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  3. Universal and quantitative detection of double-stranded RNAs as a signature of pan-virus infections using a luciferase-based biosensor

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

  • J Biol Eng. 2025 Dec 16. doi: 10.1186/s13036-025-00601-0.
Michihito Sasaki 1 2,  Eri Fujii 3,  Satoko Sasaki 3,  Takuma Ariizumi 3,  Kei Konishi 4 5,  Akihiko Sato 6 4 5,  William W Hall 7 8 9,  Hirofumi Sawa 6 4 7 9,  Yasuko Orba 10 11 12 13
Affiliations

Affiliations

  • 1 Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. [email protected].
  • 2 Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan. [email protected].
  • 3 Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan.
  • 4 Division of Anti-Virus Drug Research, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan.
  • 5 Laboratory for Drug Discovery & Disease Research, Shionogi & Co., Ltd., Osaka, Japan.
  • 6 Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan.
  • 7 International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan.
  • 8 National Virus Reference Laboratory, School of Medicine, University College of Dublin, Dublin, Ireland.
  • 9 Global Virus Network, Baltimore, MD, USA.
  • 10 Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. [email protected].
  • 11 Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan. [email protected].
  • 12 Division of Anti-Virus Drug Research, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. [email protected].
  • 13 International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. [email protected].
PMID: 41402836 DOI: 10.1186/s13036-025-00601-0
Abstract

Background: Infections with various RNA viruses and certain DNA viruses may 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 for viral dsRNA detection, which consists of protein kinase R (PKR)-derived dsRNA binding domains fused to split luciferase subunits and is available as part of a commercial system.

Results: 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 single-stranded RNA (ssRNA) viruses, dsRNA viruses, and DNA viruses. We validated the specific interaction between the dsRNA biosensor and viral RNA including subgenomic Flavivirus RNA (sfRNA) 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.

Conclusions: Our results demonstrate that the luciferase-based dsRNA biosensor offers a simple, homogeneous, and high-throughput platform for quantifying viral replication, presenting a promising alternative to antibody-based dsRNA detection methods.

Keywords

Bioluminescence; DNA virus; Flavivirus; PKR; RNA binding protein; RNA structure; RNA virus; dsRNA.

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