Differential induction of PD-L1 expression in cells infected with feline infectious peritonitis virus and feline enteric coronavirus.

Feline infectious peritonitis (FIP) is a fatal disease driven by feline coronavirus induced immune dysregulation and excessive inflammatory cytokine production. Immunomodulatory agents capable of rebalancing this response are therefore of increasing interest. Phallus indusiatus (P. indusiatus), an edible mushroom containing diverse bioactive compounds, has previously demonstrated antiviral and anti-inflammatory potential. This study investigated the immunomodulatory effects of P. indusiatus extract on peripheral blood mononuclear cells (PBMCs) from healthy cats and FIP cats and characterized its chemical constituents using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). PBMCs were isolated from whole blood and FIP fluid. Cytotoxicity screening identified 19 µg/mL as a non-toxic concentration for subsequent assays. Cytokine responses (IL-1β, IFN-γ, and IL-10) were evaluated following LPS stimulation in PBMCs from whole blood and under basal conditions in PBMCs from FIP fluid after treatment with P. indusiatus extract and dexamethasone. LC-MS/MS profiling combined with STITCH analysis was used to identify bioactive metabolites and their predicted molecular targets. PBMCs derived from FIP fluid exhibited markedly elevated IL-1β and IFN-γ, indicating strong baseline immune activation. P. indusiatus significantly reduced IL-1β and IFN-γ in PBMCs from FIP fluid and suppressed LPS-induced IL-1β and IL-10 in whole-blood PBMCs, demonstrating immunomodulatory patterns comparable to dexamethasone. LC-MS/MS analysis identified compounds including adenosine, phenylalanine, tyrosine, cystathionine, arginine, and sialic acid, which were linked to inflammatory signaling. Overall, the extract exhibited context-dependent modulation of pro- and anti-inflammatory cytokines, suggesting that P. indusiatus may serve as a promising natural adjunctive candidate for managing immune imbalance in cats with FIP.

Feline infectious peritonitis (FIP) is a major disease of cats which, unless promptly diagnosed and treated, is invariably fatal. Although it has long been recognised that the condition is the result of an aberrant immune response to infection with feline coronavirus, there remain significant gaps in our understanding of its pathogenesis. Consequently, diagnosis is complex and relies on the combined interpretation of numerous clinical signs and laboratory biomarkers, many of which are non-specific. In the case of effusive FIP, a commonly encountered acute form of the disease where body cavity effusions develop; the interpretation of fluid analysis results is key to diagnosing the condition. We hypothesised that machine learning could be applied to fluid analysis test data in order to help diagnose effusive FIP. Thus, historical test records from a veterinary laboratory dataset of 718 suspected cases of effusive disease were identified, representing 336 cases of FIP and 382 cases that were determined not to be FIP. This dataset was used to train an ensemble model to predict disease status based on clinical observations and laboratory features. Our model predicts the correct disease state with an accuracy of 96.51%, an area under the receiver operator curve of 96.48%, a sensitivity of 98.85% and a specificity of 94.12%. This study demonstrates that machine learning can be successfully applied to the interpretation of fluid analysis results to accurately detect cases of effusive FIP. Thus, this method has the potential to be utilised in a veterinary diagnostic laboratory setting to standardise and improve service provision.

Feline coronavirus (FCoV) has two biotypes: feline enteric coronavirus (FECV), which typically causes mild intestinal infections, and feline infectious peritonitis virus (FIPV), a virulent form associated with a fatal systemic and infectious disease in cats, known as feline infectious peritonitis (FIP). The differentiation between FECV and FIPV has been linked to specific mutations, most notably M1058L and S1060A, in the spike (S) protein. These mutations are believed to enhance macrophage tropism and facilitate systemic dissemination. However, the precise contribution of these mutations to FIPV virulence remains uncertain, as they have also been reported in non-FIP cases. Here, we report three FIP-positive cases (3/20; 15%) in domestic cats in Hanoi, Vietnam, in which the virus carried the M1058L mutation, while none had the S1060A substitution. These findings contribute to the ongoing debate regarding the role of spike protein mutations in FIP pathogenesis and underscore the need for continued genomic surveillance. Further research is needed to elucidate the molecular determinants involved in the FECV-to-FIPV transition, with potential implications for improving diagnosis, treatment, and prevention strategies for feline infectious peritonitis.

