Simple and quantitative detection of double-stranded RNA as a signature of pan-virus infections using a luciferase-based biosensor

Viral infections produce double-stranded RNA (dsRNA) during replication, which trigger host innate immune responses. Immunoassays using anti-dsRNA antibodies have been widely employed to detect viral dsRNA. In this study, we used a luciferase-based dsRNA biosensor which consists of protein kinase R (PKR)-derived dsRNA binding domains fused to split luciferase subunits. Here, we demonstrate the use of the dsRNA biosensor to measure viral dsRNA in RNA specimens extracted from cells infected with Japanese encephalitis virus (JEV). Moreover, the biosensor reacts to a broad-spectrum of dsRNAs from infection with representatives of various viral families including positive- and negative-sense ssRNA viruses, dsRNA viruses, and DNA viruses. We validated the specific interaction between the dsRNA biosensor and viral RNA through RNA immunoprecipitation. Additionally, we observed luminescence signals directly from lysates of JEV-infected cells after cell lysis and phase separation with Triton X-114. Finally, we used the biosensor to assess the activity of antiviral compounds. Collectively, our results demonstrate that the luciferase-based dsRNA biosensor provides a simple, homogeneous, and high-throughput platform for the quantification of viral replication, offering a promising alternative to antibody-based dsRNA detection methods.