以體外試驗評估具益生菌特性之芽孢桿菌萃取物抗貓傳染性腹膜炎病毒活性

Abstract

貓傳染性腹膜炎是一種由 feline infectious peritonitis virus （FIPV） 的冠狀病毒所引起之高致死率疾病，目前僅有極少能夠治療的藥物。本研究藉由 MTT 試驗從 Bacillus amyloliquefaciens LN（LN）、B. velezensis AC（AC）及 B. licheniformis CK1（CK1） 芽孢桿菌萃取物挑選對 fcwf-4 細胞模型較不具細胞毒性之 AC、CK1 萃取物進行三種抗 FIPV 病毒策略效用評估，包含預防、治療及聯合處理。其中 AC 與 CK1 萃取物以預防模式處理下具有提升細胞存活率的效果；而治療模式下則是 CK1 萃取物處理組有較好的效果。利用即時螢光定量技術與免疫螢光染色法觀察在上述兩種模式中的 FIPV 表現量及分布，發現經芽孢桿菌萃取物處理之細胞與未處理組相比並無降低 FIPV-N 表現及陽性率。而結合預防及治療的聯合處理中，以AC/CK1 進行的聯合處理不如 CK1/CK1 具有顯著提升存活率之效果。最後以即時螢光定量技術分析以芽孢桿菌萃取物進行預防與治療策略中以 FIPV 感染與未感染細胞之基因表現，證實具有預防效果的芽孢桿菌萃取物皆能提升 TNF-α 的表現量；而 CK1 胞內液與細胞壁在治療模式且受感染的情況下，降低了受體 fAPN 並提升了抗病毒蛋白 Mx1 的表現。綜合上述， AC 與 CK1 萃取物未能降低 FIPV 複製能力，但能夠對 fcwf-4 細胞之免疫與抗病毒相關基因產生影響。

Feline infectious peritonitis (FIP) is a viral disease of cats caused by certain strains of a virus called the feline coronavirus (FCoV). This study aimed to evaluate the antiviral efficacy of extracts from probiotic strains, including Bacillus amyloliquefaciens LN (LN), B. velezensis AC (AC), and B. licheniformis CK1 (CK1) on the fcwf-4 cell model using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Three strategies, including pre-treatment, post-treatment, and combination treatment, were assessed for their effectiveness against FIPV, focusing on the extracts from AC, CK1 intracellular fluid (INT), cell wall (CW), and exopoly-substances (EPS), which showed lower cytotoxicity to the fcwf-4 cell model. AC and CK1 extracts demonstrated improvement of cell viability in the strategy of pre-treatment, while CK1 extract performed better in the post-treatment. However, analysis of FIPV-N expression showed both strategies had no reduction in FIPV-N expression and in cells treated with Bacillus extracts compared to the untreated group. In the combination therapy, the AC/CK1 combination did not present a significant increase in survival rate compared to the CK1/CK1 combination. Analysis of gene expression in FIPV-infected and uninfected cells treated with Bacillus extracts confirmed that the preventive Bacillus extracts increased TNF-α expression. In the post-treatment with infected cells, CK1_INT and CK1_CW reduced the receptor fAPN and increased the antiviral protein Mx1 expression. In conclusion, this study demonstrated that AC and CK1 extracts did not reduce FIPV replication but had regulations to antiviral and proinflammatory genes in fcwf-4 cells.