Feline coronavirus (FCoV) infects both domestic and wild felids and has the potential to cause feline infectious peritonitis (FIP), a progressive and often fatal systemic disease. Although rapid diagnosis and treatment are crucial in cases of FIP, conventional reverse-transcription quantitative real-time PCR (RT-qPCR) requires RNA extraction and specialized equipment, limiting its use for timely testing in general veterinary practice. We evaluated the performance of a direct RT-qPCR method using the PicoGene PCR1100 system (GoFoton, Ibaraki, Japan), which omits the RNA extraction step and delivers results within ~40 min. Compared with FCoV culture supernatants and extracted RNA, we estimated the limit of detection of this direct RT-qPCR method to be 150 copies/reaction-a detection sensitivity equivalent to that of conventional RT-qPCR targeting the FCoV 3'-UTR. We observed no cross-reactivity with other feline viruses or SARS-CoV-2. We subsequently analyzed 28 pleural and abdominal effusions collected from cats suspected of having FIP to compare the direct RT-qPCR method with the conventional approach. The sensitivity of the direct RT-qPCR method was 95.5% (95% CI: [78.2, 99.2]) and the specificity was 100% (95% CI: [61.0, 100.0]), which supports the use of the PCR1100 system as a rapid and user-friendly point-of-care tool for the detection of FCoV RNA in effusion samples.

Performance evaluation of a SYBR Green-based real-time quantitative PCR for SARS-CoV-2 detection from animal oropharyngeal samples.

This study investigated the prevalence of 11 common pathogens in dogs and cats in Shenzhen, China, from January 2022 to March 2024, aiming to enhance the understanding of their epidemiological characteristics for improved disease control strategies. Diagnostic testing for the target pathogens was performed using polymerase chain reaction (PCR), colloidal gold test strips, or fluorescence immunoassay. Statistical analysis revealed that among 13,134 cats, Feline Panleukopenia Virus (FPV) showed the highest prevalence (35.83%), followed by Feline Calicivirus (FCV, 26.20%), Feline Infectious Peritonitis Virus (FIPV, 22.00%), and Feline Herpesvirus (FHV, 15.76%). Among 3626 dogs, Canine Parvovirus (CPV) and Canine Distemper Virus (CDV) were predominant, showing a prevalence of 54.55% and 42.83%, respectively. Risk factor analysis showed that most infections occurred in unvaccinated animals and young individuals (<1 year old), with higher incidences in winter and spring. Logistic regression indicated that sex, age, and season were significantly associated with FPV, FHV, and FIPV infections, while age and season were associated with FCV, CPV, and CDV infections (sex showed no association). This study contributes to the epidemiological knowledge of common infectious diseases in dogs and cats, providing a theoretical basis for disease prevention in dogs and cats.

