臺灣博碩士論文加值系統

禽傳染性支氣管炎 ( infectious bronchitis；IB ) 是由冠狀病毒所造成之雞急性呼吸道及生殖道傳染病，造成家禽產業嚴重經濟損失。台灣近年來所流行的分離株在S1基因上與疫苗株相似度不高，這會影響市售疫苗的保護效力，因此新型的疫苗開發是刻不容緩的。本研究目的是開發新型傳染性支氣管炎之疫苗，已將實驗室分離之病毒進行基因定序，以S1的基因片段與NCBI上的標準株進行比對後，確認其基因型別，並利用無特定病原菌之雞胚進行病毒的增殖及連續繼代，目前繼代10代後所測得之力價落在106.0~106.5 EID50 / 0.1ml。毒力試驗中，以十倍劑量施打後並無不良反應，解剖觀察腎臟、氣管等器官，無組織病理學上之病變，且第3週之中和抗體力價高於檢定標準。另一方面以福馬林( Formalin )不活化之死毒疫苗，其第三週之血清抗體力價未達標準。爾後將以BEI ( Binary ethylenimine ) 當作不活化劑測試是否為不活化劑影響免疫效力。並且，我們將以活毒方式免疫，來評估病毒適合的疫苗類型。綜合目前試驗結果，活毒疫苗較具開發之可能。

Avian infectious bronchitis virus (IBV), a member of the Coronaviridae family, replicate primarily in the respiratory tract and oviduct. It has a major impact on the poultry industry worldwide and causes severe economic losses. IBV was first described in the 1930s in the USA. Multiple IBV serotypes or genotypes have been identified worldwide. Although various attenuated and inactivated vaccines have been widely used in chicken farms, their efficacy is poor, and IB disease outbreaks occur frequently in vaccinated chicken flocks. Therefore, developing of a novel IBV strain vaccine is necessary. In this study, the S1 gene of isolated IBV strains were sequenced, as well as sequence analysis of the isolated IBV strains confirm genotype IBV strains. IBV isolates were propagated in SPF chicken embryos, and virus titers were determined by EID50 and Real-time PCR. After the 10th passage, titers of the allantoic fluids ranged between 106.0and 106.5 EID50 / 0.1 ml. In virulence test, SPF chickens were inoculated with ten dose of virus at 3 days of age. After the SPF chicken inoculated, none of the clinical signs or death was observed, and the neutralization indexes of serum were greater than 2. On the other hand, the inactivated vaccine used formalin inactivation, the neutralization indexes of serum were lower than 1. In the near future, we will use BEI inactivate IBV, and testing inactivants whether affects antigenicity and immunogenicity. Also, we will inoculate live virus to evaluate the suitable type of vaccine for virus. The above analysis shows that, the development of live vaccine is more likely.

