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研究生: 歐庭吟
Ou, Ting-Yin
論文名稱: 鸚鵡多瘤病毒次單位疫苗之開發
Development of Subunit Vaccine Against Avian Polyomavirus in Parrots
指導教授: 柯冠銘
Ke, Guan-Ming
學位類別: 碩士
Master
系所名稱: 獸醫學院 - 動物疫苗科技研究所
Graduate Institute of Animal Vaccine Technology
畢業學年度: 108
語文別: 中文
論文頁數: 58
中文關鍵詞: 鸚鵡多瘤病毒鸚鵡桿狀病毒表現系統次單位疫苗
外文關鍵詞: avian polyomavirus, parrot, baculovirus expression system, subunit vaccine
DOI URL: http://doi.org/10.6346/NPUST202000216
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  • 鸚鵡多瘤病毒(Avian Polyomavirus;APV)感染症在鸚鵡科鳥類中為常見的病毒性疾病。受APV感染之鸚鵡科鳥類可能出現厭食、精神不濟、羽毛掉落、皮下出血等症狀,甚至死亡。所有年齡之鸚鵡科鳥類皆可受感染,而受感染之個體年齡越小死亡率越高,屬於高傳染性病毒疾病。鸚鵡多瘤病毒歸類於乳多瘤病毒科(Papovaviridae),多瘤病毒屬(Polymavirus)之一員,為不具封套之二十面對稱體、環狀雙股DNA病毒。鸚鵡多瘤病毒具有四種結構蛋白VP1、VP2、VP3及VP4,其中VP1為病毒主要外殼蛋白,具有誘導中和抗體的抗原決定位。隨著台灣飼養鸚鵡的數量增加,加上高密度的飼養環境,病毒的擴散速度也日益提升,但市面上僅在美國地區有販售死毒APV疫苗,台灣目前仍無相關疫苗能用於預防。此外死毒APV疫苗陸續被報導出注射部位出現肌肉壞死的情形,經研究後認為此情形與死毒疫苗搭配使用之油質佐劑有關。因此本研究目的旨在製備有效之APV次單位疫苗,作為鸚鵡疾病預防之用。目前已成功轉殖VP1基因至pBacPAK8中,使用昆蟲桿狀病毒表達系統生產重組pBacPAK8_APV VP1蛋白,搭配O/W劑型之佐劑製成APV疫苗免疫於SPF小雞與虎皮鸚鵡幼鳥,透過ELISA檢測血清中之特異性抗體反應及中和試驗檢測免疫血清中和病毒之能力。結果顯示,注射APV疫苗之組別特異性抗體反應皆升高且血清具有中和病毒之能力,表示製備出的APV疫苗具有發展成商業疫苗之潛力。

    Avian polyomavirus (APV) is the common viral diseases in the Psittacidae family. Infection by APV may lead to no appetite, cutaneous hemorrhage, feather abnormalities, and even death. All age of parrots can be infected by APV and the mortality is associated with young age. APV is a highly contagious viral disease. Avian polyomavirus belongs to the family polyomavirus, which is non-enveloped, double-stranded DNA virus. The icosahedral capsid is composed of the major structural protein VP1 and minor structural proteins VP2, VP3 and VP4. Among them, VP1 has an epitope that induces neutralizing antibodies. With the increase in the number of parrots raised in Taiwan, the rate of spread of the virus also increased. However, there is no effective commercial APV vaccine available in Taiwan. Inactivated APV vaccines are only sold in the United States, but the oil-adjuvanted vaccines were however found to cause muscular necrosis. We aim to prepare an effective and safe APV vaccine for the prevention of parrot disease. So far, we have successfully cloned the VP1 gene into pBacPAK8 and expressed protein using the baculovirus expression system. Then, we used o/w adjuvant to prepare APV vaccine and immunized SPF chicks and parrot chicks. The serum antibody response was detected by ELISA and the ability of the immune serum to neutralize the virus was detected by neutralization test. The results showed that the specific antibody response of the injection groups was increased and the immune serum had the ability to neutralize the virus, indicating that the prepared APV vaccine has the potential to develop into a commercial vaccine.