Background and Aim: Feline coronavirus (FeCoV) is a widely circulating Alphacoronavirus that causes mild enteric infections and, in some cases, progresses to Feline infectious peritonitis, a fatal systemic disease. FeCoV consists of two genotypes (I and II) and two biotypes (FeCoV and feline infectious peritonitis virus [FIPV]). Despite its importance, whole-genome data, particularly for FeCoV genotype II, remain limited in Thailand. This study aimed to determine the prevalence of FeCoV in domestic cats and to genetically characterize circulating strains using whole-genome and S gene sequencing. Materials and Methods: A total of 471 rectal swabs were collected from domestic cats presented to private small animal hospitals in Bangkok and neighboring provinces from October 2022 to October 2023. FeCoV detection and genotyping were performed using one-step reverse transcription polymerase chain reaction targeting the 3′UTR and S gene, respectively. Selected FeCoV-positive samples were subjected to whole-genome sequencing (WGS) (n = 4) and complete S gene sequencing (n = 6) using Oxford Nanopore technology with Minimap2, Racon, and Medaka pipelines. Phylogenetic and genetic analyses were conducted using MEGA program. Results: FeCoV positivity was 21.87% (103/471), with higher detection in young cats (&lt;6 months; 28.46%), though age, clinical status, and season showed no significant association (p &gt; 0.05). Genotype I was overwhelmingly predominant (99.03%), whereas genotype II was rare (0.97%). Phylogenetic analysis revealed that Thai FeCoV-I strains clustered closely with Chinese and Dutch FeCoV-I strains, while the FeCoV-II strain grouped with Chinese FeCoV-II. Whole-genome pairwise comparisons showed high nucleotide and amino acid identities with their respective genotype references. No mutations were detected in the S1/S2 or S2 cleavage sites of Thai FeCoV-I, indicating conserved spike characteristics typical of FECoV biotypes. FeCoV-II exhibited the characteristic deletion and insertion patterns known for this genotype. No evidence of recombination with other coronaviruses was observed. Conclusion: This study provides updated molecular epidemiology of FeCoV in Thailand and reports the first complete FeCoV-II genome sequences from the country. The predominance of FeCoV-I and the detection of conserved spike regions highlight the need for genotype-specific surveillance and the reconsideration of vaccine strategies that currently target FeCoV-II. Expanded nationwide monitoring and detailed recombination analyses are warranted to better understand FeCoV evolution and transmission in feline populations. Keywords: domestic cats, FeCov, genotype I and II, molecular epidemiology, phylogenetic analysis, spike (S) gene, Thailand, whole-genome sequencing.

Development and validation of a recombinant N protein-based indirect ELISA for serological detection of feline infectious peritonitis virus.

Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease caused by feline coronavirus (FCoV). FIP was considered untreatable; however, GS-441524, a nucleoside analog, has become a hopeful antiviral treatment. Despite its effectiveness, survival outcomes depend on several prognostic factors, especially the type of disease and clinical presentation. This research aimed to assess the effectiveness of GS-441524 in a large population of cats with FIP in Iran, examine survival rates, and identify crucial prognostic factors affecting treatment outcomes. Additionally, it evaluates alterations in clinical, laboratory, and imaging outcomes during treatment, proposing a secure treatment protocol for veterinarians utilizing GS-441524 for FIP. This retrospective study analyzed 629 cats diagnosed with or highly suspected of having FIP in Iran between December 2020 and March 2024. Diagnosis was based on clinical signs, laboratory findings, ultrasonographic features, and therapeutic responses. Cats received GS-441524 via subcutaneous injection and/or oral administration for a minimum of 12 weeks. Dosages were adjusted according to the FIP form, and clinical, laboratory, and imaging data were collected before, during, and at the end of treatment with GS-441524. Dosages were further adjusted based on FIP form, weight gain, clinical improvement, and laboratory or imaging results. Statistical analyses comprised ANOVA, t-tests, chi-square tests, and non-parametric techniques to pinpoint important prognostic indicators and treatment effects. The survival rate reached 94.12%, with a relapse rate of 0.63%. Most reported type was Effusive forms accounted for 54.84% of the cats. Key prognostic factors associated with reduced survival included being male, over 6 years old, having neurological or mixed forms of FIP, and exhibiting fever, icterus, anemia, and thrombocytopenia (p < 0.05). Significant improvements were observed in parameters such as the A: G ratio, albumin, globulin, bilirubin levels, and changing in imaging findings. The study also assessed dosage modifications during treatment. The average starting dose for effusive forms was 6.9 mg/kg, later increasing to 10.11 mg/kg, while neurological and mixed forms initially required 9.65 mg/kg, which was then raised to 12.7 mg/kg. Treatment duration extended beyond 84 days for 17.32% of cats (109 cats) due to showing abnormalities. Imaging studies confirmed a gradual resolution of abdominal and pleural effusions, reduced lymph node size (both abdominal and mediastinal), decreased kidney size in cats of renomegaly, and regression of gallbladder edema. This research features the largest group of FIP-treated cats in Iran and ranks among the largest worldwide, showcasing impressive survival rates with GS-441524 treatment. Positive results rely on timely intervention, suitable dosage modifications, and extended treatment periods, particularly for cats showing neurological and ocular signs. Major risk factors affecting lower survival rates were fever, icterus, anemia, and low platelet count. These findings provide vital clinical insights for veterinarians, underscoring the need for customized treatment approaches and continued research to enhance FIP outcomes therapy and long-term care.

