Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Republic of Korea.
Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Republic of Korea.
Fish Shellfish Immunol. 2024 Sep;152:109767. doi: 10.1016/j.fsi.2024.109767. Epub 2024 Jul 14.
Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to global aquaculture, prompting ongoing efforts to identify potential drug candidates for disease prevention. Coumarin derivatives have recently emerged as a promising class of compounds effective against rhabdoviruses, which severely impact the aquaculture industry. In this study, we assessed the anti-VHSV activity of umbelliferone (7-hydroxycoumarin) in fathead minnow (FHM) cells and olive flounder Paralichthys olivaceus. Umbelliferone exhibited an EC of 100 μg/mL by reducing cytopathic effect, with a maximum cytotoxicity of 30.9 % at 750 μg/mL. Mechanistic analyses via a time-course plaque reduction assay revealed that direct incubation with the virus for 1 h resulted in 97.0 ± 1.8 % plaque reduction, showing excellent direct virucidal activity. Pretreatment for 4 h resulted in a 33.5 ± 7.8 % plaque reduction, which increased with longer incubation times. Cotreatment led to a 33.5 ± 2.9 % plaque reduction, suggesting interference with viral binding, whereas postinfection treatment proved less effective. Umbelliferone was prophylactically administered to the olive flounder through short-term (3 days) and long-term (14 days) medicated feeding, followed by a 4-day postinfection period. Short-term administration at 100 mg/kg body weight (bw)/day resulted in the highest relative percent survival (RPS) of 56 %, whereas long-term administration achieved a maximum RPS of 44 % at 30 mg/kg bw/day. Umbelliferone administration delayed mortality at these doses. Additionally, umbelliferone significantly inhibited the expression of the VHSV N gene during viral challenge, with no observed toxic effects in fish up to an administration dose of 30 mg/kg bw/day for 28 days. Our findings suggest that the protective mechanism of short-term administration of 100 mg umbelliferone against VHSV infection may involve the overexpression of TLR2, MDA5, STAT1, and NF-κB at 24 h postinfection (hpi). IL-8 and IFN II expression was upregulated, whereas TNF-α, IL-1β, and IFN I expression was suppressed at 24 hpi. The upregulation of ISG15 at 48 hpi may contribute to the inhibition of VHSV replication, whereas the downregulation of Caspase 3 expression at 96 hpi suggests a possible inhibition of virus-induced apoptosis at later infection stages. Overall, umbelliferone exhibited anti-VHSV activity through multiple mechanisms, with the added advantage of convenient administration via medicated feed.
病毒性出血性败血症病毒（VHSV）对全球水产养殖业构成重大威胁，促使人们不断寻找潜在的药物候选物来预防疾病。香豆素衍生物最近成为一类有效的抗弹状病毒化合物，这些化合物对水产养殖业有严重影响。在这项研究中，我们评估了伞形酮（7-羟基香豆素）在胖头鲷（FHM）细胞和橄榄石斑鱼中的抗 VHSV 活性。通过减少细胞病变效应，伞形酮的 EC 为 100μg/mL，在 750μg/mL 时最大细胞毒性为 30.9%。通过时间进程蚀斑减少测定的机制分析表明，与病毒直接孵育 1 小时可导致 97.0±1.8%的蚀斑减少，显示出极好的直接病毒杀菌活性。预处理 4 小时可导致 33.5±7.8%的蚀斑减少，随着孵育时间的延长而增加。同时处理可导致 33.5±2.9%的蚀斑减少，表明干扰病毒结合，而感染后处理效果较差。伞形酮通过短期（3 天）和长期（14 天）给药喂养方式预防性给药给橄榄石斑鱼，然后进行为期 4 天的感染后治疗。每天 100mg/kg 体重（bw）的短期给药可获得最高相对存活率（RPS）为 56%，而每天 30mg/kg bw 的长期给药可获得最高 RPS 为 44%。在这些剂量下，伞形酮给药可延迟死亡率。此外，伞形酮在病毒攻毒时显著抑制 VHSV N 基因的表达，在 28 天内每天给药 30mg/kg bw 时，在鱼类中未观察到毒性作用。我们的研究结果表明，短期 100mg 伞形酮给药预防 VHSV 感染的保护机制可能涉及感染后 24 小时（hpi） TLR2、MDA5、STAT1 和 NF-κB 的过度表达。IL-8 和 IFN II 的表达上调，而 TNF-α、IL-1β 和 IFN I 的表达下调。ISG15 在 48 hpi 的上调可能有助于抑制 VHSV 复制，而 Caspase 3 表达的下调可能表明在后期感染阶段可能抑制病毒诱导的细胞凋亡。总的来说，伞形酮通过多种机制表现出抗 VHSV 活性，通过药物饲料给药更加方便。
