具抗豬繁殖與呼吸症候群病毒能力之乳酸菌株篩選

Abstract

豬繁殖與呼吸症候群病毒(Porcine reproductive and respiratory syndrome virus， PRRSV)是豬隻嚴重的病毒性疾病。PRRSV 能夠破壞早期的先天免疫反應，引起主 要的肺臟病變，至今仍然是全球豬隻中最嚴重的疾病。益生菌被定義為具有促進健 康的微生物食品補充劑。益生菌可能帶來有益作用，包括產生抗微生物物質、調節 免疫反應和促進宿主先天防禦機制。最重要的是，已經有研究證實益生菌可以對抗 病毒。 本研究的目的是建立一個體外模型，以研究益生菌對 MARC-145 細胞中 PRRSV 的抗病毒潛力。從離乳前的小豬中分離出乳酸菌(lactic acid bacteria，LAB)菌株。 通過 MTT 試驗，排除對 MARC-145 細胞有細胞毒性的乳酸菌菌株。Plaque 試驗的結 果顯示，PRRSV 力價為 8.6×10! PFU/mL。根據 MTT 試驗搭配 TCID50，稀釋10"倍 的病毒液使 MARC-145 細胞活性低於 50%，將此濃度用於後續實驗。 根據抗病毒試驗，發現部分 LAB 菌株具有抗 PRRSV 的能力。首先，在預處理試 驗中，HH35 和 HH69 菌株顯著提高了 MARC-145 細胞的活性，兩者皆有效減少胞外 的病毒量，此結果與 ISG15、IL-8 有關。在感染後處理試驗中，與 OAS1、TGF-𝛽1、 IL-8 有關，HH09 和 HH32(1)菌株也能顯著提高細胞的活性，兩者皆有效減少胞內外 的病毒量。在競爭試驗中，與 TGF-𝛽1、TNF-𝛼、IL-6、IL-8 有關，HH09 和 HH32(1) 菌株也具有顯著的抗病毒效果，有效減少胞內外的病毒量。此外，與 TGF-𝛽1、TNF- 𝛼、IL-6、IL-8、IL-10 有關，所有 LAB 菌株在無細胞預處理試驗中均呈現有效對抗 病毒的反應，其中 HH32(1) 有效減少胞內的病毒量。這些 LAB 菌株經過 16S rDNA 序列及 API 20 strep 和 API 50 CHL 生化測試分析為 Limosilactobacillus reuteri、 Enterococcus faecium 和 Enterococcus faecalis 相關菌株。HH32(1)菌株在 pH 3 的情況 下具有抗酸特性。HH35、HH69 菌株在抗膽鹽特性上生長曲線雖顯著低於對照組， 但仍能持續生長。HH09 對 Ampicillin、Chloramphenicol、Kanamycin、Spectinomycin、 Streptomycin、Vancomycin 等抗生素不具抗性，因此較不具有抗藥性之疑慮。最後， HH09、HH32(1)、HH35、HH69 菌株皆可抵抗 Salmonella enterica BCRC 12947、 Listeria monocytogenes BCRC 15338 及 BCRC 15378 等病原菌。綜上所述，HH09、 HH32(1)、HH35、HH69 菌株都有潛力成為具有抗 PPRS 病毒效果的益生菌。

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe viral disease affecting pigs. PRRSV disrupts early innate immune responses and primarily causes lung pathology, making it the most significant disease in global swine populations. Probiotics are defined as microbial food supplements that promote health. Probiotics have shown beneficial effects, including the production of antimicrobial substances, modulation of immune responses, and enhancement of host innate defense mechanisms. Importantly, research has demonstrated the potential of probiotics to combat viral infections. The objective of this study is to establish an in vitro model to investigate the antiviral potential of probiotics against PRRSV in MARC-145 cells. Lactic acid bacteria (LAB) strains were isolated from pre-weaned piglets. LAB strains that exhibited cytotoxicity to MARC-145 cells were excluded through the MTT assay. The plaque assay confirmed a viral titer of 8.6 × 10!PFU/mL. Based on the MTT assay and TCID50, a virus dilution of 10#"was determined, resulting in MARC-145 cell viability below 50% for subsequent experiments. The antiviral assays revealed that certain LAB strains displayed significant anti-PRRSV activity. In the pretreatment assay, strains HH35 and HH69 significantly increased the viability of MARC-145 cells while effectively reducing extracellular viral load, which correlated with ISG15 and IL-8. In the post-infection assay, strains HH09 and HH32(1) exhibited a significant increase in cell viability and effectively reduced both intracellular and extracellular viral load, associated with OAS1, TGF-β1, and IL-8. In the competition assay, strains HH09 and HH32(1) demonstrated remarkable antiviral effects, correlated with TGF- β1, TNF-α, IL-6, IL-8, effectively reducing both intracellular and extracellular viral load. Additionally, in the cell-free preincubation assay, all LAB strains exhibited a positive response against the virus, correlated with TGF-β1, TNF-α, IL-6, IL-8, and IL-10, with HH32(1) effectively reducing intracellular viral load. Through 16S rDNA sequencing and biochemical tests using API 20 strep and API 50 CHL, the LAB strains were identified as Limosilactobacillus reuteri, Enterococcus faecium, and Enterococcus faecalis. Strain HH32(1) demonstrated acid resistance at pH 3. While strains HH35 and HH69 exhibited growth curves significantly lower than the control group in terms of bile salt tolerance, they still exhibited sustained growth. Strain HH09 did not exhibit resistance to antibiotics such as Ampicillin, Chloramphenicol, Kanamycin, Spectinomycin, Streptomycin, and Vancomycin, indicating a lower concern for antibiotic resistance. Finally, strains HH09, HH32(1), HH35, and HH69 demonstrated resistance against pathogenic bacteria such as Salmonella enterica BCRC 12947, Listeria monocytogenes BCRC 15338, and BCRC 15378. In conclusion, strains HH09, HH32(1), HH35, and HH69 show potential as probiotics with anti-PRRSV effects.