    中文摘要 II
    Abstract III
    謝誌 V
    目錄 VII
    圖表目錄 X
    第1章 緒言 1
    第2章 文獻回顧 3
    2.1 鸚鵡多瘤病毒簡介 3
    2.2鸚鵡多瘤病毒之結構特性 4
    2.2.1鸚鵡多瘤病毒之基因結構 4
    2.2.2鸚鵡多瘤病毒之蛋白質結構 5
    2.3 鸚鵡多瘤病毒病徵與感染途徑 7
    2.3.1鸚鵡多瘤病毒感染症狀 7
    2.3.2鸚鵡多瘤病毒傳染途徑 8
    2.4 鸚鵡多瘤病毒之檢測方式 9
    2.4.1組織病理學檢測 9
    2.4.2血清學檢測(serological) 10
    2.4.3分子生物學檢測 10
    2.5鸚鵡多瘤病毒之預防與控制 11
    第3章 材料方法 13
    3.1 實驗架構 13
    3.2 病材收集 15
    3.3 病毒檢測與培養 15
    3.3.1 萃取臟器、羽毛樣本之DNA 15
    3.3.2 萃取血液樣本之DNA 16
    3.3.3即時定量聚合酶鏈鎖反應之檢測 17
    3.3.4病毒之培養 17
    3.3.5 半數組織培養感染劑量(50% Tissue culture infective dose, TCID50) 18
    3.4 建構重組pBacPAK8_APV VP1基因 18
    3.4.1 APV VP1基因取得 18
    3.4.2 製備重組pBacPAK8載體 18
    3.4.3萃取重組pBacPAK8與APV VP1質體 19
    3.4.4限制酶切割pBacPAK8與APV VP1 20
    3.4.5接合pBacPAK8和APV VP1基因 20
    3.5桿狀病毒表現系統生產重組pBacPAK8_APV VP1蛋白 21
    3.5.1昆蟲細胞培養初代病毒 21
    3.5.2 以SF9細胞擴增重組桿狀病毒 21
    3.5.3以High Five細胞生產重組蛋白 22
    3.5.4重組pBacPAK8_APV VP1蛋白質處理 22
    3.5.4.1不可溶蛋白之處理 22
    3.5.4.2蛋白質之純化 23
    3.5.5 SDS PAGE蛋白質電泳分析 23
    3.5.6 Western blot 24
    3.6動物試驗設計與效力檢測 24
    3.6.1第一次動物試驗 24
    3.6.2第二次動物試驗 25
    3.6.3第三次動物試驗 25
    3.6.4酵素免疫分析法 25
    3.6.4.1 免疫對象-SPF雞隻 25
    3.6.4.2 免疫對象-鸚鵡 26
    3.6.5中和試驗 26
    第4章 結果 28
    4.1 建立qPCR檢驗方式 28
    4.2 APV病毒之分離與培養 28
    4.3 偵測APV VP1之引子設計 29
    4.4 製備重組pBacPAK8_APV VP1基因結果 29
    4.5以桿狀病毒生產系統生產重組pBacPAK8_APV VP1蛋白 29
    4.5.1重組pBacPAK8_APV VP1蛋白生產結果 29
    4.5.2重組pBacPAK8_APV VP1蛋白純化結果 30
    4.6 APV疫苗效力評估結果 30
    4.6.1第一次動物試驗-血清抗體反應 30
    4.6.2第二次動物試驗-血清抗體反應 30
    4.6.3第三次動物試驗-血清抗體反應 31
    4.6.4中和試驗 31
    第5章 討論 48
    第6章 參考文獻 51
    作者簡介 58

    [1] Altan E, Eravci E, Cizmecigil UY, Yildar E, Aydin O, Turan N, et al. Detection and phylogeny of beak and feather disease virus and avian polyomavirus in psittacine pet birds in Turkey. 2016;25:280-7.