中文摘要 I
Abstract II
謝誌 IV
目錄 V
圖表目錄 VIII
第1章 緒言 1
第2章 文獻回顧 4
2.1 雞傳染性支氣管炎簡介 4
2.1.1歷史背景 4
2.1.2 IB流行病學 5
2.1.3預防及控制 6
2.2 IBV之分類 7
2.3 IB病毒的型態及物理化學特性、結構及功能 7
2.3.1病毒的型態及物理化學特性 7
2.3.2病毒的結構及功能 8
2.4 IB之病毒株分型 10
2.4.1 IB之基因型別鑑定方法 10
2.4.2 IBV之血清型分型方法 10
2.4.3 IBV之病毒中和試驗 11
2.5 IB之診斷方法 12
2.5.1 IBV之分離 12
2.5.2 IB之血清學診斷方法 12
2.6疫苗研究發展 13
第3章 材料方法 15
3.1雞傳染性支氣管炎病毒來源及分離 15
3.1.1病材及組織來源 15
3.1.2 IBV病毒株分離 15
3.2病毒之純化、增殖及力價測定 15
3.2.1 IB病毒純化 15
3.2.2 IB病毒增殖 16
3.2.3 IB病毒力價測定 16
3.3病毒之檢測及定量 17
3.3.1病毒核酸之萃取 17
3.3.2增幅IBV-S1基因之引子 17
3.3.3聚合酶鏈連鎖反應 (Polymerase Chain Reaction, PCR) 18
3.3.4膠體電泳分析 18
3.3.5 PCR產物純化(膠體回收) 19
3.3.6 PCR產物之分子選殖 19
3.3.7質體之純化 20
3.3.8核苷酸序列及胺基酸序列比較、親源關係分析 20
3.3.9 Real-Time PCR (qPCR ) 20
3.3.10重組DNA萃取及鑑定 21
3.3.11病毒定量 21
3.4死毒、活毒疫苗配製 21
3.4.1不活化病毒(BEI、Formalin) 21
3.4.2疫苗配製 22
3.4.3活毒疫苗製備 22
3.5疫苗免疫動物實驗 22
3.5.1試驗雞隻及實驗動物物種來源 22
3.5.2毒力測試 23
3.5.3免疫試驗及實驗動物組別設定 23
3.5.4攻毒試驗 24
3.6酵素免疫分析法（Enzyme-linked immunosorbent assay；ELISA） 24
3.7血清中和試驗 25
第4章 結果 26
4.1多重聚合酶鏈反應( Multiple PCR )野外病毒分離株之純潔度試驗 26
4.2 RT-PCR 26
4.3 IB病毒增殖 26
4.4序列比對及親緣關係分析 27
4.5 Multiplex PCR檢測各代QX-VM207病毒株之純潔度試驗 27
4.6 qPCR與蛋胚檢測病毒力價 27
4.7 IBV病毒力價( EID50 )與qPCR的定量曲線 28
4.8免疫血清之抗體力價分析 28
第5章討論 46
參考文獻 50
作者簡介 65

Adzhar, A., R. E. Gough, D. Haydon, K. Shaw, P. Britton and D. Cavanagh (1997). "Molecular analysis of the 793/B serotype of infectious bronchitis virus in Great Britain." Avian Pathol 26(3): 625-640.

Albassam, M. A., R. W. Winterfield and H. L. Thacker (1986). "Comparison of the nephropathogenicity of four strains of infectious bronchitis virus." Avian Dis 30(3): 468-476.

Alexander, D. J. and R. E. Gough (1977). "Isolation of avian infectious bronchitis virus from experimentally infected chickens." Res Vet Sci 23(3): 344-347.

Andrade, L. F., P. Villegas and O. J. Fletcher (1983). "Vaccination of day-old broilers against infectious bronchitis: effect of vaccine strain and route of administration." Avian Dis 27(1): 178-187.

Archetti, I. and F. L. Horsfall, Jr. (1950). "Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum." J Exp Med 92(5): 441-462.

Arvidson, Y., G. A. Tannock, M. Zerbes and J. Ignjatovic (1991). "Efficacy of Australian vaccines against recent isolates of avian infectious bronchitis viruses." Aust Vet J 68(6): 211-212.

Avellaneda, G. E., P. Villegas, M. W. Jackwood and D. J. King (1994). "In vivo evaluation of the pathogenicity of field isolates of infectious bronchitis virus." Avian Dis 38(3): 589-597.

Beaudette, F. and C. Hudson (1937). "Cultivation of the virus of infectious bronchitis. ." JAVMA 90: 51-60.

Bijlenga, G., J. K. A. Cook, J. J. Gelb and J. J. d. Wit (2004). "Development and use of the H strain of avian infectious bronchitis virus from the Netherlands as a vaccine: a review." Avian Pathology 33(6): 550-557.

Binns, M. M., M. E. Boursnell, D. Cavanagh, D. J. Pappin and T. D. Brown (1985). "Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV." J Gen Virol 66 ( Pt 4): 719-726.
Bochkov, Y. A., G. V. Batchenko, L. O. Shcherbakova, A. V. Borisov and V. V. Drygin (2006). "Molecular epizootiology of avian infectious bronchitis in Russia." Avian Pathol 35(5): 379-393.

Bosch, B. J., R. van der Zee, C. A. de Haan and P. J. Rottier (2003). "The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex." J Virol 77(16): 8801-8811.

