State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, the Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China.
Viruses. 2021 Sep 18;13(9):1866. doi: 10.3390/v13091866.
Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.
猪流行性腹泻病毒（PEDV）是导致新生仔猪急性、高度传染性肠病的主要病原体。目前尚无针对 PEDV 感染的批准药物。在这里，我们报告了一种基于纳米荧光素酶（NLuc）的高通量筛选（HTS）平台的开发，以鉴定新型抗 PEDV 化合物。我们构建了一株细胞适应型 PEDV 菌株 YN150 的全长 cDNA 克隆。通过反向遗传学，我们用 NLuc 基因替换了病毒基因组中的开放阅读框 3（ORF3），从而构建了表达 NLuc 的重组 PEDV（rPEDV-NLuc）。rPEDV-NLuc 在体外产生与野生型 PEDV 相似的蚀斑形态，并表现出相似的生长动力学。值得注意的是，rPEDV-NLuc 感染细胞中的荧光素酶活性可稳定检测到，并与病毒滴度呈强正相关。鉴于 NLuc 表达代表 PEDV 复制的直接读数，可通过定量 NLuc 活性轻松鉴定抗 PEDV 化合物。使用该平台，我们从 803 种天然产物文库中筛选出抗 PEDV 化合物，并鉴定出 25 种可显著抑制 PEDV 复制的化合物。有趣的是，在鉴定出的 25 种化合物中，有 7 种是天然抗氧化剂，包括桦木酸、熊果酸、七叶苷、石胆酸、去甲二氢愈创木酸、咖啡酸苯乙酯和葡萄籽提取物。正如预期的那样，所有的抗氧化剂都能有效地减少 PEDV 诱导的氧自由基产生，从而以剂量依赖的方式抑制 PEDV 复制。总之，我们的研究结果为快速筛选针对 PEDV 感染的有前途的治疗性化合物提供了一个强大的平台。
