Development and Application of a Dual-Fluorescent Reporter Assay for Feline Infectious Peritonitis Virus RNA-Dependent RNA Polymerase Inhibitor Screening.
- 1Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.
- 2Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.
- 3Graduate Program in Animal Health and Biomedical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.
Abstract
Feline infectious peritonitis virus (FIPV) causes a fatal disease in cats that remains challenging to treat due to the absence of effective vaccines and limited antiviral therapies. Given its central role in viral replication and transcription, RNA-dependent RNA polymerase (RdRp) represents a highly promising and strategic target for antiviral drug development. To enable live-cell monitoring of RdRp activity without external substrates or cell disruption, we developed a biologically validated, cell-based dual-fluorescent reporter assay using green fluorescent protein and red fluorescent protein to evaluate RdRp-dependent reporter activity. Following validation with the well-characterized RdRp inhibitor ribavirin, the dual-reporter platform achieved a Z-factor of 0.54 and a Z'-factor of 0.61. The system was further utilized to assess the antiviral potential of fifteen alkaloid compounds. Among compounds evaluated, lycorine and lycorine hydrochloride exhibited potent inhibition of RdRp-dependent reporter activity and yielded IC₅₀ values of 2.83 ± 0.45 μM and 1.58 ± 0.20 μM, respectively. Further evaluation revealed that both compounds significantly reduced FIPV replication in infected cell. These findings identify lycorine and lycorine hydrochloride as promising anti-FIPV candidates and support further investigation of lycorine-class alkaloids as anti-coronaviral scaffolds. Together, the validated cell-based screening platform and whole-virus antiviral activity provide a foundation for advancing RdRp-targeted antiviral discovery against FIPV.
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BibTeX
@article{semkum2026development,
title = {{Development and Application of a Dual-Fluorescent Reporter Assay for Feline Infectious Peritonitis Virus RNA-Dependent RNA Polymerase Inhibitor Screening.}},
author = {Ploypailin Semkum and Varanya Lueangaramkul and Supichaya Jiwattanasap and Natjira Mana and Nantawan Phetcharat and Porntippa Lekcharoensuk and Sirin Theerawatanasirikul},
journal = {International journal of molecular sciences},
year = {2026},
month = sep,
doi = {10.3390/ijms27198682},
url = {https://europepmc.org/article/MED/42883927},
note = {CC BY},
}
RIS
TY - JOUR
TI - Development and Application of a Dual-Fluorescent Reporter Assay for Feline Infectious Peritonitis Virus RNA-Dependent RNA Polymerase Inhibitor Screening.
AU - Ploypailin Semkum
AU - Varanya Lueangaramkul
AU - Supichaya Jiwattanasap
AU - Natjira Mana
AU - Nantawan Phetcharat
AU - Porntippa Lekcharoensuk
AU - Sirin Theerawatanasirikul
PY - 2026
DA - 2026/09/28
JO - International journal of molecular sciences
DO - 10.3390/ijms27198682
UR - https://europepmc.org/article/MED/42883927
AB - Feline infectious peritonitis virus (FIPV) causes a fatal disease in cats that remains challenging to treat due to the absence of effective vaccines and limited antiviral therapies. Given its central role in viral replication and transcription, RNA-dependent RNA polymerase (RdRp) represents a highly promising and strategic target for antiviral drug development. To enable live-cell monitoring of RdRp activity without external substrates or cell disruption, we developed a biologically validated, cell-based dual-fluorescent reporter assay using green fluorescent protein and red fluorescent protein to evaluate RdRp-dependent reporter activity. Following validation with the well-characterized RdRp inhibitor ribavirin, the dual-reporter platform achieved a Z-factor of 0.54 and a Z'-factor of 0.61. The system was further utilized to assess the antiviral potential of fifteen alkaloid compounds. Among compounds evaluated, lycorine and lycorine hydrochloride exhibited potent inhibition of RdRp-dependent reporter activity and yielded IC₅₀ values of 2.83 ± 0.45 μM and 1.58 ± 0.20 μM, respectively. Further evaluation revealed that both compounds significantly reduced FIPV replication in infected cell. These findings identify lycorine and lycorine hydrochloride as promising anti-FIPV candidates and support further investigation of lycorine-class alkaloids as anti-coronaviral scaffolds. Together, the validated cell-based screening platform and whole-virus antiviral activity provide a foundation for advancing RdRp-targeted antiviral discovery against FIPV.
SN - 1422-0067
ER -