具抗虹彩病毒能力之益生菌篩選及其阻止病毒進入石斑魚腎臟細胞之機制探討

Abstract

石斑魚為國內主要的養殖魚種，吋魚時期易受病毒感染而造成高死亡率。石斑魚虹彩病毒(Grouper iridovirus, GIV)為其主要的威脅之一。在過去研究中顯示水產養殖所使用的益生菌，除了能增進魚隻生長、提升免疫力外，部分菌株甚至具有抗病毒的效果。本研究中以石斑魚腎臟細胞（grouper kidney cell, GK cell）作為篩選具抗GIV病毒的潛力菌株之平台，除了評估菌株之益生菌特性外，並更進一步探討GIV進入宿主細胞的途徑。以16S rRNA與Gyrase B基因進行菌種鑑定後，確認從石斑魚腸道中分離出的菌株分別為Kocuria assamensis KA、Bacillus altitudinis BS、Bacillus altitudinis BA。隨後在預防（pre-treatment）、共培養（co-incubation）、預培養（pre-incubation）的策略下，檢測各菌株萃取物抗GIV的潛力以及GK細胞的免疫與抗病毒基因表現。結果顯示以B. altitudinis BS、B. altitudinis BA菌株的胞內萃取液 （IF）在三種策略中均能提升細胞存活率，但在基因表現方面，BS-IF能調降預防策略下第48小時的發炎反應與GK細胞內GIV病毒基因表現量；BS-IF在共培養策略下能同時提升第48小時的發炎與抗病毒反應；而在預培養策略下，BS-IF會先促進24小時後的發炎反應，隨後在48小時才提升抗病毒反應。在免疫染色結果中驗證BS-IF在預處理下能減少GK細胞內病毒累積量。而B. altitudinis BA的萃取物在基因表現的調節較不顯著也無明顯GIV病毒減少，因此B. altitudinis BS是最具抗GIV病毒潛力的菌株，其中萃取物IF最有效。在益生菌評估中，B. altitudinis BS具有耐膽鹽、抗病原菌、抗生素敏感等特性，但在pH 2.5至3的酸性環境下不易生長，且呈現β-hemolysis的溶血反應，由此不完全符合益生菌的規範，雖可被視為有益菌株但並非益生菌。最後在GIV進入機制的探討中確認網格蛋白介導的內吞作用（clathrin-mediated endocytosis）為其進入宿主細胞的途徑之一。

Groupers, the main-cultured and economic fish species in Taiwan, easily faced high mortality in the larvae period due to the virus infection. Grouper iridovirus (GIV) is one of the virus challenges to groupers. In past studies, probiotics were normally used in the aquaculture industry as an additive to elevate the growth index and immune system of fish. In addition, some of them were proven with anti-viral activity. In this study, we used Grouper kidney cell (GK cell) for GIV infection to select the ideal bacterial strains with anti-GIV potential and evaluate their probiotic characteristics. Furthermore, we explored the GIV entry mechanism to the GK host cell. Firstly, we successfully identified the isolated strains from the groupers through 16S rRNA and Gyrase B sequencing method, they were Kocuria assamensis KA, Bacillus altitudinis BS, and Bacillus altitudinis BA. Subsequently, under the three deigned strategies, pre-treatment, co-incubation, and pre-incubation, the results indicated that the intracellular fraction (IF) extraction of Bacillus altitudinis BS, Bacillus altitudinis BA were both able to elevate the cell viability of GIV-infected GK cell. However, only BS-IF significantly regulated the immune-related gene expression. In pre-treatment strategy, BS-IF decreased the inflammatory reaction and the GIV gene expression in GK cells； In the co-incubation strategy, BS-IF increased the inflammatory and anti-viral reactions at the same period； In the pre-incubation strategy, BS-IF stimulated the inflammation before the anti-viral reaction. The immune-staining results also displayed the same phenomenon that BS-IF decreased the GIV accumulation in GK cells. In conclusion, Bacillus altitudinis BS was the most efficient isolated strain against GIV infection. Then, in probiotic tests, Bacillus altitudinis BS had bile salt tolerance, anti-pathogen, and antibiotic sensitivity features but without acid tolerance and appeared β-hemolysis. Thus, Bacillus altitudinis BS is not suitable as a probiotic. What’s more, in the virus entry mechanism, we figured out that GIV used clathrin-mediated endocytosis as one of the pathways to enter the host cell.