石斑魚鰭細胞株GF-3 Mx蛋白質抗魚類野田病毒的機制

神經壞死病毒是魚類神經壞死病毒症的病原。罹患神經壞死病毒症之後的殘活魚，其中樞神經系統以及鰭部位皆可測到大量病毒核酸。為瞭解神經壞死症病毒與石斑魚先天性免疫防禦機制的交互關係，本實驗以石斑魚鰭細胞株GF-3為系統，研究干擾素誘導的抗病毒蛋白質Mx與神經壞死症病毒複製之間的關係。當GF-3細胞感染神經壞死病毒或轉染Poly I:C後，皆會誘發Mx蛋白質的表現，並且Mx蛋白具有抗神經壞死病毒的活性。在神經壞死病毒感染的GF-3 細胞中，Mx蛋白會隨著感染時間而表現量會上升，但病毒的RdRp (RNA-dependent RNA polymerase) 表現量則是下降。在感染神經壞死病毒 24小時的細胞中，用免疫螢光染色法可觀察到Mx 蛋白與在粒線體進行複製的NNV RdRp一起座落在細胞質中，同時間RdRp與lysosome的分佈位置也有重疊，因此推測，石斑魚細胞中的Mx蛋白可能藉由與RdRp結合並運送到lysosome進行降解以干擾病毒的複製。以低病毒感染劑量感染GF-3細胞，會形成病毒持續性感染的細胞株,每代細胞上清液中的病毒力價平均為105 TCID50 ml-1，但每代只有0.1% 的細胞有表現病毒蛋白，因此推測，GF-3細胞上清液存在類干擾素的細胞激素，且所誘導細胞表現的抗病毒Mx蛋白，可在GF-3細胞株的NNV持續性感染機制中扮演其中一個重要角色。

關鍵字

石斑魚； Mx蛋白質；魚類野田病毒

並列摘要

Nervous Necrosis virus (NNV) is the causative agent of Viral Nervous Necrosis (VNN) disease among many species of fish. Groupers survived from VNN show high viral copies in the central nervous system and fin. However, the interplay between betanodavirus and the innate system defense of host remains unclear. The GF-3 cell line derived from grouper fin tissue was used to examine the interaction of interferon (IFN)-induced Mx protein and betanodavirus replication. The expression of grouper Mx Ⅱ was found after the infection with NNV or transfection with Poly I:C. IFN response and Mx protein showed antiviral activity in GF-3 cells. In addition, the expression of grouper Mx protein increased while viral RdRp (RNA-dependent RNA polymerase) decreased through the time courses of NNV life cycle. Grouper Mx was found in the cytoplasm at 24 h post infection (hpi) and co-localized with RdRp which replicates at mitochondria. Meanwhile, RdRp was found co-localized with lysosome. It is therefore suggested that Mx protein may interfere with NNV replication via binding with viral RdRp at mitochondria and translocate together to the lysosome for degradation. Besides, low multiplicity of NNV infection induced IFN response and Mx in GF-3 cells, and then the cells became NNV persistent infection (PI). Only 0.1% of the cells showed positive signal in the immunoflourescent staining with anti-capsid polyclonal antibodies, and the viral titer in the PI cell supernatant of each subculture was about 105 TCID50 ml-1. Therefore, the mechanism of NNV-PI in GF-3 cells is suggested to relate to IFN response and Mx protein.

並列關鍵字

grouper ； Mx protein ； betanodavirus ； NNV

參考文獻

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石斑魚鰭細胞株GF-3 Mx蛋白質抗魚類野田病毒的機制

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