利用細胞生產高病原性的傳染性華氏囊病毒並且開發成活毒疫苗

Cell culture produce highly pathogenic infectious bursal disease virus and develop attenuated vaccine

簡裕峰(Yu-Feng Chien)

指導教授 ： 柯冠銘

國立屏東科技大學/農學院/動物疫苗科技研究所/碩士(2015年)

https://doi.org/10.6346/NPUST.2015.00229

摘要

傳染性華氏囊病病毒 ( Infectious bursal disease virus; IBDV ) 會引發幼齡雞隻造成高度免疫障礙性傳染病，是雞隻中重大疾病之一。IBDV屬於Birnaviridae科Avibirnavirus屬的病毒。IBDV的基因組片段A ( Segment A ) 編碼含有結構 ( VP2、VP3和VP4 ) 以及非結構 ( VP5 )蛋白，而片段B ( Segment B ) 編碼病毒RNA-dependent RNA polymerase ( VP1 )。VP2是主要的外膜蛋白，能誘發宿主產生特異性的中和抗體且與病毒吸附並進入細胞有關。在本研究中利用超強毒的傳染性華氏囊病病毒 ( vvIBDV ) 之野外分離株 ( V512 ) 在綠猴腎細胞 ( Vero cells ) 進行連續培養20代。馴化過程以細胞半數致死劑量 ( 50% tissue culture infective dose; TCID50 ) 及即時定量聚合酶連鎖反應 ( Real-time polymerase chain reation; Real-time PCR ) 來定量vvIBDV之病毒力價，並以SPF雞隻接種分析其華氏囊/體重比 ( Bursa/Body ratio; BBR )、華氏囊肉眼病變 ( Bursa gross lesion; BGL ) 以及華氏囊組織學病變 ( Bursa histological lesion; BHL ) 來判定病毒毒力變化情形。V512於細胞增殖之力價由第1代無任何力價至第20代可達106.5 TCID50/ml。本研究以第20代病毒製成活毒疫苗進行SPF雞隻免疫，可見其病毒中和性試驗 ( viral neutralization; VN ) 最高可達1024。研究成功將vvIBDV馴化於細胞達到高病毒力價，並使其毒力下降，由於V512來源為vvIBDV，其抗原性與野外強毒株相近，因此製成活毒疫苗後可誘發良好的免疫保護力。本疫苗以細胞培養生產抗原可降低以雞胚胎接種製成產生之免疫副作用並降低雞胚廢棄物處理成本。

關鍵字

傳染性華氏囊病病毒 ； VP2 ； 綠猴腎細胞 ； 活毒疫苗

並列摘要

Infectious bursal disease ( IBD ) is one of the major diseases on the poultry and cause immune disorders in young chickens. IBDV belongs to the genus Birnaviridae families Avibirnavirus virus. IBDV genome segment A (Segment A) contains structure (VP2, VP4 and VP3) and non-structural proteins, while fragment B (Segment B) encodes viral RNA-dependent RNA polymerase (VP1). VP2 is the major outer membrane proteins, can induce the host to produce specific neutralizing antibodies and affect the virus to enter cells. In this study, we use the very virulence infectious bursal disease virus (vvIBDV) of field isolates (V512) in green monkey kidney cells (Vero cells) 20 generations of continuous subculture. To check the degrees of domestication using cell lethal dose (50% tissue culture infective dose; TCID50) and quantitative real-time polymerase chain reaction (Real-time polymerase chain reation; Real-time PCR) to quantify vvIBDV virus titer, and to evaluate the virulence vary by analysising SPF chickens bursa / body weight ratio ( Bursa / Body ratio; BBR )、( Bursa gross lesion; BGL ) and ( Bursa histological lesion; BHL ). V512 titer from first generation has no force up to 20 generations has 106 TCID50 / 100ml. In this study, using 20th-generation virus made the live vaccine to immunizise SPF chickens, virus neutralization test (viral neutralization; VN) reach very high antibody titer. In this study successfully domesticates the vvIBDV virus in the cell can achieve high viral titer and low virulence. Since V512 antigenicity similar to the field virulent of vvIBDV, made it to attenuated vaccine can induce good immune protection. This Vaccine made from cell culture can decrease the side effects of chicken embryonic antigen vaccination and the costs of chicken embryonic waste treatment.

並列關鍵字

infectious bursal disease virus ； VP2 ； Vero cell ； live vaccine

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