Box, P. G., H. C. Holmes, P. M. Finney and R. Froymann (1988). "Infectious bronchitis in laying hens: the relationship between haemagglutination inhibition antibody levels and resistance to experimental challenge." Avian Pathol 17(2): 349-361.

Bumstead, N., M. B. Huggins and J. K. Cook (1989). "Genetic differences in susceptibility to a mixture of avian infectious bronchitis virus and Escherichia coli." Br Poult Sci 30(1): 39-48.

Capua, I., Z. Minta, E. Karpinska, K. Mawditt, P. Britton, D. Cavanagh and R. E. Gough (1999). "Co-circulation of four types of infectious bronchitis virus (793/B, 624/I, B1648 and Massachusetts)." Avian Pathology 28(6): 587-592.
Cavanagh, D. (1983). "Coronavirus IBV: structural characterization of the spike protein." J Gen Virol 64 ( Pt 12): 2577-2583.

Cavanagh, D. (2002). Coronaviruses and Toroviruses. Principles and Practice of Clinical Virology, John Wiley & Sons, Ltd: 345-356.

Cavanagh, D. (2003). "Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus." Avian Pathol 32(6): 567-582.

Cavanagh, D. (2005). Coronaviridae: a review of coronaviruses and toroviruses. Coronaviruses with Special Emphasis on First Insights Concerning SARS. A. Schmidt, O. Weber and M. H. Wolff. Basel, Birkhäuser Basel: 1-54.

Cavanagh, D. (2005). "Coronaviruses in poultry and other birds." Avian Pathol 34(6): 439-448.

Cavanagh, D. (2007). "Coronavirus avian infectious bronchitis virus." Vet Res 38(2): 281-297.

Cavanagh, D., J. H. Darbyshire, P. Davis and R. W. Peters (1984). "Induction of humoral neutralising and haemagglutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus." Avian Pathol 13(3): 573-583.

Cavanagh, D., P. J. Davis, J. K. Cook, D. Li, A. Kant and G. Koch (1992). "Location of the amino acid differences in the S1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus." Avian Pathol 21(1): 33-43.

Cavanagh, D., P. J. Davis and A. P. Mockett (1988). "Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes." Virus Res 11(2): 141-150.

Cavanagh, D., M. M. Elus and J. K. Cook (1997). "Relationship between sequence variation in the S1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo." Avian Pathol 26(1): 63-74.

Cavanagh, D. G., J. (2008). "Infectious bronchitis." 117-133.
Cook, J. K. and M. B. Huggins (1986). "Newly isolated serotypes of infectious bronchitis virus: their role in disease." Avian Pathol 15(1): 129-138.

Cook, J. K., S. J. Orbell, M. A. Woods and M. B. Huggins (1999). "Breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes." Avian Pathol 28(5): 477-485.

Cook, J. K., H. W. Smith and M. B. Huggins (1986). "Infectious bronchitis immunity: its study in chickens experimentally infected with mixtures of infectious bronchitis virus and Escherichia coli." J Gen Virol 67 ( Pt 7): 1427-1434.

Crinion, R. A. and M. S. Hofstad (1972). "Pathogenicity of two embryo-passage levels of avian infectious bronchitis virus for the oviduct of young chickens of various ages." Avian Dis 16(5): 967-973.

Cubillos, A., J. Ulloa, V. Cubillos and J. K. Cook (1991). "Characterisation of strains of infectious bronchitis virus isolated in Chile." Avian Pathol 20(1): 85-99.

Cumming, R. B. (1969). "The control of avian infectious bronchitis/nephrosis in Australia." Aust Vet J 45(4): 200-203.
Darbyshire, J. H., J. G. Rowell, J. K. Cook and R. W. Peters (1979). "Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures." Arch Virol 61(3): 227-238.

Davelaar, F. G. and B. Kouwenhoven (1977). "Influence of maternal antibodies on vaccination of chicks of different ages against infectious bronchitis." Avian Pathol 6(1): 41-50.

