicity, pathogenicity and immunopgenicity of small and large plaque infectious bursal disease virus clones. Avian Diseases, 40, 832-836. 1996.

He CQ, Ma LY, Wang D, Li GR, Ding NZ. Homologous recombination is apparent in infectious bursal disease virus. Virology 384: 51–58.2009.

Hirai K, Calnek BW. In vitro replication of infectious bursal disease virus in established lymphoid cell lines and chicken B lymphocytes. Infection and Immunity 25: 964-970. 1979.

Hirai K, Shimakura S. Structure of infectious bursal disease virus. Journal of Virology 14: 957-964. 1974.

Hon CC, Lam TY, Drummond A, Rambaut A, Lee YF, Yip CW, Zeng
F, Lam PY, Ng PT, Leung FC. Phylogenetic analysis reveals a correlation between the expansion of very virulent infectious bursal disease virus and reassortment of its genome segment B. Journal of Virology 80 (17), 8503–8509. 2006.

Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, Hasebe F, Morita K. Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. Journal of Clinical Microbiology 42(5):1956-61. 2004.

Hudson PJ, McKern NM, Power BE, Azad AA. Genomic structure of the large RNA segment of infectious bursal disease virus. Nucleic Acid Research 14: 5001-5012. 1986.

Inoue M, Yamamoto H, Matuo K, Hihara H. Susceptibility of chicken monocytic cell lines to infectious bursal disease virus. Journal of Veterinary and medical Science 54: 575-577. 1992.

Jackwood DH, Saif YM. Antigenic diversity of infectious bursal disease viruses. Avian Diseases 31: 766-770. 1987.
Jackwood DJ, Byerley EH, Sommer SE. Use of a genetic marker for wild-type potentially pathogenic infectious bursal disease viruses. Avian Diseases 45: 701-705. 2001.

Jackwood DJ, Sommer-Wagner SE, Pharo HJ. The use of gamma irradiation for the inactivation of infectious bursal disease viruses. Avian Disease. 51: 606-8. 2007.

Kataria RS, Tiwari AK, Butchaiah G, Kataria JM, Skinner MA. Sequence analysis of the VP2 gene hypervariable region of infectious bursal disease viruses from India. Avian Pathology 30: 501-507. 2001.

Kibenge FSB, Dhillon AS, Russel RG. Biochemistry and immunology of infectious bursal disease virus. Journal of General Virology 69: 1757-1775. 1988.

Kibenge FSB, Jackwood J, Mercado CC. Nucleotide sequence analysis of genome segment A of infectious bursal disease virus. Journal of General Virology 71: 569-577. 1990.

Lam KM. Alteration of chicken heterophil and macrophage function by the infectious bursal disease virus. Microbiological Pahtogenesis 25: 147-155. 1998.

Lana DP, Beisel CE, Silva RF. Genetic mechamisms of antigenitc variation in infectious bursal disease virus: analysis of a naturally occurring variant virus. Virus Genes 3: 247-259. 1992.

Le Nouen C, Rivallan G, Toquin D, Darlu P, Morin Y, Beven V, de Boisseson C, Cazaban C, Comte S, Gardin Y, Eterradossi N. Very virulent infectious bursal disease virus: reduced pathogenicity in a rare natural segment-B-reassorted isolate. J. Gen. Virol. 87 (Pt 1), 209-216.2006.

Lee JW, Yook HS, Lee KH, Kim JH, Kim WJ, Byun MW. Conformational changes of myosin by gamma irradiation. Radiation physics and chemistry 58: 271-277, 2000.

Lee LH, Lukert PD. 1986. Adaptation and antigenic variation of infectious bursal disease virus. Journal of the Chinese Society of Veterinary Science 12: 297-304.

Lin Z, Kato A, Otaki Y, Nakamura T, Sasmaz E, Ueda S. Sequence comparison of a highly virulent infectious bursal disease virus prevalent in Japan. Avian Diseases 37: 315-323. 1993.

Liu HJ, Giambrone JJ, Dormitorio T. Detection of genetic variations in serotype I isolates of infectious bursal disease virus using polymerase chain reaction and restriction endonuclease analysis. Journal of Virology Methods 48: 281-291. 1994.

