Stephanie DeWitte-Orr, Wilfrid Laurier University

Daniels, D1, Carr S1, Semple S, DeWitte-Orr SJ1,2 1Department of Biology, Faculty of Science, Wilfrid Laurier University, Waterloo, Ontario, Canada; 2Department of Health Sciences, Faculty of Science, Wilfrid Laurier University, Waterloo, Ontario, Canada

Fighting & Responding

Long double-stranded RNA (LdsRNA; >30 bp) is generated during viral replication and is widely regarded as a potent inducer of the type I interferon (IFN) response. IFN signalling establishes an antiviral state by inducing interferon-stimulated genes (ISGs), recruiting immune cells, activating adaptive immunity, and contributing to inflammation, tissue damage, and energetic costs. However, emerging evidence from our group suggests that LdsRNA engages additional innate pathways depending on its intracellular abundance. We propose a model in which LdsRNA shapes antiviral immunity in a concentration-dependent manner. During early infection, when viral titres and LdsRNA levels are low, LdsRNA is preferentially processed by Dicer into small interfering RNAs (siRNAs), activating the RNA interference pathway (dsRNAi) to suppress viral replication. If this response is insufficient and LdsRNA accumulates, cytoplasmic pattern recognition receptors such as RIG-I and MDA5 are engaged, initiating IFN production. Rather than functioning as mutually exclusive pathways, our findings support the existence of a transitional state in which dsRNAi and IFN responses co-exist and potentially act synergistically. At higher intracellular LdsRNA levels, consistent with uncontrolled infection, viral LdsRNA can be packaged into extracellular vesicles (EVs). These LdsRNA-containing EVs transfer antiviral signals to neighbouring naïve cells, promoting a pre-emptive immune response. Together, this model reframes LdsRNA as a dynamic regulator of innate antiviral immunity, sequentially engaging RNAi, IFN, and EV-mediated signalling as infection progresses.