Davelaar, F. G. and B. Kouwenhoven (1980). "Vaccination of 1-day-old broilers against infectious bronchitis by eye drop application or coarse droplet spray and the effect of revaccination by spray." Avian Pathol 9(4): 499-510.
De Wit, J. J. (2000). "Detection of infectious bronchitis virus." Avian Pathol 29(2): 71-93.

de Wit, J. J., J. Nieuwenhuisen-van Wilgen, A. Hoogkamer, H. van de Sande, G. J. Zuidam and T. H. Fabri (2011). "Induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype D388 (genotype QX) by maternally derived antibodies and by early vaccination." Avian Pathol 40(5): 463-471.

Dolz, R., J. Pujols, G. Ordonez, R. Porta and N. Majo (2008). "Molecular epidemiology and evolution of avian infectious bronchitis virus in Spain over a fourteen-year period." Virology 374(1): 50-59.

Ducatez, M. F., A. M. Martin, A. A. Owoade, I. O. Olatoye, B. R. Alkali, I. Maikano, C. J. Snoeck, A. Sausy, P. Cordioli and C. P. Muller (2009). "Characterization of a new genotype and serotype of infectious bronchitis virus in Western Africa." J Gen Virol 90(Pt 11): 2679-2685.

el Houadfi, M. and R. C. Jones (1985). "Isolation of avian infectious bronchitis viruses in Morocco including an enterotropic variant." Vet Rec 116(16): 445.

Fabricant, J. (1998). "The early history of infectious bronchitis." Avian Dis 42(4): 648-650.

Fingerut, E., B. Gutter, G. Gallili, A. Michael and J. Pitcovski (2003). "A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein." Vaccine 21(21-22): 2761-2766.

Ganapathy, K., M. Wilkins, A. Forrester, S. Lemiere, T. Cserep, P. McMullin and R. C. Jones (2012). "QX-like infectious bronchitis virus isolated from cases of proventriculitis in commercial broilers in England." Vet Rec 171(23): 597.
Gelb, J., Jr. and S. S. Cloud (1983). "Effect of serial embryo passage of an Arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity." Avian Dis 27(3): 679-687.

Gelb, J., Jr., R. L. Lunt, A. L. Metz and P. A. Fries (1991). "Attenuation of avian infectious bronchitis virus by cold-adaptation." Avian Dis 35(4): 847-853.

Gelb, J., Jr., J. B. Wolff and C. A. Moran (1991). "Variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens." Avian Dis 35(1): 82-87.

Gonzalez, J. M., P. Gomez-Puertas, D. Cavanagh, A. E. Gorbalenya and L. Enjuanes (2003). "A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae." Arch Virol 148(11): 2207-2235.

Gough, R. E., W. J. Cox, C. E. Winkler, M. W. Sharp and D. Spackman (1996). "Isolation and identification of infectious bronchitis virus from pheasants." Vet Rec 138(9): 208-209.

Gough, R. E., C. J. Randall, M. Dagless, D. J. Alexander, W. J. Cox and D. Pearson (1992). "A 'new' strain of infectious bronchitis virus infecting domestic fowl in Great Britain." Vet Rec 130(22): 493-494.

Grattan, D. A. (1968). "Infectious bronchitis in laying fowls." Vet Rec 83(6): 157.

Guy, J. S. (2000). "Turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: A review." Avian Pathology 29(3): 207-212.

Handberg, K. J., O. L. Nielsen, M. W. Pedersen and P. H. Jorgensen (1999). "Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique." Avian Pathol 28(4): 327-335.

Hofstad, M. S. (1984). "Avian infectious bronchitis." IA: 429-443.

Hofstad, M. S. and H. W. Yoder, Jr. (1966). "Avian infectious bronchitis--virus distribution in tissues of chicks." Avian Dis 10(2): 230-239.

Hopkins, S. R. (1974). "Serological comparisons of strains of infectious bronchitis virus using plaque-purified isolants." Avian Dis 18(2): 231-239.
Huang, Y. P., W. C. Chang and C. H. Wang (2006) "Evaluation of an attenuated TW II infectious bronchitis vaccine." 123-128.

