Polymicrobial Septic Peritonitis Caused by Enterococcus faecium and Enterococcus casseliflavus following Uterine Rupture in a Goat.

A one-year-old female miniature goat was presented to an emergency service after calving a dead goatling. Physical and ultrasonographic examination revealed the presence of a viable fetus; therefore, the goat was submitted to an emergency cesarean section. In the postoperative period, the animal had septic peritonitis caused by Enterococcus faecium and Enterococcus casseliflavus. Both bacterial strains showed contrasting antimicrobial resistance profiles. Laparohysterectomy and abdominal cavity lavage were performed, but, once the animal had adhesions and necrotic lesions in abdominal organs, euthanasia was executed. A post-mortem examination revealed fibrino-necrotic septic peritonitis secondary to uterine rupture. To the authors' knowledge, this is the first detailed report of polymicrobial septic peritonitis in a miniature goat and the first report of septic peritonitis caused by E. faecium and E. casseliflavus.

First analytical confirmation of drug-induced crystal nephropathy in felines caused by GS-441524, the active metabolite of Remdesivir.

GS-441524 is an adenosine nucleoside antiviral demonstrating significant efficacy in the treatment of feline infectious peritonitis (FIP), an otherwise fatal illness, resulting from infection with feline coronavirus. However, following the emergence of COVID-19, veterinary development was halted, and Gilead pursued clinical development of a GS-441524 pro-drug, resulting in the approval of Remdesivir under an FDA emergency use authorization. Despite lack of regulatory approval, GS-441524 is available without a prescription through various unlicensed online distributors and is commonly purchased by pet owners for the treatment of FIP. Herein, we report data obtained from the analytical characterization of two feline renal calculi, demonstrating the propensity for GS-441524 to cause renal toxicity through drug-induced crystal nephropathy in vivo. As definitive diagnosis of drug-induced crystal nephropathy requires confirmation of the lithogenic material to accurately attribute a mechanism of toxicity, renal stone composition and crystalline matrix were characterized using ultra-performance liquid chromatography photodiode array detection (UPLC-PDA), ultra-performance liquid chromatography mass spectrometry (LCMS), nuclear magnetic resonance (NMR) spectroscopy, X-ray powder diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). This work serves to provide the first analytical confirmation of GS-441524-induced crystal nephropathy in an effort to support toxicologic identification of adverse renal effects caused by administration of GS-441524 or any pro-drug thereof.

Antiviral activity of Vigna radiata extract against feline coronavirus in vitro.

Feline infectious peritonitis (FIP) is a fatal illness caused by a mutated feline coronavirus (FCoV). This disease is characterized by its complexity, resulting from systemic infection, antibody-dependent enhancement (ADE), and challenges in accessing effective therapeutics. Extract derived from Vigna radiata (L.) R. Wilczek (VRE) exhibits various pharmacological effects, including antiviral activity. This study aimed to investigate the antiviral potential of VRE against FCoV, addressing the urgent need to advance the treatment of FIP. We explored the anti-FCoV activity, antiviral mechanism, and combinational application of VRE by means of in vitro antiviral assays. Our findings reveal that VRE effectively inhibited the cytopathic effect induced by FCoV, reduced viral proliferation, and downregulated spike protein expression. Moreover, VRE blocked FCoV in the early and late infection stages and was effective under in vitro ADE infection. Notably, when combined with VRE, the polymerase inhibitor GS-441524 or protease inhibitor GC376 suppressed FCoV more effectively than monotherapy. In conclusion, this study characterizes the antiviral property of VRE against FCoV in vitro, and VRE possesses therapeutic potential for FCoV treatment.

Effect of Nucleic Acid Analog Administration on Fluctuations in the Albumin-to-Globulin Ratio in Cats with Feline Infectious Peritonitis.

