芽孢桿菌抗病毒活性之研究

Abstract

現今畜牧業仍飽受各種動物疾病的侵擾，造成嚴重的經濟損失，而又以病毒性疾病較為棘手，目前是以疫苗施打來預防病毒性疾病的問題，但此方法會有病毒專一性、成本考量、施打的安全性及疫苗預防效率等諸多問題，因此本研究希望能以益生菌來輔助疫苗作為動物抵抗病毒疾病的方法。本研究選擇芽孢桿菌屬的益生菌作為研究對象，芽孢桿菌能夠在極端的環境下以內孢子的方式度過，所以生存能力較佳，所選擇的芽孢桿菌有枯草芽孢桿菌AC（Bacillus subtilis AC）、地衣芽孢桿菌CK（B. licheniformis CK），以及液化澱粉芽孢桿菌LN（B. amyloliquefaciens LN），三者分別經由生長曲線實驗得知所須培養時間，並將處理組分為胞內萃取物、胞外分泌物、細胞壁及活菌四個不同的處理方法進行共培養實驗。實驗所選用的第一個病毒為辛德比病毒（Sindbis virus, SBV)，細胞平台分別為倉鼠腎臟細胞（baby hamster kidney cell, BHK）與人類腸道上皮細胞（colon epithelial cells, Caco-2），結果發現與地衣桿菌CK之胞內萃取物、胞外分泌物及活菌共培養的組別皆能顯著降低細胞受病毒感染的百分比，且發現CK的胞內萃取物及活菌分別能在攻毒後提升ISG56及MX1的表現量，但CK胞外分泌物所作用機制仍尚未明確。第二個病毒為豬流行性下痢病毒（porcine epidemic diarrhea virus, PEDV），所使用的細胞平台為恆河猴腎臟細胞（Chlorocebus aethiops kidney cell, Vero cell），在此實驗結果三種菌皆未能改善細胞受感染的情形，且與CK的細胞壁及活菌共培養的組別還使受感染情況加重，其原因可能為細胞經共培養處理後慢性的死亡，也可能因為細胞模型的不同，其表面上具不同受體，所觸發的反應不利於細胞生存，但還須經實驗加以證實。綜合上述，地衣芽孢桿菌CK能藉由提升抗病毒基因表現來抵抗SBV感染，雖然在抵抗PEDV並沒有一樣的保護效果，但由於細胞平台的不同，其共培養的效果可能不盡相同，未來能以地衣芽孢桿菌CK為實驗對象，針對其抗病毒能力設計相關實驗加以證實。

Due to the occurrence of respiratory and enteric viruses, the development of novel antiviral-agents is a growing public health concern across the globe. As Bacillus strains have been widely used as probiotic agents in animal feed industry, the aim of this study is to evaluate the potential of three Bacillus strains, including Bacillus licheniformis CK (CK), B. subtilis AC (AC), and B. amyloliquefaciens LN (LN), as antiviral agents. The cytotoxicity of the bacterial preparations, including the extracellular supernatant (ES), intracellular extract (IN), cell wall fraction (CW), and viable whole cell (VC) of the Bacillus strains, toward baby hamster kidney (BHK) cells, human colon epithelial Caco-2 cells, and Chlorocebus aethiops kidney cell (Vero cell) were determined. The antiviral activities of the bacterial preparations were evaluated by determining their inhibitory effect against Sindbis virus (SBV) replication in BHK or Caco-2 cells and against Porcine epidemic diarrhea virus (PEDV) in Vero cells. In the BHK cell model, the IN of CK showed no cytotoxicity but exhibited anti-SBV activity. In the Caco-2 cell model, the ES, IN, and VC of CK showed no cytotoxicity but exhibited anti-SBV activities. The mRNA expression levels of antiviral factors, including IFN-β, IL-6, MX1, and ISG56, of the virus-infected Caco-2 cells were further determined. The results indicated that EX and VC of CK significantly increased the IL-6 expression levels, IN of CK significantly increased the ISG56 expression levels, and VC of CK significantly increased the MX1 expression levels in the SBV-infected Caco-2 cells. Therefore, the inhibitory effect of CK against SBV replication in Caco-2 cells could be attributable to the fact that CK could enhance the expression of cell-intrinsic antiviral factors. However, all the bacterial preparations could not decrease the PEDV infection rate in Vero cell model. These results suggested that further experiments will be need to verify the antiviral activity of B. licheniformis CK before it developed as feed additives with antiviral activity.