Huang, Y. P., H. C. Lee, M. C. Cheng and C. H. Wang (2004). "S1 and N gene analysis of avian infectious bronchitis viruses in Taiwan." Avian Dis 48(3): 581-589.

Huang, Y. P. and C. H. Wang (2006). "Development of attenuated vaccines from Taiwanese infectious bronchitis virus strains." Vaccine 24(6): 785-791.

Ignjatovic, E. J. and F. Ashton (1996). "Detection and differentiation of avian infectious bronchitis viruses using a monoclonal antibody-based." Avian Pathol 25(4): 721-736.

Ignjatovic, J. and L. Galli (1993). "Structural proteins of avian infectious bronchitis virus: role in immunity and protection." Adv Exp Med Biol 342: 449-453.

Ignjatovic, J. and S. Sapats (2000). "Avian infectious bronchitis virus." Rev Sci Tech 19(2): 493-508.

Ignjatovic, J., S. I. Sapats and F. Ashton (1997). "A long-term study of Australian infectious bronchitis viruses indicates a major antigenic change in recently isolated strains." Avian Pathol 26(3): 535-552.

Irvine, R. M., W. J. Cox, V. Ceeraz, S. M. Reid, R. J. Ellis, R. M. Jones, J. Errington, A. M. Wood, C. McVicar and M. I. Clark (2010). "Detection of IBV QX in commercial broiler flocks in the UK." Vet Rec 167(22): 877-879.
Jackwood, M. W. (2012). "Review of infectious bronchitis virus around the world." Avian Dis 56(4): 634-641.

Jackwood, M. W., H. M. Kwon and D. A. Hilt (1992). "Infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a DNA probe." Avian Dis 36(2): 403-409.

Johnson, R. B. and W. W. Marquardt (1975). "The neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures." Avian Dis 19(1): 82-90.

Kant, A., G. Koch, D. J. van Roozelaar, J. G. Kusters, F. A. Poelwijk and B. A. van der Zeijst (1992). "Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide." J Gen Virol 73 ( Pt 3): 591-596.

King, D. J. (1984). "Observations on the preparation and stability of infectious bronchitis virus hemagglutination antigen from virus propagated in chicken embryos and chicken kidney cell cultures." Avian Dis 28(2): 504-513.

Koch, G., L. Hartog, A. Kant and D. J. van Roozelaar (1990). "Antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions." J Gen Virol 71 ( Pt 9): 1929-1935.

Koegl, C., E. Wolf, N. Hanhoff, H. Jessen, K. Schewe, M. Rausch, J. Goelz, A. Goetzenich, H. Knechten, H. Jaeger, W. Becker, I. Becker-Boost, D. Berzow, B. Beiniek, J. Brust, D. Shcuster, S. Dupke, S. Fenske, H. J. Gellermann, R. Gippert, P. Hartmann, B. Hintsche, H. Jaeger, E. Jaegel-Guedes, H. Jessen, J. Golz, J. Koelzsch, E. B. Helm, G. Knecht, H. Knechten, I. Lochet, P. Gute, S. Mauruschat, S. Mauss, V. Miasnikov, F. A. Mosthaf, M. Rausch, M. Freiwald, B. Reuter, H. M. Schalk, B. Schappert, E. Schnaitmann, I. Schneider, W. Schuler-Maue, C. Schuler, T. Seidel, W. Starke, A. Ulmer, M. Muller, I. Weitner, K. Schewe, C. Zamani, A. Hanmond, K. Ross, A. Bottlaender, C. Hoffmann, A. Dix, A. Schneidewind, M. Lademann and D. A. G. S. G. Ac (2009). "Treatment during primary HIV infection does not lower viral set point but improves CD4 lymphocytes in an observational cohort." Eur J Med Res 14(7): 277-283.

Kusters, J. G., E. J. Jager, J. A. Lenstra, G. Koch, W. P. Posthumus, R. H. Meloen and B. A. van der Zeijst (1989). "Analysis of an immunodominant region of infectious bronchitis virus." J Immunol 143(8): 2692-2698.