BACKGROUND: feline infectious peritonitis (FIP) is a fatal disease in cats classified as either effusive ('wet'), non-effusive ('dry'), or a mixture of both forms ('mixed'). The anti-FIP therapeutic effects of Mutian and molnupiravir, two drugs with a nucleic acid analog as an active ingredient, have been confirmed recently. METHODS: Of the cats with FIP, we observed a total of 122 and 56 cases that achieved remission after the administration of Mutian and molnupiravir as routine treatments, respectively. Changes in clinical indicators suggested to be correlated with FIP remission (weight, hematocrit, and albumin-to-globulin ratio) before and after the administration of each drug and during follow-up observation were statistically compared for each FIP type. RESULTS: In all three FIP types, the administration of either Mutian or molnupiravir resulted in statistically significant increases in these indicators. Furthermore, the effect of Mutian on improving the albumin-to-globulin ratio was not observed at all in wet FIP, as compared with that of molnupiravir, but statistically significant in mixed and dry (p < 0.02 and p < 0.003, respectively). The differences in albumin-to-globulin ratio were all due to those of circulating globulin levels. CONCLUSIONS: These results indicate that slight inflammatory responses might be elicited continuously by a residual virus that persisted through molnupiravir treatments.

Detection of Feline Coronavirus in Bronchoalveolar Lavage Fluid from Cats with Atypical Lower Airway and Lung Disease: Suspicion of Virus-Associated Pneumonia or Pneumonitis.

The premortem understanding of the role of feline coronavirus (FeCoV) in the lungs of cats is limited as viruses are seldom inspected in the bronchoalveolar lavage (BAL) specimens of small animal patients. This study retrospectively analyzed the prevalence of FeCoV in BAL samples from cats with atypical lower airway and lung disease, as well as the clinical characteristics, diagnostic findings, and follow-up information. Of 1162 clinical samples submitted for FeCoV RT-nPCR, 25 were BAL fluid. After excluding 1 case with chronic aspiration, FeCoV was found in 3/24 (13%) BAL specimens, with 2 having immunofluorescence staining confirming the presence of FeCoV within the cytoplasm of alveolar macrophages. The cats with FeCoV in BAL fluid more often had pulmonary nodular lesions (66% vs. 19%, p = 0.14) and multinucleated cells on cytology (100% vs. 48%, p = 0.22) compared to the cats without, but these differences did not reach statistical significance due to the small sample size. Three cats showed an initial positive response to the corticosteroid treatment based on the clinical signs and radiological findings, but the long-term prognosis varied. The clinical suspicion of FeCoV-associated pneumonia or pneumonitis was raised since no other pathogens were found after extensive investigations. Further studies are warranted to investigate the interaction between FeCoV and lung responses in cats.

Prevalence and Risk Factors Associated with Feline Infectious Peritonitis (FIP) in Mainland China between 2008 and 2023: A Systematic Review and Meta-Analysis.

To evaluate the overall prevalence of FIP infection in cats in mainland China and associated risk factors, studies on the prevalence of FIP conducted from 1 January 2008 to 20 December 2023 were retrieved from five databases-CNKI, Wanfang, PubMed, Web of Science, and ScienceDirect-and comprehensively reviewed. The 21 studies selected, with a total of 181,014 samples, underwent a rigorous meta-analysis after quality assessment. The results revealed a 2% prevalence of FIP (95% CI: 1-2%) through the random-effects model, showing considerable heterogeneity (I2 = 95.2%). The subsequent subgroup analysis revealed that the age and gender of cats are significant risk factors for FIP infection in mainland China. In order to effectively reduce and control the prevalence of FIP on the Chinese mainland, we suggest improving the immunity of cats, with special attention given to health management in kittens and intact cats, and continuously monitoring FIPV.

Feline infectious peritonitis: A comprehensive evaluation of clinical manifestations, laboratory diagnosis, and therapeutic approaches.

Objective: This study aimed to investigate the clinical and laboratory characteristics of naturally occurring feline infectious peritonitis (FIP) and estimate the median survival time of FIP cats treated with prednisolone to guide further therapeutic planning. Materials and Methods: In this retrospective study, data from a total of 116 cats with effusion were fully recorded. Forty-five FIP-diagnosed cats were enrolled for analysis. Results: The study findings indicate that FIP was a disease affecting cats aged 1-2 years and was highly prevalent among male cats. Clinical manifestations of FIP affected the digestive (60%), hematological (53.3%), respiratory (33.3%), neurological (6.7%), and ocular (4.4%) systems. Blood profiles revealed mild anemia, lymphopenia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, and an albumin to globulin ratio of 0.4. Fluid analysis and cytology of FIP cats demonstrated a transparent yellow fluid with a protein content of 6 gm/dl and a total nucleated cell count of approximately 5,000-10,000 cells. During the observation period, FIP cats treated with prednisolone exhibited a median survival time of 31 days. Conclusion: Confirming FIP cases can be challenging; therefore, a tentative diagnosis of FIP must be made with care. This study provided practical diagnostic tools to diagnose FIP based on clinical signs and multiple abnormalities, which allowed for more efficient and rapid detection.