Liu HJ, Huang PH, Wu YH, Lin MY, Liao MH. Molecular characterization of very virulent infectious bursal disease viruses in Taiwan. Research in Veterinary Science 70: 139-147. 2001.

Lombardo E, Maraver A, Casten JR, Rivera J, Ferandez-Arias A, Serrano A, Carrascosa, JLN, Rodriguez JF. VP1, the putative RNA-dependent RNA polymerase of infectious bursal disease virus, forms complexes with the capsid protein VP3, leading to efficient encapsidation into virus-like particles. Journal of Virology 73: 6973-6983. 1999.

Liu Y, Wei YG, Wu XF, Yu L. Preparation of ChIL-2 and IBDV VP2 fusion protein by baculovirus expression system. Chinese Society of Immunology 2(3): 231-235. 2005.

Lowy RJ, Vavrina GA, LaBarre DD. Comparison of gamma and neutron radiation inactivation of influenza A virus. Antiviral Research 52(3) :261-73. 2001

Lu YL, Shieh HK. Infectious bursal disease in Taiwan. Journal of the Chinese Society of Veterinary Science 9: 61-66. 1983.

May JC, Rey L, Lee CJ. Evaluation of selected vaccines and other biological products irradiated by gamma irradiation, electron beams and X-rays. Radiation physics and chemistry 63: 709-711. 2002.

Matloubi H, Afaki F, Hadjiezadegan M. Effect of γ-irradiation on amino acids content of baby food proteins. Journal of food composition and analysis 17:133-139. 2004.

Meir R, Jackwood DJ, Weisman Y. Molecular typing of infectious bursal disease virus of Israeli field and vaccine strains by the reverse transcription/polymerase chain reaction /restriction fragment length polymorphism assay. Avian Diseases 45: 223-228. 2001.

Michelle MC, Giambrone JJ, Dormitorio TV. Detection of infectious bursal disease vaccine virus in lymphoid tissues after in ovo vaccination of specific-pathogen-free embryos. Avian Diseases 45: 897-905. 2001.

Muller H, Nischke R. The two segments of the infectious brusal disese virus genome are circularized by a 90,000-Da protein. Virology 159: 174-177. 1987.

Mundt E. Tissue culture infectivity of different strains of infectious bursal disease virus ins determined by distinct amino acids in VP2. Journal of General Virology 80: 2067-2076. 1999.

Okoye JOA, Uzoukwu M. An outbreak of infectious bursal disease among chickens between 16 and 20 weeks old. Avian Diseases 25: 1034-1038. 1981.

Pitcovski J, Goldberg D, Levi BZ, Di-Castro D, Azriel A, Krispel S, Maray T, Sahaaltiel Y. Coding region of segment A seaquence of a very virulent isolate of IBDV- comparison with isolates from different countries and virulence. Avian Diseases 42: 497-506. 1998.

Pitcovski J, Goldberg, Di-Castro D, Azriel AB, Gutter S, Krispel S, Maray T, Sahaaltiel Y. Insect cell-derived VP2 of infectious bursal virus confers protection against the disease in chickens. Avian Diseases 40: 753-761. 1996.

Plotkin SA. Vaccines : past , present and future. Nature Medicine 11(4) :S5- 11. 2005.

Ramamoorthy S, Lindsay DS, Schurig GG, Boyle SM, Duncan RB, Vemulapalli R, Sriranganathan N. Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum. The Journal of eukaryotic microbiology 53(2):151-6. 2006.

Saif YM. Immunosuppression induced by infectious bursal disease virus. Veterinary Immunology and Immunopathology 30: 45-50. 1991.

Shi LX, Li HB, Ma GP, Zhou JY, Hong LI, Zheng XJ, Wu YP, Wang YZ, Yan Y. Competitive replication of different genotypes of infectious bursal
disease virus on chicken embryo fibroblasts. Virus Genes. 2008.

Smiley JR, Jackwood DJ. Genetic stability of the VP2 hypervariable region of four infectious bursal disease virus isolates after serial passage in specific-pathogen-free chicken embryos. Avian Diseases 45: 1-8. 2001.