Larghi, O. P. and A. E. Nebel (1980). "Rabies virus inactivation by binary ethylenimine: new method for inactivated vaccine production." J Clin Microbiol 11(2): 120-122.

Lai, M. M. and D. Cavanagh (1997). "The molecular biology of coronaviruses." Adv Virus Res 48: 1-100.

Lai, M. M. C. (2001). Coronaviridae: The Viruses and Their Replication.

Lambrechts, C., M. Pensaert and R. Ducatelle (1993). "Challenge experiments to evaluate cross-protection induced at the trachea and kidney level by vaccine strains and Belgian nephropathogenic isolates of avian infectious bronchitis virus." Avian Pathol 22(3): 577-590.

Li, D. and D. Cavanagh (1992). "Coronavirus IBV-induced membrane fusion occurs at near-neutral pH." Arch Virol 122(3-4): 307-316.

Lister, S. A., J. V. Beer, R. E. Gough, R. G. Holmes, J. M. Jones and R. G. Orton (1985). "Outbreaks of nephritis in pheasants (phasianus colchicus) with a possible coronavirus aetiology." Vet Rec 117(23): 612-613.

Liu, S. and X. Kong (2004). "A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in China." Avian Pathol 33(3): 321-327.

Liu, S., X. Zhang, Y. Wang, C. Li, Z. Han, Y. Shao, H. Li and X. Kong (2009). "Molecular characterization and pathogenicity of infectious bronchitis coronaviruses: complicated evolution and epidemiology in china caused by cocirculation of multiple types of infectious bronchitis coronaviruses." Intervirology 52(4): 223-234.

Lohr, J. E. (1976). "Serologic Differences between Strains of Infectious Bronchitis Virus from New Zealand, Australia, and the United States." Avian Diseases 20(3): 478-482.

Luo, H., J. Qin, F. Chen, Q. Xie, Y. Bi, Y. Cao and C. Xue (2012). "Phylogenetic analysis of the S1 glycoprotein gene of infectious bronchitis viruses isolated in China during 2009-2010." Virus Genes 44(1): 19-23.

M'Rini, C., G. Cheng, C. Schweitzer, L. L. Cavanagh, R. T. Palframan, T. R. Mempel, R. A. Warnock, J. B. Lowe, E. J. Quackenbush and U. H. von Andrian (2003). "A novel endothelial L-selectin ligand activity in lymph node medulla that is regulated by alpha(1,3)-fucosyltransferase-IV." J Exp Med 198(9): 1301-1312.

Macnaughton, M. R., H. J. Hasony, M. H. Madge and S. E. Reed (1981). "Antibody to virus components in volunteers experimentally infected with human coronavirus 229E group viruses." Infect Immun 31(3): 845-849.

Mondal, S. P. and S. A. Naqi (2001). "Maternal antibody to infectious bronchitis virus: its role in protection against infection and development of active immunity to vaccine." Vet Immunol Immunopathol 79(1-2): 31-40.

Moore, K. M., M. W. Jackwood and D. A. Hilt (1997). "Identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus." Arch Virol 142(11): 2249-2256.

Nakamura, K., J. K. Cook, K. Otsuki, M. B. Huggins and J. A. Frazier (1991). "Comparative study of respiratory lesions in two chicken lines of different susceptibility infected with infectious bronchitis virus: histology, ultrastructure and immunohistochemistry." Avian Pathol 20(2): 241-257.

Nix, W. A., D. S. Troeber, B. F. Kingham, C. L. Keeler, Jr. and J. Gelb, Jr. (2000). "Emergence of subtype strains of the Arkansas serotype of infectious bronchitis virus in Delmarva broiler chickens." Avian Dis 44(3): 568-581.

Pensaert, M. and C. Lambrechts (1994). "Vaccination of chickens against a Belgian nephropathogenic strain of infectious bronchitis virus B1648 using attenuated homologous and heterologous strains." Avian Pathol 23(4): 631-641.

Raj, G. D. and R. C. Jones (1997). "Infectious bronchitis virus: Immunopathogenesis of infection in the chicken." Avian Pathol 26(4): 677-706.

Reed, L. J. and H. Muench (1938). "A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS12." American Journal of Epidemiology 27(3): 493-497.

