Feline infectious peritonitis (FIP) is a highly fatal disease caused by a virulent feline coronavirus in domestic and wild cats. We have previously reported the synthesis of potent coronavirus 3C-like protease (3CLpro) inhibitors and the efficacy of a protease inhibitor, GC376, in client-owned cats with FIP. In this study, we studied the effect of the amino acid changes in 3CLpro of feline coronavirus from a feline patient who received antiviral treatment for prolonged duration. We generated recombinant 3CLpro containing the identified amino acid changes (N25S, A252S or K260 N) and determined their susceptibility to protease inhibitors in the fluorescence resonance energy transfer assay. The assay showed that N25S in 3CLpro confers a small change (up to 1.68-fold increase in the 50% inhibitory concentration) in susceptibility to GC376, but other amino acid changes do not affect susceptibility. Modelling of 3CLpro carrying the amino acid changes was conducted to probe the structural basis for these findings. The results of this study may explain the observed absence of clinical resistance to the long-term antiviral treatment in the patients.

[This corrects the article DOI: 10.1371/journal.ppat.1005531.].

Objectives The safety and efficacy of the 3C-like protease inhibitor GC376 was tested on a cohort of client-owned cats with various forms of feline infectious peritonitis (FIP). Methods Twenty cats from 3.3-82 months of age (mean 10.4 months) with various forms of FIP were accepted into a field trial. Fourteen cats presented with wet or dry-to-wet FIP and six cats presented with dry FIP. GC376 was administered subcutaneously every 12 h at a dose of 15 mg/kg. Cats with neurologic signs were excluded from the study. Results Nineteen of 20 cats treated with GC376 regained outward health within 2 weeks of initial treatment. However, disease signs recurred 1-7 weeks after primary treatment and relapses and new cases were ultimately treated for a minimum of 12 weeks. Relapses no longer responsive to treatment occurred in 13 of these 19 cats within 1-7 weeks of initial or repeat treatment(s). Severe neurologic disease occurred in 8/13 cats that failed treatment and five cats had recurrences of abdominal lesions. At the time of writing, seven cats were in disease remission. Five kittens aged 3.3-4.4 months with wet FIP were treated for 12 weeks and have been in disease remission after stopping treatment and at the time of writing for 5-14 months (mean 11.2 months). A sixth kitten was in remission for 10 weeks after 12 weeks of treatment, relapsed and is responding to a second round of GC376. The seventh was a 6.8-year-old cat with only mesenteric lymph node involvement that went into remission after three relapses that required progressively longer repeat treatments over a 10 month period. Side effects of treatment included transient stinging upon injection and occasional foci of subcutaneous fibrosis and hair loss. There was retarded development and abnormal eruption of permanent teeth in cats treated before 16-18 weeks of age. Conclusions and relevance GC376 showed promise in treating cats with certain presentations of FIP and has opened the door to targeted antiviral drug therapy.

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.