Synder DB, Lana DP, Cho BR, Marquardt WW. Group and strain-specific neutralization sites of infectious bursal disease viruses defined with monoclonal antibodies. Avian Diseases 32: 527-534. 1988a.

Tano Y, Shimizu H., Martin J, Nishimura Y, Simizu B, Miyamura T.Antigenic characterization of a formalin-inactivated poliovirus vaccine derived fromlive-attenuated Sabin strains. Vaccine 25 (2007) 7041–7046

Toroghi R, Kataria JM, Verma KC, Kataria RS, Tiwari AK. Amino acid changes in the variable region of VP2 in three infectious bursal disease viruses with different virulence, originating from a common ancestor. Avian Pathology 30: 667-673. 2001.

Tsai HJ, Saif YM. Effect of cell-culture passage on the pathogenicity and immunogenicity of two variant strains of infectious bursal disease virus. Avian Diseases 36: 415-422. 1992.

Vakharia VN, He J, Ahamed B, Snyder DB.. Molecular basis of antigenic variation in IBDV. Virus Research 31: 265-273. 1994b

Vakharia VN, Synder DB, Lutticken D, Mengel-Whereat SA, Savage PK, Edwards GH, Goodwin MA. Active and passive protection against variant and classic infectious bursal disease virus strains induced by baculovirus-expressed structural proteins. Vaccine 123: 452-456. 1994a.

van den Berg TP, Gonze M, Meulemans G. Acute infectious bursal disease in poultry: isolation and characterisation of a highly virulent strain. Avian Pathology 20: 133-143. 1991.

van den Berg TP, Gonze M, Morales D, Meulemans G. Acute infectious bursal disease in poultry: immunological and molecular basis of antigenicity of a highly virulent strain. Avian Pathology 25: 751-768. 1996.

van den Berg TP, Meulemans G. Acute infectious bursal disease in poultry: protection afforded by maternally derived antibodies and interference with live vaccination. Avian Pathology 20: 409-421. 1991.

van der Marel P, Snyder D, Lutticken D. Antigenic characterization of IBDV field isolates by their reactivity with a panel of monoclonal antibodies. Deutsch Tierarzil Wochenschrift 97: 81-83. 1990.

WHO. High-dose irradiation: wholesomeness of food irradiated with doses above 10 kGy, report of a joint FAO/IAEA/WHO study group. WHO technical Report Series 890, World Health Organization, Geneva. 1999.

Wu CC, Rubinelli AP, Lin TL. Molecular detection and differentiation of infectious bursal disease virus. Avian Diseases 51: 515-526. 2007.

Xu J, Zhang Z, Yin Y, Cui S, Xu S, Guo Y, Li J, Wang J, Liu X, Han L. Development of reverse-transcription loop-mediated isothermal amplification for the detection of infectious bursal disease virus. Journal of Virology Methods. 2009

Yamaguchi T, Setiyono A, Kobayashi M, Takigami S, Fukushi H, Hirai K. Infectious bursal disease live vaccines: changes in the virus population during serial passage in chickens and embryo fibroblast cell. Avian Diseases 44: 284-290. 2000.

Yamaguchi T, Wgawa M, Inoshima Y, Miyoshi M, Fukushi H, Hirai K. Identification of sequence changes responsible for the attenuation of highly virulent infectious bursal disease virus. Virology 223: 219-223. 1996b.

Yao K, Vahkaria VN. Induction of apoptosis in vitro by the 17-kDa nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis. Virology 285: 50-58. 2001.

Yu LJR. Li YW. Huang J, Dikki RD. Molecular characteristics of full-length genomic segment A of three infectious bursal disease viruses in China: two attenuated strains and one virulent field strain. Avian Disease 45: 862-874. 2001.

Zierenberg K, Nieper H, van den Berg TP, Ezeokoli CD, Voss M, Muller H. The VP2 variable region of two German and 11 African isolates of infectious bursal disease viruses ( IBDVs ): comparison of the amino acid sequences with very virulent, “classical” virulent and attenuated tissue culture-adapted strains. Archives of Virology 145: 113-125. 2000.