Oberhoffner Shayne J, Daniels Dominique E, Cooper Erin, Ijaz Aizah, Richardson Starla A, DeWitte-Orr Stephanie J
Department of Biology, Wilfrid Laurier University, 75 University Avenue West, Waterloo, ON, N2L 3C5, Canada.
Department of Health Sciences, Wilfrid Laurier University, Waterloo, Canada.
In Vitro Cell Dev Biol Anim. 2025 Jan 9. doi: 10.1007/s11626-024-01002-1.
Long dsRNA induces the expression of type I interferons (IFNs) and IFN-stimulated genes (ISGs) to establish an antiviral state. When induced prophylactically, this antiviral state can reduce the severity and mortality of viral infections. One of the limiting factors in delivering dsRNA in animal models is the lack of an effective carrier that protects the dsRNA from degradation in the extracellular space. In this study, commercially available cationic liposomes composed of stearylamine, L-α-phosphatidylcholine, and cholesterol were analyzed for their ability to encapsulate and deliver a 621-bp dsRNA sequence. This encapsulated dsRNA was delivered to two Oncorhynchus mykiss cell lines, RTG-2 and RTgill-W1, to activate the IFN pathway and reduce chum salmon reovirus (CSV) infection. EMSA analysis revealed that the liposomes effectively encapsulated 55 and 800 µg/mL doses of dsRNA, remained stable when stored at 4°C and - 20°C, and protected the encapsulated dsRNA from degradation by RNase III. Cell viability assays determined that liposomes loaded with dsRNA were highly cytotoxic after 24 h of exposure. A shorter exposure of 2 h resulted in reduced cytotoxicity and enhanced expression of the ISG Mx1 in both dsRNA alone and dsRNA-liposome-treated cells; however, the elevated Mx1 induction was not sufficient in the dsRNA-liposome treatment group to provide protection against viral infection. Meanwhile, the unencapsulated dsRNA significantly reduced the CSV titer and amount of syncytia formation. Thus, while dsRNA represents an important immune modulator in fish cells, this liposome formulation is too toxic for antiviral applications.
长双链RNA（dsRNA）可诱导I型干扰素（IFN）和干扰素刺激基因（ISG）的表达，从而建立抗病毒状态。预防性诱导时，这种抗病毒状态可降低病毒感染的严重程度和死亡率。在动物模型中递送dsRNA的一个限制因素是缺乏有效的载体来保护dsRNA在细胞外空间不被降解。在本研究中，分析了由硬脂胺、L-α-磷脂酰胆碱和胆固醇组成的市售阳离子脂质体对621 bp dsRNA序列的包封和递送能力。这种包封的dsRNA被递送至两种虹鳟鱼细胞系RTG-2和RTgill-W1，以激活IFN途径并减少鲑鱼呼肠孤病毒（CSV）感染。电泳迁移率变动分析（EMSA）显示，脂质体有效地包封了55和800 µg/mL剂量的dsRNA，在4°C和-20°C储存时保持稳定，并保护包封的dsRNA不被核糖核酸酶III降解。细胞活力测定表明，装载dsRNA的脂质体在暴露24小时后具有高细胞毒性。2小时的较短暴露导致细胞毒性降低，并且单独dsRNA处理和dsRNA-脂质体处理的细胞中ISG Mx1的表达均增强；然而，dsRNA-脂质体处理组中Mx1诱导的升高不足以提供针对病毒感染的保护。同时，未包封的dsRNA显著降低了CSV滴度和多核体形成量。因此，虽然dsRNA是鱼类细胞中的一种重要免疫调节剂，但这种脂质体制剂对于抗病毒应用毒性太大。
