Alpinone is a flavonoid obtained from the resinous exudate of Heliotropium huascoense. This flavonoid shows antiviral activity against the infectious salmon anemia virus (ISAV), which causes severe disease in farmed Atlantic salmon. Here, we aim to elucidate mechanisms underlying the antiviral effects of the flavonoid. In this regard, we evaluated whether Alpinone can act upregulating the pattern-recognition receptor genes, i.e., the RIG-I-like, TLR3, and TLR9 genes, and the genes of the downstream signaling pathways. Transcriptional expression of the genes was analyzed using real-time PCR after 8, 24, and 48 h treatment of salmon kidney adherent cells with 15 μg/mL of Alpinone. First, we showed that Alpinone induced IFNa expression in the kidney adherent cells, indicating that this type of salmon cells is in part responsible for the effects previously reported in vivo. Upregulation of the IFN-induced myxovirus resistance (Mx) gene was also observed in the head kidney cells in response to the treatment. Overexpression reached a maximum level at 24 h post-treatment. Interestingly, Alpinone also induced upregulation of the cytosolic receptors of ssRNA, named Retinoic acid-inducible gene I (RIG-I) and Melanoma Differentiation-Associated protein 5 (MDA5), but there were no effects on the transcriptional expression of the TLR3 and TLR9 endosomal receptors. In addition, Alpinone upregulated the expression of genes encoding the main components of the RIG-I/MDA5 signaling pathways, such as the mitochondrial antiviral-signaling protein (MAVS), TNF Receptor Associated Factor 3 (TRAF3), TANK-binding kinase 1 (TBK1), I-kappaB kinase ε (IKKε), the transcription factors IRF-3, and IRF7. The increased expression of all these genes is consistent with the upregulation of IFNa and Mx mRNAs. Because BX795 completely prevents Alpinone-dependent upregulation of IFNa and IRF3, the flavonoid targets seem to be upstream of the kinases TBK1 and IKKε. Altogether, this study contributes to elucidating the mechanisms involved in Alpinone antiviral activity in fish. Alpinone can be used to counteract virus mechanisms of evasion where the onset of interferon-mediated response is prevented or delayed.

Alpinone 是从 Heliotropium huascoense 的树脂渗出物中获得的类黄酮。这种类黄酮对传染性鲑鱼贫血病毒 (ISAV) 具有抗病毒活性，这种病毒会导致养殖的大西洋鲑鱼患上严重疾病。在这里，我们旨在阐明黄酮类化合物抗病毒作用的潜在机制。在这方面，我们评估了Alpinone是否可以上调模式识别受体基因，即RIG-I样、TLR3和TLR9基因，以及下游信号通路的基因。在用 15 μg/mL Alpinone 处理鲑鱼肾贴壁细胞 8、24 和 48 小时后，使用实时 PCR 分析基因的转录表达。首先，我们发现 Alpinone 在肾贴壁细胞中诱导 IFNa 表达，在体内。在响应治疗的头肾细胞中也观察到干扰素诱导的粘病毒抗性（Mx）基因的上调。过表达在治疗后 24 小时达到最高水平。有趣的是，Alpinone 还诱导 ssRNA 的胞质受体上调，称为视黄酸诱导基因 I (RIG-I) 和黑色素瘤分化相关蛋白 5 (MDA5)，但对 TLR3 和 TLR9 的转录表达没有影响内体受体。此外，Alpinone 上调了编码 RIG-I/MDA5 信号通路主要成分的基因的表达，如线粒体抗病毒信号蛋白 (MAVS)、TNF 受体相关因子 3 (TRAF3)、TANK 结合激酶 1 ( TBK1)，I-kappaB 激酶ε(IKKε)、转录因子 IRF-3 和 IRF7。所有这些基因的表达增加与 IFNa 和 Mx mRNAs 的上调一致。因为 BX795 完全阻止了依赖于 Alpinone 的 IFNa 和 IRF3 上调，黄酮类化合物靶标似乎位于激酶 TBK1 和 IKKε 的上游。总而言之，这项研究有助于阐明鱼中 Alpinone 抗病毒活性所涉及的机制。Alpinone 可用于抵消病毒的逃避机制，其中干扰素介导的反应的发生被阻止或延迟。