    [2] Bernier G, Morin M, Marsolais G. A generalized inclusion body disease in the budgerigar (Melopsittacus undulatus) caused by a papovavirus-like agent. Avian diseases. 1981;25:1083-92.
    [3] Bert E, Tomassone L, Peccati C, Navarrete MG, Sola SC. Detection of beak and feather disease virus (BFDV) and avian polyomavirus (APV) DNA in psittacine birds in Italy. Journal of veterinary medicine B, Infectious diseases and veterinary public health. 2005;52:64-8.
    [4] Bozeman LH, Davis RB, Gaudry D, Lukert PD, Fletcher OJ, Dykstra MJ. Characterization of a papovavirus isolated from fledgling budgerigars. Avian diseases. 1981;25:972-80.
    [5] Calvignac-Spencer S, Feltkamp MC, Daugherty MD, Moens U, Ramqvist T, Johne R, et al. A taxonomy update for the family Polyomaviridae. Arch Virol. 2016;161:1739-50.
    [6] Daniels R, Sadowicz D, Hebert DN. A very late viral protein triggers the lytic release of SV40. PLoS pathogens. 2007;3:e98.
    [7] Davis RB, Bozeman LH, Gaudry D, Fletcher OJ, Lukert PD, Dykstra MJ. A viral disease of fledgling budgerigars. Avian diseases. 1981;25:179-83.
    [8] Dolz G, Sheleby-Elías J, Romero-Zu JJ, Vargas-Leitón B, Gutiérrez-Espeleta G, Madriz-Orde K. Prevalence of psittacine beak and feather disease virus and avian polyomavirus in captivity psittacines from Costa Rica. 2013.
    [9] Doneley B. Avian medicine and surgery in practice: companion and aviary birds: CRC press; 2016.
    [10] Enders F, Gravendyck M, Gerlach H, Kaleta EF. Fatal avian polyomavirus infection during quarantine in adult wild-caught red-faced lovebirds (Agapornis pullaria). Avian diseases. 1997;41:496-8.
    [11] Fungwitaya P, Bunlertcharoensuk A, Uttamaburana W, Sariya L, Chaichoune K, Ratanakorn P, et al. Prevalence of psittacine beak and feather disease and avian plyomavirus disease infection in captive psittacines in the central part of Thailand by Multiplex Polymerase Chain Reaction. 2009;2.
    [12] Garcia A, Latimer KS, Niagro FD, Ritchie BW, Campagnoli RP. Diagnosis of polyomavirus-induced hepatic necrosis in psittacine birds using DNA probes. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 1994;6:308-14.
    [13] Gaskin JJPAAV, Houston. The serodiagnosis of psittacine viral infections. 1988:7-10.
    [14] González-Hein GA, González CM, Huaracán BRJAjovs. Fatal dual infection of avian polyomavirus and psittacine beak and feather disease virus in Chile. 2017;49:59-61.
    [15] Graham DL, Calnek BWJAd. Papovavirus infection in hand-fed parrots: virus isolation and pathology. 1987:398-410.
    [16] Halami MY, Dorrestein GM, Couteel P, Heckel G, Muller H, Johne R. Whole-genome characterization of a novel polyomavirus detected in fatally diseased canary birds. J Gen Virol. 2010;91:3016-22.
    [17] Hirai K, Nonaka H, Fukushi H, Shimakura S, Masegi T, Mizoguchi T. Isolation of a papovavirus-like agent from young budgerigars with feather abnormalities. Nihon juigaku zasshi The Japanese journal of veterinary science. 1984;46:577-82.
    [18] Hsu CM, Ko CY, Tsaia HJ. Detection and sequence analysis of avian polyomavirus and psittacine beak and feather disease virus from psittacine birds in Taiwan. Avian diseases. 2006;50:348-53.