Pathological findings and patterns of feline infectious peritonitis in the respiratory tract of cats.

Feline infectious peritonitis (FIP) is an important cause of death in cats. Thoracic manifestations are less common than abdominal manifestations, and FIP-associated respiratory disease is poorly documented. This study aimed to investigate pathological findings in the respiratory tract of cats with FIP and the occurrence and distribution of feline coronavirus antigen in the respiratory tract using immunohistochemistry. A retrospective study was carried out on 112 cats with FIP, of which 66 had inflammatory histological lesions in the respiratory tract (58.9%) and were included in this study. Three major gross patterns were defined: marked fibrin deposition in the thoracic cavity with lung atelectasis; marked fibrin deposition in the thoracic cavity with lung pyogranulomas; and lung pyogranulomas without thoracic effusion. Histological analysis revealed primary lesions in the visceral pleura and lung parenchyma at a similar frequency, with multifocal to diffuse presentations. Marked lesions were commonly observed. Five major histological patterns were defined: pleuritis; pleuritis and vasculitis/perivascular injury in the lung parenchyma; pleuritis and pneumonia; perivascular injury in the parenchyma without pleuritis; and pneumonia without pleuritis. In the pleura and pulmonary parenchyma, FIP virus antigen was detected in perivascular and peribronchial macrophages and in macrophages within bronchial-associated lymphoid tissue and foci of necrosis and inflammation in the pleura and lung parenchyma. Co-infections with retroviruses were detected in 47 cats (71.2%), mainly with feline leukemia virus (62.2%). Although FIP is a systemic disease, some cats developed significant lesions in the thoracic cavity, including involvement of the upper respiratory tract and presenting respiratory signs, without other classic signs of FIP. This work advances our knowledge of FIP in the respiratory system, helping veterinarians to recognize the various presentations of this disease.

Compound C inhibits the replication of feline coronavirus.

Feline Coronavirus (FCoV) is a viral pathogen of cats and a highly contagious virus. Cats in a cattery can be infected by up to 100%, and even household cats are infected by 20-60%. Some strains of FCoV are known to induce a fatal disease in cats named Feline Infectious Peritonitis (FIP). However, no effective treatments are available. We demonstrated that compound C (dorsomorphin) can potentially inhibit feline coronavirus replication. Compound C treatment decreased the FCoV-induced plaque formation and cytopathic effect in FCoV-infected cells. Compound C treatment also significantly reduced the amount of viral RNA and viral protein in the cells in a dose-dependent manner. Our findings suggest that compound C is potentially useful for feline coronavirus-related diseases.

Unlicensed antiviral products used for the at-home treatment of feline infectious peritonitis contain GS-441524 at significantly different amounts than advertised.

OBJECTIVE: To analyze the content of unlicensed GS-441524-like products being used as a largely successful at-home treatment for cats suspected to have FIP. The remdesivir content and pH were also measured. SAMPLE: 127 injectable and oral samples from 30 of the most popular brands of black market producers. METHODS: Unlicensed GS-441524-like products were procured through donations and tested for GS-441524 and remdesivir content by liquid chromatography with tandem mass spectrometry. A pH meter measured the pH of injectable samples. RESULTS: Of the 87 injectable formulations, 95% contained more (on average 39% more) GS-441524 than expected based on the producer's marketed concentrations. The average pH (1.30 pH) was well below the physiologic pH conditions recommended for SC injections. The oral formulations were more variable, with 43% containing more GS-441524 (on average 75% more) than expected and 58% containing less (on average 39% less) than the expected content. There was minimal variability in GS-441524 content between replicate samples in the injectables formulations (measured by coefficient of variation). One injectable and 2 oral samples additionally contained remdesivir. CLINICAL RELEVANCE: All unlicensed products used for the at-home treatment of FIP that we tested contain GS-441524. The injectables generally contain significantly more drug than advertised at a below-physiologic pH. Unlicensed oral products vary more widely in drug content and suffer from unconventional dosing and labeling. These data should highlight the need for regulation of these products and the development of legal pathways to procure GS-441524.

