摘要
細胞為病毒學研究及病毒性疫苗生產之主要生物材料，但魚類品種繁多且病毒往往具有物種特異性，必須有相應細胞才能進行病毒之體外培養。目前在全世界生物資源保存中心能夠提供研究的魚類細胞種類很少，因此本研究希望能夠建立多種魚類細胞株包括鱘龍魚、虹鱒及龍虎石斑等永生化細胞，做為日後魚類病毒學研究及疫苗開發之用。本研究選取龍虎石斑腦、鰭、鰓等組織建立細胞株；鱘龍魚外加肝、脾、腎等組織建立細胞株；虹鱒再外加心臟組織建立細胞株，經連續繼代來確認初代細胞繼代極限及生長速率。在測試細胞對病毒感受性方面：利用鱘龍魚及龍虎石斑嘗試用神經壞死病毒(Nervous necrosis virus , NNV)、錦鯉皰疹病毒(Koi herpesvirus , KHV)進行細胞攻毒試驗，每日觀察病毒及細胞病變，且利用即時定量聚合酶連鎖反應(Real-time Quantitative Polymeras Chain Reaction; Real-time PCR)測定病毒生長之力價。結果顯示鱘龍魚的細胞感染NNV及KHV測定對病毒感受性，尤其腦細胞受NNV感染後第三天即可觀察到CPE現象，則鱘龍魚其他細胞皆對NNV及KHV並無感受性。龍虎石斑的細胞感染NNV及KHV測定細胞對病毒的感受性，尤其是龍虎石斑細胞在感染NNV後第三天皆可觀察到明顯的CPE現象。藉由結果證明龍虎石斑所建立的細胞株可增值NNV並產生高病毒力價，日後可做為病毒分離及疫苗開發之潛力。
並列摘要
Cell-culture is the main biological tool for virology research and vaccine development. However, the specificity of virus culture mainly depends on the host cell of fish types. Currently, there are only few options of fish cells for virus culture that are provided by the Bioresource Collection and Research Centers (BCRC), resulted in restricting the development of fish vaccines. Therefore, this study aims to establish variety of fish cell lines including Acipenser sinensis, Oncorhynchus mykiss and Epinephelus fuscoguttatus for future fish virological research and vaccine development. In this study, we selected brain, fins, gill (Epinephelus fuscoguttatus, Acipenser sinensis ,Oncorhynchus mykiss) ,liver, spleen, kidney (Acipenser sinensis, Oncorhynchus mykiss) ,heart (Oncorhynchus mykiss). After cells of Acipenser sinensis and grouper were infected with nerve necrosis virus (NNV) and Koi herpes virus (KHV) ,cytopathic effect (CPE) and real-time polymerase chain reaction (RT-RCR) were performed to investigate virus replication. The results demonstrated of NNV and KHV susceptibility to Acipenser sinensis cells. Only the brain cells were susceptible to NNV, and the CPE could be observed on the third day. Other cells were insensitive to the virus. Epinephelus fuscoguttatus cells were susceptible to NNV while CPE being observed on the third day. The results showed that the grouper cells was suitable for proliferating NNV with high viral titers, suggesting commercial usage virus isolation and vaccine development in the future.
參考文獻
延伸閱讀
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