    [19] Johne R, Buck CB, Allander T, Atwood WJ, Garcea RL, Imperiale MJ, et al. Taxonomical developments in the family Polyomaviridae. Arch Virol. 2011;156:1627-34.
    [20] Johne R, Jungmann A, Muller H. Agnoprotein 1a and agnoprotein 1b of avian polyomavirus are apoptotic inducers. J Gen Virol. 2000;81:1183-90.
    [21] Johne R, Muller H. The genome of goose hemorrhagic polyomavirus, a new member of the proposed subgenus Avipolyomavirus. Virology. 2003;308:291-302.
    [22] Johne R, Muller H. Nuclear localization of avian polyomavirus structural protein VP1 is a prerequisite for the formation of virus-like particles. J Virol. 2004;78:930-7.
    [23] Johne R, Muller H. Polyomaviruses of birds: etiologic agents of inflammatory diseases in a tumor virus family. J Virol. 2007;81:11554-9.
    [24] Katoh H, Ogawa H, Ohya K, Fukushi H. A review of DNA viral infections in psittacine birds. J Vet Med Sci. 2010;72:1099-106.
    [25] Katoh H, Ohya K, Une Y, Yamaguchi T, Fukushi H. Molecular characterization of avian polyomavirus isolated from psittacine birds based on the whole genome sequence analysis. Vet Microbiol. 2009;138:69-77.
    [26] Ke GM, Cheng HL, Ke LY, Ji WT, Chulu JL, Liao MH, et al. Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus. J Virol Methods. 2006;133:6-13.
    [27] Khan MS, Johne R, Beck I, Pawlita M, Kaleta EF, Muller H. Development of a blocking enzyme-linked immunosorbent assay for the detection of avian polyomavirus-specific antibodies. J Virol Methods. 2000;89:39-48.
    [28] Kingston RS. Budgerigar fledgling disease (papovavirus) in pet birds. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 1992;4:455-8.
    [29] Kou Z, Zhang Z, Chen S, Fan Z, Tang S, Zhao L, et al. Molecular characterizations of avian polyomavirus isolated from budgerigar in China. Avian diseases. 2008;52:451-4.
    [30] Li J, Liu Q, Muller H, Hobom G. Avian polyomavirus expression patterns of bicistronic late mRNAs. Virology. 2009;388:42-8.
    [31] Mamom T, Dumrongsoonthornchai P, Trongwongsa LJTTJoVM. Avian polyomavirus infection in non-budgerigar psittacine birds in Thailand-A case report. 2010;40:75-80.
    [32] Mato T, Penzes Z, Rueda P, Vela C, Kardi V, Zolnai A, et al. Recombinant subunit vaccine elicits protection against goose haemorrhagic nephritis and enteritis. Avian pathology : journal of the WVPA. 2009;38:233-7.
    [33] Miller REJSLES. Fowler’s zoo and wild animal medicine current therapy, vol. 7. 2012.
    [34] Moens U, Krumbholz A, Ehlers B, Zell R, Johne R, Calvignac-Spencer S, et al. Biology, evolution, and medical importance of polyomaviruses: An update. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2017;54:18-38.
    [35] Ogawa H, Chahota R, Hagino T, Ohya K, Yamaguchi T, Fukushi H. A survey of avian polyomavirus (APV) infection in imported and domestic bred psittacine birds in Japan. J Vet Med Sci. 2006;68:743-5.
    [36] Özmen Ö, Dorrestein GMJTJoV, Sciences A. Avian polyomavirus infection in budgerigars (Melopsittacus undulatus) in Turkey. 2004;28:409-14.
    [37] Padzil F, Mariatulqabtiah A, Abu JJJVM. AVIAN POLYOMAVIRUS: A RECENT UPDATE. 2017;29:9-13.