An RNA replicon system to investigate promising inhibitors of feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal feline disease, caused by a feline coronavirus (FCoV), namely feline infectious peritonitis virus (FIPV). We produced a baby hamster kidney 21 (BHK) cell line expressing a serotype I FCoV replicon RNA with a green fluorescent protein (GFP) reporter gene (BHK-F-Rep) and used it as an in vitro screening system to test different antiviral compounds. Two inhibitors of the FCoV main protease (Mpro), namely GC376 and Nirmatrelvir, as well as the nucleoside analog Remdesivir proved to be effective in inhibiting the replicon system. Different combinations of these compounds also proved to be potent inhibitors, having an additive effect when combined. Remdesivir, GC376, and Nirmatrelvir all have a 50% cytotoxic concentration (CC50) more than 200 times higher than their half-maximal inhibitory concentrations (IC50), making them important candidates for future in vivo studies as well as clinically implemented drug candidates. In addition, results were acquired with a virus infection system, where Felis catus whole fetus 4 (Fcwf-4) cells were infected with a previously described recombinant GFP-expressing FIPV (based on the laboratory-adapted serotype I FIPV strain Black) and treated with the most promising compounds. Results acquired with the replicon system were comparable to the results acquired with the virus infection system, demonstrating that we successfully implemented the FCoV replicon system for antiviral screening. We expect that this system will greatly facilitate future screens for anti-FIPV compounds and provide a non-infectious system to study and evaluate drug-resistant mutations that may emerge in the FIPV genome.IMPORTANCEFIPV is of great significance in the cat population around the world, causing 0.3%-1.4% of feline deaths in veterinary practices (2). As there are neither effective preventive measures nor approved treatment options available, there is an urgent need to identify antiviral drugs against FIPV. Our FCoV replicon system provides a valuable tool for drug discovery in vitro. Due to the lack of cell culture systems for serotype I FCoVs (the serotype most prevalent in the feline population) (2), a different system is needed to study these viruses. A viral replicon system is a valuable tool for studying FCoVs. Overall, our results demonstrate the utility of the serotype I feline coronavirus replicon system for antiviral screening as well as to study this virus in general. We propose several compounds representing promising candidates for future clinical trials and ultimately with the potential to save cats suffering from FIP.

Feline infectious peritonitis virus ORF7a is a virulence factor involved in inflammatory pathology in cats.

A hyperinflammatory response is a prominent feature of feline infectious peritonitis (FIP), but the mechanisms behind the feline infectious peritonitis virus (FIPV)-induced cytokine storm in the host have not been clarified. Studies have shown that coronaviruses encode accessory proteins that are involved in viral replication and associated with viral virulence, the inflammatory response and immune regulation. Here, we found that FIPV ORF7a gene plays a key role in viral infection and host proinflammatory responses. The recombinant FIPV strains lacking ORF7a (rQS-79Δ7a) exhibit low replication rates in macrophages and do not induce dramatic upregulation of inflammatory factors. Furthermore, through animal experiments, we found that the rQS-79Δ7a strain is nonpathogenic and do not cause symptoms of FIP in cats. Unexpectedly, after three vaccinations with rQS-79Δ7a strain, humoral and cellular immunity was increased and provided protection against virulent strains in cats, and the protection rate reaches 40%. Importantly, our results demonstrated that ORF7a is a key virulence factor that exacerbates FIPV infection and inflammatory responses. Besides, our findings will provide novel implications for future development of live attenuated FIPV vaccines.

Assessing in vitro stability of remdesivir (GS-5734) and conversion to GS-441524 in feline plasma and whole blood.

