Investigating the production of viral double-stranded RNA and its effects on the innate antiviral immune response in fish

我的研究计划的长期目标是研究水生动物对病毒感染的先天免疫反应。养鱼业是加拿大经济的重要贡献者；然而，近年来，它一直受到无法治愈的病毒爆发的困扰，这些病毒导致整个人口死亡，造成重大经济损失。虽然鱼类有免疫系统来保护自己免受病毒感染，但由于病毒逃避机制，它本身并不总是有效的。一种很有希望的治疗策略是触发动物体内已经存在的先天免疫防御来阻止病毒复制。本研究旨在了解鱼类对病毒双链RNA （ds）的先天免疫反应。所有病毒在细胞内复制时都会产生dsRNA，这种dsRNA对细胞起着“标志”的作用，表明它被病毒感染了。细胞感知dsRNA时产生I型干扰素（IFN）；这些干扰素诱导干扰素刺激基因（ISGs）的表达，其作用是通过阻断病毒复制来保护细胞。先前已有研究表明，鱼细胞在合成的dsRNA分子poly IC的作用下产生IFN和isg。然而，没有人表征鱼病毒在鱼细胞中产生的实际天然dsRNA分子，这些分子有可能成为抗病毒反应的最重要调节因子。这项拨款的目的是明确表明鱼类病毒产生dsRNA，并开始对其进行表征。我们还将研究dsRNA如何进入鱼细胞，以及它是否能够诱导ifn和isg，类似于之前用poly IC描述的情况。从对病毒dsRNA知之甚少的情况来看，它的作用似乎与poly IC不同。这一点很重要，因为基于poly IC的药物在临床试验中有严重的副作用。更好地了解病毒dsRNA可以为设计副作用更小的更好的dsRNA疗法提供信息。综上所述，这一建议有助于开发对抗鱼类病毒感染的新策略，并为人类抗病毒机制的进化提供见解。在加拿大，最近在两海岸和五大湖发生的病毒性鱼类死亡事件说明了鱼类病毒感染的重要性。**********

The long-term goal of my research program is to study the innate immune response to virus infections in aquatic animals. Fish farming is an important contributor to Canada's economy; however, in recent years it has been plagued by untreatable viral outbreaks that destroy entire populations causing significant economic loss. Although fish have an immune system to protect themselves from virus infections, it is not always effective on its own due to virus evasion mechanisms. A promising therapy strategy is to trigger innate immune defenses already in place in the animal to block virus replication. The present research proposal aims to understand the fish innate immune response to viral double-stranded (ds)RNA. All viruses make dsRNA when they replicate in a cell, and this dsRNA acts as a 'flag' to the cell, to indicate that it is infected with a virus. Cells make type I interferon (IFN) when they sense dsRNA; these IFNs induce the expression of interferon-stimulated genes (ISGs) whose actions protect the cell by blocking virus replication. It has been previously shown that fish cells make IFN and ISGs in response to a synthetic dsRNA molecule, poly IC. However, no one has characterized the actual native dsRNA molecule produced by a fish virus in a fish cell, and these molecules have the potential to be the most important regulators of antiviral responses. This grant is proposing to show definitively that fish viruses produce dsRNA and to begin its characterization. We will also investigate how dsRNA enters a fish cell and whether it is able to induce IFNs and ISGs similar to what has been previously described with poly IC. From what little is known of viral dsRNA, it appears to act differently than poly IC. This is important because poly IC-based drugs have severe side effects in clinical trials. A better understanding of viral dsRNA could provide information for designing better dsRNA therapeutics with fewer side effects. In summary, this proposal can contribute to the development of new strategies for combating virus infections in fish and provide insight into the evolution of antiviral mechanisms in humans. In Canada the importance of fish viral infections has been illustrated by recent incidents of viral fish kills on both coasts as well as in the Great Lakes. **********