Beyond macrophages: FIPV tropism includes T and B lymphocytes.

Development of rapid and simple FCoV RNA detection systems using RT-PCR and RT-RPA combined with STH-PAS to diagnose FIP in cats.

Diagnosis of feline infectious peritonitis using the cell tube block technique - a pilot study.

IntroductionElectrophoretic analytical techniques provide extremely important information about an animal’s clinical condition. They are recommended in every case, including in screening tests of animals showing no concerning clinical symptoms. Such tests can detect subclinical conditions, such as inflammation, antigen stimulation or certain forms of cancer. The aim of the study was to determine the suitability of native serum protein electrophoresis and the comet assay for assessing the health status of cats.Material and MethodsElectrophoresis was performed on serum samples from 125 cats. On sera with abnormalities in electropherograms (25 individuals), the following additional analyses were performed: haematological analysis, microscopic examination of a blood smear, plate tests detecting antibodies against feline infectious peritonitis (FIP) and feline immunodeficiency virus (FIV), a plate test detecting feline leukaemia virus (FeLV) surface antigen and a comet assay in peripheral blood lymphocytes.ResultsNative protein electrophoresis enabled the identification of latent disease conditions in individuals assessed as good for overall condition on the basis of clinical examination. Some cats thus assessed had an abnormal electropherogram and were carriers of FIV, FeLV or FIP. In addition, the comet assay identified increased instability in the genetic material of cats with electropherogram abnormalities.ConclusionElectrophoretic techniques can be successfully used as a tools for identifying latent conditions and evaluating the overall health status of cats.

Establishment of an indirect ELISA for feline coronavirus antibody detection and serotype discrimination.

ObjectivesThe present study retrospectively examined effusive feline infectious peritonitis (FIP) cases to investigate whether baseline viral RNA loads and serum biomarkers are associated with treatment responses and to identify early prognostic indicators for clinical decision-making.MethodsFifteen cats with effusive FIP that presented to a primary care veterinary hospital in Japan between August 2024 and August 2025 were included. Diagnosis followed the European Advisory Board on Cat Diseases guidelines, combining clinical presentation, laboratory findings, and feline coronavirus (FCoV) RNA detection by RT-qPCR. Antiviral treatment included GS-441524, remdesivir, molnupiravir, or adjunctive nirmatrelvir. Cats were retrospectively classified as high responders (HR), low responders (LR), or non-responders (NR) based on blood FCoV N gene RNA load 2 weeks after treatment initiation. LR and NR cats were combined (LR/NR, n=10). Viral RNA loads in ascitic fluid and blood, hematology, acute-phase proteins, and serum protein fractions were compared between groups.ResultsAt treatment initiation, the LR/NR group had significantly higher blood N gene RNA loads (p<0.01) and ascitic fluid RNA loads (p<0.05) than the HR group. In contrast, no differences were detected in M gene loads. Hematological markers showed higher total protein, globulin (Glb), and lactate dehydrogenase in the LR/NR group, with no significant differences in albumin (Alb), bilirubin, or SAA. A serum protein fraction analysis revealed distinct profiles: the HR group had higher Alb-to-Glb (A/G) ratios and higher Alb, α1-, α2-, and β-Glb fractions, while the LR/NR group had a markedly higher γ-Glb fraction. Persistence of blood viral RNA 2 weeks after treatment, together with opposing changes in α2- and γ-Glb fractions, emerged as predictors of treatment outcomes.Conclusions and relevanceBaseline blood N gene RNA loads and serum Glb fractions have potential as early prognostic indicators in effusive FIP. These results support combining viral and host biomarkers to improve predictions and monitoring.