Feline infectious peritonitis (FIP) is a potentially fatal coronavirus-driven disease of cats. Treatment with nucleoside analogue GS-441524 and or prodrug remdesivir (RDV) have produced remission in both experimentally induced and naturally occurring FIP, yet information regarding metabolism of RDV into GS-441524 in cats is scarce. This study assessed possible phase I metabolism of RDV in cats, utilising an in vitro feline microsome model with in vitro t1/2 and in vitro Clint calculated using the substrate depletion method. A previously validated high-performance liquid chromatography (HPLC) fluorescence method was utilised for detection and analysis of RDV and GS-441524. Qualitative yield of RDV and intermediate metabolite GS-441524 were determined following microsome incubation, then compared to whole blood and plasma incubations. In vitro microsome incubation resulted in rapid depletion of RDV, though it did not appear to resemble a conventional phase I-dependent reaction in cats, as it is in humans and dogs. Depletion of RDV into GS-441524 was demonstrated in whole blood in vitro, suggesting cats convert RDV to GS-441524, likely via blood esterases, as observed in mice and rats. RDV metabolism is unlikely to be impacted by impaired liver function in cats. Furthermore, as RDV depletes within minutes, whereas GS-441524 is very stable, whole blood or plasma GS-441524 concentrations, rather than plasma RDV concentrations, are more appropriate for therapeutic drug monitoring (TDM) in cats receiving RDV.

An Aptamer-Based Proteomic Analysis of Plasma from Cats (Felis catus) with Clinical Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.

Analysis of spike and accessory 3c genes mutations of less virulent and FIP-associated feline coronaviruses in Beijing, China.

Mutations in S and 3c genes of feline coronavirus (FCoV) have been associated with the development of feline infectious peritonitis (FIP). In the present study, FCoV S and 3c genes mutations were analyzed in healthy and FIP cats. M1058L mutation was found in 13.64% (3/22) feces from FIP cats, but not in feces from healthy cats (0/39). The intact 3c gene was found in feces from both healthy cats (19/19) and FIP cats (12/12). All parenteral samples from FIP cats carried one or more of the M1058L mutation, S1060A mutation and mutated 3c gene. FCoV reverse-transcriptase polymerase chain reaction (RT-PCR) of parenteral samples (including ascites, pleural effusions and tissue) is recommended as the gold standard for clinical diagnosis of FIP rather than detection of the M1058L mutation, but when cats have severe gastrointestinal symptoms and lesions, detection of the M1058L mutation in feces may be helpful in diagnosing FIP.

Abdominal ultrasonographic findings of cats with feline infectious peritonitis: an update.

OBJECTIVES: The aim of this study was to describe the abdominal ultrasonographic findings in cats with confirmed or presumed feline infectious peritonitis (FIP). METHODS: This was a retrospective study performed in an academic veterinary hospital. The diagnosis of FIP was reached on review of history, signalment, clinical presentation, complete blood count, biochemistry panel, peritoneal fluid analysis, cytology and/or histopathology results from abnormal organs, and/or molecular testing (immunohistochemical or FIP coronavirus [FCoV] RT-PCR). Cats with confirmed FIP by molecular testing or with a highly suspicious diagnosis of FIP were included. Abdominal ultrasound examination findings were reviewed. RESULTS: In total, 25 cats were included. Common clinical signs/pathology findings included hyperglobulinemia (96%), anorexia/hyporexia (80%) and lethargy (56%). Abdominal ultrasound findings included effusion in 88% and lymphadenopathy in 80%. Hepatic changes were noted in 80%, the most common being hepatomegaly (58%) and a hypoechoic liver (48%). Intestinal changes were noted in 68% of cats, characterized by asymmetric wall thickening and/or loss of wall layering, with 52% being ileocecocolic junction and/or colonic in location. Splenic changes were present in 36% of cats, including splenomegaly, mottled parenchyma and hypoechoic nodules. Renal changes were present in 32%, encompassing a hypoechoic subcapsular rim and/or cortical nodules. Mesenteric and peritoneal abnormalities were seen in 28% and 16% of cats, respectively. Most cats (92%) had two or more locations of abdominal abnormalities on ultrasound. CONCLUSIONS AND RELEVANCE: The present study documents a wider range and distribution of ultrasonographic lesions in cats with FIP than previously reported. The presence of effusion and lymph node, hepatic and/or gastrointestinal tract changes were the most common findings, and most of the cats had a combination of two or more abdominal abnormalities.

