Investigation of monotherapy and combined anticoronaviral therapies against feline coronavirus serotype II in vitro.

OBJECTIVES: Feline infectious peritonitis (FIP), caused by genetic mutants of feline enteric coronavirus known as FIPV, is a highly fatal disease of cats with no currently available vaccine or US Food and Drug Administration-approved cure. Dissemination of FIPV in affected cats results in a range of clinical signs, including cavitary effusions, anorexia, fever and lesions of pyogranulomatous vasculitis and perivasculitis, with or without central nervous system or ocular involvement. The objectives of this study were to screen an array of antiviral compounds for anti-FIPV (serotype II) activity, determine cytotoxicity safety profiles of identified compounds with anti-FIPV activity and strategically combine identified monotherapies to assess compound synergy against FIPV in vitro. Based upon clinically successful combination treatment strategies for human patients with HIV and hepatitis C virus infections, we hypothesized that a combined anticoronaviral therapy approach featuring concurrent multiple mechanisms of drug action would result in an additive or synergistic antiviral effect. METHODS: This study screened 90 putative antiviral compounds for efficacy and cytotoxicity using a multimodal in vitro strategy, including plaque bioassays, real-time RT-PCR viral inhibition and cytotoxicity assays. RESULTS: Through this process, we identified 26 compounds with effective antiviral activity against FIPV, representing a variety of drug classes and mechanisms of antiviral action. The most effective compounds include GC376, GS-441524, EIDD2081 and EIDD2931. We documented antiviral efficacy for combinations of antiviral agents, with a few examined drug combinations demonstrating evidence of limited synergistic antiviral activity. CONCLUSIONS AND RELEVANCE: Although evidence of compound synergy was identified for several combinations of antiviral agents, monotherapies were ultimately determined to be the most effective in the inhibition of viral transcription.

An Optimized Bioassay for Screening Combined Anticoronaviral Compounds for Efficacy against Feline Infectious Peritonitis Virus with Pharmacokinetic Analyses of GS-441524, Remdesivir, and Molnupiravir in Cats.

Feline infectious peritonitis (FIP) is a fatal disease of cats that currently lacks licensed and affordable vaccines or antiviral therapeutics. The disease has a spectrum of clinical presentations including an effusive ("wet") form and non-effusive ("dry") form, both of which may be complicated by neurologic or ocular involvement. The feline coronavirus (FCoV) biotype, termed feline infectious peritonitis virus (FIPV), is the etiologic agent of FIP. The objective of this study was to determine and compare the in vitro antiviral efficacies of the viral protease inhibitors GC376 and nirmatrelvir and the nucleoside analogs remdesivir (RDV), GS-441524, molnupiravir (MPV; EIDD-2801), and ß-D-N4-hydroxycytidine (NHC; EIDD-1931). These antiviral agents were functionally evaluated using an optimized in vitro bioassay system. Antivirals were assessed as monotherapies against FIPV serotypes I and II and as combined anticoronaviral therapies (CACT) against FIPV serotype II, which provided evidence for synergy for selected combinations. We also determined the pharmacokinetic properties of MPV, GS-441524, and RDV after oral administration to cats in vivo as well as after intravenous administration of RDV. We established that orally administered MPV at 10 mg/kg, GS-441524 and RDV at 25 mg/kg, and intravenously administered RDV at 7 mg/kg achieves plasma levels greater than the established corresponding EC50 values, which are sustained over 24 h for GS-441514 and RDV.