石斑魚腦細胞株魚類結病毒持續性感染之研究

The persistent infection of fish nodavirus in grouper brain cell line

詹茹潔(Ju-Chien Chan)

指導教授 ： 齊肖琪

國立臺灣大學/生命科學院/動物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02196

摘要

急性感染神經壞死病毒 (Nervous Necrosis Virus, NNV)之後的殘活石斑魚會形成持續性帶原現象。為瞭解石斑魚腦組織Mx蛋白質與NNV帶原的關係，本實驗以殘活石斑腦組織建立GB 細胞株，並以RT-PCR確認GB細胞有NNV持續性感染，且免疫化學染色結果顯示GB細胞的帶原特徵是少數細胞的細胞質有大量NNV蛋白表現，顯示GB細胞株只有少數細胞有病毒急性感染，大多數細胞則類似被保護狀態。以抗NNV的多源抗體連續處理GB細胞幾代後，得到一株無NNV帶原之cGB (cured GB)細胞株。GB細胞、感染NNV 之後的cGB細胞以及轉染poly I:C後的cGB細胞皆會表現干擾素下游的Mx蛋白質，但未經任何處理的cGB細胞則無Mx表現，顯示 (1) GB細胞中有干擾素反應，cGB細胞則無； (2) NNV感染或poly I:C轉染皆可誘發干擾素反應及Mx蛋白質表現。因poly I:C轉染後的cGB細胞感染NNV後的子代病毒力價比未轉染的細胞低，又GB細胞在低劑量NNV感染後，會再現GB細胞持續性感染的特徵，因此推測GB細胞持續性感染和寄主細胞抗NNV的機制與干擾素及其下游的Mx 有關。cGB細胞感染NNV 24小時後， NNV RNA-dependent RNA polymerase (RdRp)與Mx蛋白會共同座落在核周圍，病毒鞘蛋白質則無此現象，因此推測Mx蛋白可能經由與NNV RdRp結合來干擾NNV的複製。

關鍵字

神經壞死病毒: 持續性感染 ； 干擾素 ； Mx 蛋白 ； 石斑魚腦細胞株

並列摘要

Grouper survivors after nervous necrosis virus (NNV) acute infection will become persistently infected. In order to reveal the relationship of grouper Mx protein and the persistent infection of NNV, a cell line GB was established from the brain tissue of grouper survivors. NNV was found in GB cells by RT-PCR, and NNV protein was only detectable in the cytoplasm of a few GB cells by immunohistochemistry staining, suggesting that only a few cells were acutely infected and most cells were likely protected. After serial treatments of NNV-specific polyclonal antibodies, GB cells became a NNV-free cell line which was named as cured GB (cGB). The expression of grouper Mx (GMx), a downstream protein of interferon response, was detected in GB cells, NNV-infected cGB cells and poly I:C-transfected cells, but was abscent in cGB cells, indicating that IFN response existed in GB cells, and IFN response and Mx expression was inducible in cGB cells by NNV infection and poly I:C transfection. When cGB cells were infected by NNV with low MOI, all characteristics of persistent infection in GB cells reappeared in infected cGB cells. In addition, NNV titer proliferated in poly I:C-transfected cGB cells were lower than that in cGB cells, indicating that the mechansim of NNV persistence in GB cells possibly mediated through IFN and Mx protein. By the way, NNV. At 24 h post-infection, only viral RNA-dependent RNA polymerase (RdRp) was found to be colocalized with Mx at perinuclear area, suggesting that Mx might interfere with NNV replication by interaction with RdRp.

並列關鍵字

Nervous necrosis virus (NNV) ； Persistent infection ； Interferon ； Mx protein ； Grouper Brain cell line(GB cell line)

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