Comparison of the Performance of Bioresonance, Electrophoresis and RT-PCR in the Diagnosis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) continues to be one of the most researched infectious diseases of cats. The diagnosis of FIP is challenging, and diverse techniques have been developed for its accurate diagnosis. However, they have some limitations. The present study was conducted to investigate the efficacy of specific modulation frequency (SMF), compared to other routine diagnostic methods for detecting feline coronavirus. Blood samples were collected from 30 diseased cats suspected of having FIP based on clinical signs. Electrophoresis, polymerase chain reaction (PCR), and SMF tests were performed for each sample. The sensitivity and specificity of each test, as well as the agreement between the tests and the gold standard (the combination of PCR, electrophoresis, and bioresonance results), were calculated using the Kappa coefficient method. The sensitivity and specificity of electrophoresis, PCR, and SMF for the diagnosis of FIP were 70.6%, 70.6%, 100%, and 100%, 72.7%, 81.8%, respectively. According to the findings of the present study, SMF is effective and safe in FIP diagnosis, which is a challenge in veterinary medicine diagnosis.

Chitinase-1 Activity in Serum of Cats with FIP.

BACKGROUND: Chitotriosidase (chitinase 1 or CHIT1) is secreted by activated macrophages. Macrophages are involved in the pathogenesis of feline infectious peritonitis (FIP). No reports on CHIT1 activity in cats with FIP are available. OBJECTIVE: To preliminarily investigate the possible changes in serum CHIT1 activity in cats with FIP. METHODS: CHIT1 activity was measured in serum samples from clinically healthy cats (n = 17), cats with FIP (n = 19) and cats with diseases potentially characterized by macrophage activation (n = 20), after a preliminary assessment of the imprecision and linearity of the method. RESULTS: The highest CHIT1 activity was found in cats with FIP, followed by sick cats and clinically healthy cats. The magnitude of the differences between groups was higher than the intra- and inter-assay imprecision of the method (<5% and >57%, respectively). Based on receiver operating characteristic (ROC) curves, CHIT1 may differentiate sick from clinically healthy cats and, to a lesser extent, cats with FIP from cats without FIP. CONCLUSIONS: CHIT1 activity may identify sick cats and, within the appropriate clinical context, cats with FIP, although larger and more standardized studies, coupled with additional information on analytical performances of the method, are required to fully explore the diagnostic or prognostic potential of this test for FIP.

Quantification of GS-441524 concentration in feline plasma using high performance liquid chromatography with fluorescence detection.

The adenosine analogue GS-441524 has demonstrated efficacy in treatment of feline infectious peritonitis (FIP). With no commercially registered formulations of GS-441524 available, global focus shifted to its pro-drug remdesivir, as it became more accessible throughout the COVID-19 pandemic. This study developed and validated a simple liquid chromatography equipped with a fluorescence detector to quantify plasma concentrations of GS-441524 applicable for routine therapeutic monitoring of remdesivir or GS-441524 therapy for FIP infected cats. A Waters X-Bridge C18, 5 µm, 150 × 4.6 mm, column was used and mixtures of 20 mM ammonium acetate (pH 4.5) with acetonitrile of 5% and 70% were prepared for gradient mobile phase. With a simple protein precipitation using methanol to clean plasma sample, GS-441524 was monitored at excitation and emission wavelengths of 250 nm and 475 nm, respectively. Using an external standard, the lowest and highest limits of quantification were 19.5 ng/mL to 10,000 ng/mL, respectively. The intra- and inter day trueness of the quality controls (QCs) were within 10% of their nominal concentrations and intra- and inter day precision of the QCs (expressed as the coefficient of variation) ranged from 1.7 to 5.7%, This assay was able to quantify plasma trough levels of GS-441524 (23.7-190.1 ng/mL) after the administration of remdesivir (9.9-15.0 mg/kg BW, IV or SC) in FIP cats (n = 12). Accordingly, this study generated an alternative and cost-effective way to quantify GS-441524 in feline biological fluids at least up to 24 hr after administrations of remdesivir.