- 學位論文
具抗貓傳染性腹膜炎病毒活性之乳酸菌株篩選
Screening of lactic acid bacteria strains with anti-feline infectious peritonitis virus activity
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摘要
貓傳染性腹膜炎（feline infectious peritonitis, FIP）是貓最重要的傳染病之一，由貓傳染性腹膜炎病毒（feline infectious peritonitis virus, FIPV）引起，FIPV是貓冠狀病毒（feline coronavirus, FCoV）的高毒力突變體。 FIP 的死亡率接近 100%，但目前尚無有效治療方法。 克菲爾是一種發酵乳飲料，由複雜的微生物群組成，是多種乳酸菌 (lactic acid bacteria, LAB) 的共生組合。 這些 LAB 菌株可以調節免疫系統以抑制病毒感染。 LAB 的抗病毒機制涉及直接與病毒相互作用、產生抗病毒相關分子和對於免疫系統的調節。 本研究的目的是建立體外模型，研究乳酸菌在 felis catus whole fetus-4 （fcwf-4） 細胞中對 FIPV 的抗病毒潛力。 從克菲爾中分離出 29 株 LAB 菌株，並研究這些菌株胞內萃取物對 fcwf-4 細胞的細胞毒性。 在細胞毒性試驗中，菌株胞內萃取物添加濃度為50 μg/mL，其中11株菌株無細胞毒性。 以病毒空斑與TCID50測定病毒力價。TCID50結果顯示10-3的FIPV病毒液使fcwf-4細胞活力低於50%，病毒空斑測定之病毒力價為4 x 104 PFU/mL。後續實驗將以MOI=0.06進行感染。 透過檢測LAB 菌株細胞內萃取物的抗 FIPV 活性。 我們發現YPK14和YPK25菌株在預處理試驗中顯示出抗FIPV能力且下調tumor necrosis factor-alpha （TNF-α）and interleukin-6 （IL-6）和FIPV-N基因表現，YPK26菌株在感染後試驗中顯示出抗FIPV能力且上調IL-10基因表現，而YPK6菌株在競爭試驗中顯示出抗FIPV能力且上調interferon-stimulated gene 15 （ISG15）基因表現。 這些 LAB 菌株透過 16S rDNA 和 rpoA 定序鑑定為Lentilactobacillus kefiri和Leuconostoc mesenteroides subsp. mesenteroides。 此外，也研究了乳酸菌菌株的益生菌特性，包括抗病原菌活性、對酸、膽鹽的耐受性和抗生素敏感性。 結果表明，YPK6、YPK 14、YPK25和YPK 26對低pH值具有耐受性。 透過分析 FIPV 感染的 fcwf-4 細胞中抗發炎、促發炎細胞因子和 I 型干擾素相關基因，如Mx1、ISG15、TNF-α、IL-6和 IL-10的表達，闡明了 LAB 菌株的抗病毒活性機制。綜上所述，Lentilactobacillus keferi YPK6、YPK 14、YPK 26 和 Leuconostoc mesenteroides subsp. mesenteroides YPK25 具有抗 FIPV 活性和益生菌潛力。 期能將上述4株菌開發為具有抗病毒活性之益生菌。
並列摘要
Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats with high mortality. It is caused by the feline infectious peritonitis virus (FIPV), a highly virulent mutant form of feline coronavirus (FCoV). The fatality rate of FIP is close to 100% and there are no known effective treatments for cats. Kefir is a fermented milk drink and is composed of complex microbiota, which is a symbiotic association of a variety of lactic acid bacteria (LAB). These LAB strains can modulate the immune system to suppress infections from viruses. The antiviral mechanisms of the LABs involve enhancement of macrophage production, increasing phagocytosis, and boosting production of cytokines. The purpose of the study is to establish an in vitro model to investigate the antiviral potential of LAB for FIPV in felis catus whole fetus-4 (fcwf-4) cell. A total of 29 LAB strains were isolated from kefir milk and the cytotoxicity of these strains to fcwf-4 cell was investigated. Under the cytotoxicity test, the extract was added at a concentration of 50 μg/mL, and 11 strains had no cytotoxicity. According to TCID50 assay, 10-3 of FIPV dilution ratio caused fcwf-4 cell viability lower than 50%. The results of plaque assay showed that the viral titer used in this study was 4 x 104 PFU/mL. Thus, FIPV infection will be conducted with MOI=0.06. The anti-FIPV activity of the intracellular extracts of LAB strains was examined. We found that YPK14 and YPK25 strains showed anti-FIPV ability and downregulated tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and FIPV-N genes expression in prophylactic model, YPK26 strain showed anti-FIPV ability and upregulated IL-10 gene expression in therapeutic model, while YPK06 strain showed anti-FIPV ability and upregulated interferon-stimulated gene 15 (ISG15) gene expression in direct- inhibitory model. These LAB strains were identified by 16S rDNA and rpoA sequencing as Lentilactobacillus kefiri and Leuconostoc mesenteroides subsp. mesenteroides. Moreover, the probiotic characteristics, including anti-pathogen activity, tolerance of acid, bile salt, and antibiotics sensitivity of the LAB strains were investigated. The results showed that the LAB strains were tolerant to low pH value. The mechanisms of the antiviral activities of the LAB strains were clarified by analysis of the expressions of proinflammatory cytokines and type 1 interferon related genes, such as Mx1, ISG15, TNF-α, IL-6and IL-10 in the FIPV-infected fcwf-4 cells by using qPCR. In conclusion, Lentilactobacillus keferi YPK6, YPK14, YPK26 and Leuconostoc mesenteroides subsp. mesenteroides YPK25 were found to possess anti-FIPV activity and probiotic potential. Thus, they are expected to be used as a probiotic antiviral agent.
參考文獻
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