    [38] Perez-Losada M, Christensen RG, McClellan DA, Adams BJ, Viscidi RP, Demma JC, et al. Comparing phylogenetic codivergence between polyomaviruses and their hosts. J Virol. 2006;80:5663-9.
    [39] Phalen D, Ambrus S, Graham D. The avian urinary system: form, function, diseases. Proc Assoc Avian Vet: Assoc Avian Vet Publications; 1990. p. 44-57.
    [40] Phalen DN, Radabaugh CS, Dahlhausen RD, Styles DK. Viremia, virus shedding, and antibody response during natural avian polyomavirus infection in parrots. Journal of the American Veterinary Medical Association. 2000;217:32-6.
    [41] Phalen DN, Wilson BG, Graham DL. Production of avian polyomavirus seronegative budgerigars (Melopsittacus undulatus) from seropositive adults. Avian diseases. 1995;39:897-9.
    [42] Phalen DN, Wilson VG, Graham DL. Organ distribution of avian polyomavirus DNA and virus-neutralizing antibody titers in healthy adult budgerigars. Am J Vet Res. 1993;54:2040-7.
    [43] Piasecki T, Wieliczko AJBVIP. Detection of beak and feather disease virus and avian polyomavirus DNA in psittacine birds in Poland. 2010;54:141-6.
    [44] Rahaus M, Wolff MH. A survey to detect subclinical polyomavirus infections of captive psittacine birds in Germany. Vet Microbiol. 2005;105:73-6.
    [45] Riaz A, Yousaf A, Moaeen-Ud-Din M, Shah MAA, Zainab T, Masood S, et al. First detection and molecular characterization of avian polyomavirus in young parrots in Pakistan. Veterinary research communications. 2019;43:197-202.
    [46] Ritchie B, Gregory C, Latimer K, Pesti D, Campagnoli R, Lukert PJIZY. A review of the most common viruses affecting Psittaciformes 1. 2000;37:257-73.
    [47] Ritchie BW, Vaughn SB, Leger JS, Rich GA, Rupiper DJ, Forgey G, et al. Use of an inactivated virus vaccine to control polyomavirus outbreaks in nine flocks of psittacine birds. Journal of the American Veterinary Medical Association. 1998;212:685-90.
    [48] Stoll R, Hobom G, Muller H. Host restriction in the productive cycle of avian polyomavirus budgerigar fledgling disease virus type 3 depends on a single amino acid change in the common region of structural proteins VP2/VP3. J Gen Virol. 1994;75 ( Pt 9):2261-9.
    [49] Stoll R, Luo D, Kouwenhoven B, Hobom G, Muller H. Molecular and biological characteristics of avian polyomaviruses: isolates from different species of birds indicate that avian polyomaviruses form a distinct subgenus within the polyomavirus genus. J Gen Virol. 1993;74 ( Pt 2):229-37.
    [50] Szweda M, KoŁodziejska A, Szarek J, Babińska IJMW. Avian polyomavirus infections in Amazon parrots. 2011;67:147-50.
    [51] Tomasek O, Kubicek O, Tukac VJVM-P-. Unusual fatal avian polyomavirus infection in nestling cockatiels (Nymphicus hollandicus) detected by nested polymerase chain reaction. 2007;52:193.
    [52] Wainright PO, Lukert PD, Davis RB, Villegas P. Serological evaluation of some psittaciformes for budgerigar fledgling disease virus. Avian diseases. 1987;31:673-6.
    [53] Zanon Z, Abu J, Najihah N, Mariatulqabtiah AJPJoTAS. Prevalence of Avian Polyomavirus in Psittacine Birds in the Klang Valley. 2018;41.
    [54] Zhuang Q, Chen J, Mushtaq MH, Chen J, Liu S, Hou G, et al. Prevalence and genetic characterization of avian polyomavirus and psittacine beak and feather disease virus isolated from budgerigars in Mainland China. Arch Virol. 2012;157:53-61.

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