具抗石斑魚虹彩病毒活性之乳酸菌篩選

Abstract

目前石斑魚在臺灣的水產養殖業中仍然佔有重要的經濟地位，但由於近年來高密度的養殖方法，使得病毒的傳染變得非常容易又快速，其中最盛行且具有高感染力的病毒之一是石斑魚虹彩病毒（Grouper iridovirus, GIV），GIV屬於Ranavirus屬，當幼魚感染GIV時，死亡率甚至可高達60%。先前的研究指出，益生菌具有顯著的抗病毒效果，然而目前對於克菲爾（一種發酵乳產品），其是否具有抗GIV效果的研究仍然相當有限。 因此本研究使用實驗室先前於克菲爾粒分離出的29株乳酸菌（lactic acid bacteria, LAB）進行抗GIV活性的評估，利用石斑魚腎臟細胞（grouper kidney cells, GK cells）進行病毒感染的試驗。細胞毒性試驗的結果顯示，所有的LAB萃取物，包含胞內萃取物（intracellular fraction, IF）、胞外液（extracellular fraction, EF）與細胞壁（cell wall, CW），皆不具有細胞毒性。在預防模式下，所有LAB的EF、19株LAB的IF與11株LAB的CW，在細胞存活率試驗中皆具有抗GIV活性，其中具有最高抗GIV活性的菌株為YPK25與YPK30，兩株菌皆屬於Leuconostoc mesenteroides。而在基因表現量分析方面，雖然大部份促發炎相關基因的表現量在經由LAB萃取物預處理再感染GIV後沒有顯著差異，但與單純只有感染GIV的組別相比，大部份干擾素相關基因的表現量顯著地上升，因此推測Ln. mesenteroides在受GIV感染的GK細胞中主要是透過調節干擾素訊號，而不是藉由調節促發炎激素來抑制病毒的活性，因此未來研究干擾素相關基因的調節可能是個可行的方向。

Groupers remain an economically important species in Taiwan's aquaculture industry. However, high-density aquafarming practices have facilitated rapid and widespread viral transmission. Among the most prevalent and highly infectious viruses is the Grouper iridovirus (GIV), a major Ranavirus pathogen that can cause mortality rates up to 60%. Previous studies have indicated that probiotics possess significant antiviral properties. Nevertheless, the antiviral potential of kefir, a fermented dairy product, against GIV remains largely unexplored. In this study, 29 lactic acid bacteria (LAB) strains previously isolated from kefir grains by our laboratory were evaluated for their antiviral activity against GIV using grouper kidney (GK) cells as an in vitro infection model. Results from cytotoxicity assays showed that the intracellular fraction (IF), extracellular fraction (EF), and cell wall (CW) of all LAB strains, were non-cytotoxic. In the subsequent evaluation of antiviral activity, a pretreatment model was employed. All EF samples, along with 19 IF samples and 11 CW samples, significantly improved cell viability following GIV infection, indicating antiviral potential. Among these, two strains, YPK25 and YPK30, both identified as Leuconostoc mesenteroides, exhibited the strongest antiviral effects. Gene expression analysis further demonstrated that cells pretreated with LAB extracts prior to GIV infection exhibited a significant upregulation of most interferon-related genes, whereas the expression levels of pro-inflammatory cytokines remained largely unchanged when compared with the GIV group. These findings suggest that Ln. mesenteroides may exert its antiviral effects in GIV-infected GK cells primarily through modulation of the interferon signaling pathway rather than by pro-inflammatory cytokines. Thus, future studies investigating the regulation of interferon-related genes may provide promising directions for anti-GIV research.