Development and Application of a Dual-Fluorescent Reporter Assay for Feline Infectious Peritonitis Virus RNA-Dependent RNA Polymerase Inhibitor Screening

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.