Siddell, S., H. Wege and V. Ter Meulen (1983). "The biology of coronaviruses." J Gen Virol 64 (Pt 4): 761-776.

Smith, H. W., J. K. Cook and Z. E. Parsell (1985). "The experimental infection of chickens with mixtures of infectious bronchitis virus and Escherichia coli." J Gen Virol 66 ( Pt 4): 777-786.

Song, C. S., Y. J. Lee, J. H. Kim, H. W. Sung, C. W. Lee, Y. Izumiya, T. Miyazawa, H. K. Jang and T. Mikami (1998). "Epidemiological classification of infectious bronchitis virus isolated in Korea between 1986 and 1997." Avian Pathol 27(4): 409-416.

Spaan, W., D. Cavanagh and M. C. Horzinek (1988). "Coronaviruses: structure and genome expression." J Gen Virol 69 ( Pt 12): 2939-2952.

Stern, D. F., L. Burgess and B. M. Sefton (1982). "Structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus." J Virol 42(1): 208-219.

Sturman, L. S., K. V. Holmes and J. Behnke (1980). "Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid." J Virol 33(1): 449-462.

Tamura, K., J. Dudley, M. Nei and S. Kumar (2007). "MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0." Mol Biol Evol 24(8): 1596-1599.

Terregino, C., A. Toffan, M. S. Beato, R. De Nardi, M. Vascellari, A. Meini, G. Ortali, M. Mancin and I. Capua (2008). "Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the Ma5 and 4/91 serotypes." Avian Pathol 37(5): 487-493.

Toro, H. (2010). "Infectious bronchitis virus: dominance of ArkDPI-type strains in the United States broiler industry during the last decade." Revista Brasileira de Ciência Avícola 12: 79-86.

Van Roekel, H., M. K. Clarke, K. L. Bullis, O. M. Olesiuk and F. G. Sperling (1951). "Infectious bronchitis." Am J Vet Res 12(43): 140-146.

Vandekerchove, D., P. D. Herdt, H. Laevens, P. Butaye, G. Meulemans and F. Pasmans (2004). "Significance of interactions between Escherichia coli and respiratory pathogens in layer hen flocks suffering from colibacillosis-associated mortality." Avian Pathol 33(3): 298-302.

Wadey, C. N. and J. T. Faragher (1981). "Australian infectious bronchitis viruses: identification of nine subtypes by a neutralisation test." Res Vet Sci 30(1): 70-74.

Wang, C. H., M. C. Hsieh and P. C. Chang (1996). "Isolation, pathogenicity, and H120 protection efficacy of infectious bronchitis viruses isolated in Taiwan." Avian Dis 40(3): 620-625.

Wang, C. H. and Y. C. Huang (2000). "Relationship between serotypes and genotypes based on the hypervariable region of the S1 gene of infectious bronchitis virus." Arch Virol 145(2): 291-300.

Wang, C. H. and C. T. Tsai (1996). "Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan." Arch Virol 141(9): 1677-1688.

Wang, L., D. Junker and E. W. Collisson (1993). "Evidence of Natural Recombination within the S1 Gens of Infectious Bronchitis Virus." Virology 192(2): 710-716.

Wang, Y., Y. Wang, Z. Zhang, G. Fan, Y. Jiang, E. Liu Xiang, J. Ding and S. Wang (1998). "Isolation and identification of glandular stomach type IBV (QX IBV) in chickens." Chinese Journal of Animal Quarantine 15(1): 1-3.

Weiss, S. R. and S. Navas-Martin (2005). "Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus." Microbiol Mol Biol Rev 69(4): 635-664.

Wunderwald, C. and R. K. Hoop (2002). "Serological monitoring of 40 Swiss fancy breed poultry flocks." Avian Pathol 31(2): 157-162.

Yan, F., Y. Zhao, W. Yue, J. Yao, L. Lihua, W. Ji, X. Li, F. Liu and Q. Wu (2011). "Phylogenetic analysis of S1 gene of infectious bronchitis virus isolates from China." Avian Dis 55(3): 451-458.