具益生菌特性之芽孢桿菌的抗病毒活性評估

Abstract

豬繁殖與呼吸綜合症病毒 ( porcine reproductive and respiratory syndrome virus, PRRSV ) 的感染和傳播對全球養豬業構成嚴重威脅。無論是滅活疫苗還是減毒疫苗都不能有效預防和控制PRRSV的感染和傳播。因此，有必要拓寬新視野並製訂有效的預防策略。先前研究指出芽孢桿菌具有對哺乳動物病毒感染的抑制活性。然而，目前仍未有應用芽孢桿菌萃取物用於抗PRRSV活性相關報導。因此，本研究的目的是評估芽孢桿菌的體外抗PRRSV活性，包括貝萊斯芽孢桿菌AC ( Bacillus velezensis AC )、解澱粉芽孢桿菌LN ( Bacillus amyloliquefaciens LN ) 和地衣芽孢桿菌CK1 ( Bacillus licheniformis CK1 )。在評估芽孢桿菌菌株的體外抗病毒活性時，使用 LN、AC和CK1的細胞內提取物和細胞壁分別來測試它們的抗PRRSV活性。通過3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( MTT ) 測定法測定細胞活力，以證明芽孢桿菌提取物通過直接與病毒結合抑制病毒複製的作用。結果表明，LN、AC和CK1提取物通過抑制病毒貼附、進入有效地抑制了MARC-145細胞中PRRSV的感染，並且通過共培養病毒空斑測試，發現芽孢桿菌萃取物能有效降低病毒的效價，從而降低了病毒對細胞的感染。此外，通過處理芽孢桿菌萃取物後能有效降低因PRRSV 感染而引起的細胞中發炎細胞因子的 mRNA 表現量。總之，芽孢桿菌菌株LN、AC和CK1具有抑制PRRSV感染的潛力，並具有用作抗病毒飼料添加劑的潛力。

The infection and spread of the porcine reproductive and respiratory syndrome virus (PRRSV) has an important influence on the global pig industry. Neither inactivated nor attenuated vaccines can effectively prevent and reduce the infection and spread of PRRSV. There is a necessary need to investigate a new strategy to prevent the PRRSV infection. Previous reports have indicated the ability of probiotic Bacillus to lower the risk of vial infection response in mammals. However, the ability of anti-PRRSV infection with probiotic Bacillus was not reported. Therefore, the aim of this study was to evaluate the anti-PRRSV activities with Bacillus strains in vitro, including Bacillus velezensis AC, Bacillus amyloliquefaciens LN, and Bacillus licheniformis CK1. In the evaluation of antiviral activity of Bacillus strains, the intracellular extracts and cell wall fractions of LN, AC and CK1 from Bacillus were used to measure their anti-PRRSV activity. The cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for the effect of Bacillus extracts on inhibition of viral replication, through directly binding to the virus, internalization, or inhibitory of viral replication. The results showed that LN, AC, CK1 extracts effectively inhibited PRRSV infection in MARC-145 cells by suppressing the viral binding and internalization. On the other hand, the co-culture virus plaque test indicated that the Bacillus extract can effectively reduce the titer and the ability of infection with PRRSV. Moreover, the mRNA expression levels of the pro-inflammatory cytokines in PRRSV-infected MARC-145 cells were decreased by the addition of Bacillus strains LN, AC, and CK1. In conclusion, Bacillus strains LN, AC, and CK1 may have the ability to inhibit PRRSV infection and can be used as an antiviral feed additive.