- 學位論文
探討BHK-21亞細胞株之牛流行熱病毒感受性研究
Investigation of different susceptibility of BHK-21 subclones of bovine ephemeral fever virus (BEFV) infection
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摘要
牛流行熱病毒(BEFV)屬單股負鏈RNA之彈狀病毒科，僅一種血清型，其症狀常伴隨急性高燒、口眼鼻異常分泌、肌肉僵硬及泌乳量下降等，造成畜牧業經濟損失重大。現今市售疫苗以不活化疫苗為主，幼倉鼠腎細胞(BHK-21)為可培養BEFV之細胞株並應用於疫苗製程上，如何提高並穩定病毒生產效率一直是疫苗製程所追求的目標。先前有研究指出，小鼠胚胎成纖細胞(MEF)的老化現象對水泡性口炎病毒(VSV)產生保護作用，然而BHK-21細胞老化對BEFV感染可能出現類似的抗病毒機轉尚未被研究。因此，本研究首先將BHK-21細胞分離成不同亞細胞株，透過細胞老化染色進行篩選，將不同老化程度之亞細胞株進行病毒傳代感染，觀察病毒力價和細胞存活率之趨勢。將病毒感染傳至三代並進行三重複，發現到老化程度高之亞細胞株生產的病毒力價會逐漸下降，而老化程度低之亞細胞株及BHK-21細胞生產的病毒力價則可維持穩定生產，推測BHK-21細胞老化會影響BEFV生產的穩定性。此外，將不同老化程度之亞細胞株及BHK-21細胞生產的病毒經BEI不活化後製成疫苗免疫小鼠，利用中和試驗檢測血清中抗體保護力價，發現在免疫後28天老化程度高之亞細胞株其組別的抗體力價相較於老化程度低之亞細胞株及BHK-21細胞組別皆來得低。本研究結果顯示，BHK-21細胞老化在BEFV疫苗製程中可能會造成潛在的不良影響，會降低BEFV生產的穩定性及其中和抗體保護效力。
並列摘要
Bovine ephemeral fever virus (BEFV) is a negative-sense single-stranded RNA virus with only one serotype in the family Rhabdoviridae. It infects cattle and causes symptoms such as acute fever, oral and nasal discharges, stiffness, and decreases in milk production, etc., which makes a great economical loss in animal husbandry. Nowadays commercial vaccines are mainly inactivated virus vaccines. The BHK-21 cells are one of the cell lines that can culture BEFV in vaccine production. How to improve and stabilize virus production efficiency has always been the goal for vaccine manufacturing study. Previous studies indicated that the cellular aging of mouse embryonic fibroblasts (MEF) showed a protective effect against vesicular stomatitis virus (VSV). However, the cellular aging of BHK-21 cells with similar antiviral mechanisms against BEFV infection has not been studied. In this study, BHK-21 cells were separated into subclones and selected by cellular aging staining. Virus passage infection of subclones with different aging degree was conducted to observe changes in virus titer and cell viability. The virus was cultured for three passages and repeated three times. It was found that the virus titer produced by subclone with a higher aging degree gradually decreased. But the virus titer in subclone with a lower aging degree and the BHK-21 cells maintained stable production. It showed that the aged BHK-21 subclone could not support BEFV growth to high titer. In addition, viruses produced by different subclones and BHK-21 cells were made into vaccines to immunize mice after BEI inactivation, and the neutralization assay was used to detect their effect on antibody titer in sera. It was found that at 28 days post immunization, the group of subclone with a higher aging degree had a lower neutralization antibody compared with the BHK-21 cell group. In conclusion, the cellular aging of BHK-21 cells may cause potential adverse effects on the BEFV vaccine manufacturing, reducing the stability of BEFV production and the protective efficacy of anti-BEFV neutralizing antibodies.
參考文獻
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