First study on in vitro antiviral and virucidal effects of flavonoids against feline infectious peritonitis virus at the early stage of infection.

Background and Aim: Feline infectious peritonitis (FIP), one of the most important infectious diseases in cats is caused by FIP virus (FIPV), a mutated variant of feline coronavirus. Feline infectious peritonitis has a negative impact on feline health, with extremely high mortality in clinical FIP-infected cats, particularly young cats. There are no approved drugs for FIP treatment, and therapeutic possibilities for FIP treatment are limited. This study aimed to utilize nature-derived bioactive flavonoids with antiviral properties to inhibit FIPV infection in Crandell-Rees feline kidney (CRFK) cells. Materials and Methods: The cytotoxicity of 16 flavonoids was evaluated on CRFK cells using a colorimetric method (MTS) assay. Viral kinetics of FIPV at 50 tissue culture infectious dose (TCID50)/well was determined during the first 24-h post-infection (HPI). Antiviral activity was evaluated based on the replication steps of the virus life cycle, including pre-compound, attachment, penetration, post-viral entry, and virucidal assays. The antiviral efficacy of flavonoids against FIPV was determined based on positive FIPV-infected cells with the immunoperoxidase monolayer assay and viral load quantification using reverse transcription-quantitative polymerase chain reaction. Results: Two flavonoids, namely, isoginkgetin and luteolin, inhibited FIPV replication during post-viral entry in a dose-dependent manner, with 50% maximal effective concentrations = 4.77 ± 0.09 and 36.28 ± 0.03 µM, respectively. Based on viral kinetics, both flavonoids could inhibit FIPV replication at the early stage of infection at 0-6-HPI for isoginkgetin and 2-6-HPI for luteolin using a time-of-addition assay. Isoginkgetin exerted a direct virucidal effect that reduced the viral titers by 2 and 1.89 log10 TCID50/mL at 60 and 120 min, respectively. Conclusion: Isoginkgetin interfered with FIPV replication during both post-viral infection and virucidal experiments on CRFK cells, whereas luteolin inhibited the virus after infection. These results demonstrate the potential of herbal medicine for treating FIP.

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses.

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 µM to 25.90 ± 1.40 µM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 µM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 µM and 1.99 ± 0.30 to 4.03 ± 0.60 µM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.

In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus.

The computational search of chemical libraries has been used as a powerful tool for the rapid discovery of candidate compounds. To find small molecules with anti-feline infectious peritonitis virus (FIPV) properties, we utilized a virtual screening technique to identify the active site on the viral protease for the binding of the available natural compounds. The protease 3CL (3CLpro) plays an important role in the replication cycle of FIPV and other viruses within the family Coronaviridae. The 15 best-ranked candidate consensus compounds, based on three docking tools, were evaluated for further assays. The protease inhibitor assay on recombinant FIPV 3CLpro was performed to screen the inhibitory effect of the candidate compounds with IC50 ranging from 6.36 ± 2.15 to 78.40 ± 2.60 µM. As determined by the cell-based assay, the compounds NSC345647, NSC87511, and NSC343256 showed better EC50 values than the broad-spectrum antiviral drug ribavirin and the protease inhibitor lopinavir, under all the test conditions including pre-viral entry, post-viral entry, and prophylactic activity. The NSC87511 particularly yielded the best selective index (>4; range of SI = 13.80-22.90). These results indicated that the natural small-molecular compounds specifically targeted the 3CLpro of FIPV and inhibited its replication. Structural modification of these compounds may generate a higher anti-viral potency for the further development of a novel therapy against FIP.