Abstract Personal protective equipment (PPE) is important in protecting healthcare workers and individuals from infections, especially in high-risk settings. This study explores the development of advanced antimicrobial coatings for PPE fabrics, incorporating polyethylene glycol and zinc oxide (ZnO) nanoparticles. The antibacterial efficacy was evaluated against Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ), while antiviral performance was assessed using feline coronavirus. Various coating formulations with ZnO concentrations of 0.2, 0.5, 0.75, and 1 wt% were prepared, with the 0.75 wt% formulation demonstrating the highest antimicrobial effectiveness. This coating exhibited 99.9968% antiviral activity and, antibacterial inhibition zones of 30.3 ± 0.6 mm for S. aureus and 29.3 ± 0.6 mm for E. coli . Surface morphology and elemental composition analyzed using SEM, EDX, and AFM, confirmed the uniform distribution of ZnO across the surface. UV-Vis spectroscopy revealed that the coatings retained their transparency, while thermal stability was confirmed through TGA analysis.

Feline herpesvirus type-1 (FHV-1), a double-stranded DNA virus, which is a highly infectious upper respiratory tract infection of felids, particularly in kittens. Droplet digital PCR (ddPCR) provides an absolute quantification method with high sensitivity and accuracy. This study aimed to develop a highly sensitive and accurate ddPCR assay for the detection of FHV-1. We designed primers and a probe targeting the FHV-1 glycoprotein D (gD) gene and evaluated the assay's limit of detection (LOD), sensitivity, repeatability, and specificity in comparison to quantitative real-time PCR (qPCR). The developed ddPCR assay demonstrated a strong linear dynamic range (R2 ≥ 0.99) and an exceptionally low LOD of 0.18 copies/μL, which was significantly more sensitive than the method qPCR (LOD ~10 copies/μL). Additionally, the assay exhibited high specificity with no cross-reactivity against other common feline pathogens (feline calicivirus, FCV; feline panleukopenia virus, FPV; feline infectious peritonitis virus, FIPV; Bordetella bronchiseptica and Chlamydia felis) and displayed outstanding repeatability (inter-run CV < 1.35). When applied to 118 clinical samples, the ddPCR assay achieved a significantly higher positive detection rate (27.4%) compared to qPCR (14.8%). In conclusion, we have successfully established a reliable ddPCR assay for the absolute quantification of FHV-1, providing a superior tool for laboratory diagnosis and research.

Background: The minimized pan-coronavirus (CoV) vaccine-1 developed by our laboratory contained pDNA sequences of feline coronavirus serotype-1 (FCoV1) and SARS-CoV2 (SCoV2) spike B-cell epitopes plus FCoV/SCoV2-conserved, CoV-specific polymerase cytotoxic T-lymphocyte (CTL) epitopes formulated in lipid nanoparticle (LNP). Only FCoV2 infects feline cell lines needed for developing native challenge inoculum that causes feline infectious peritonitis (FIP). Hence, Pilot Study 1 evaluated the therapeutic efficacy and safety of the pan-CoV vaccine-1 in feline immunodeficiency virus (FIV)-infected cats, with or without FCoV1 coinfection. Pilot Study 2 evaluated the cross-protective effect of pan-CoV vaccines in specific-pathogen-free (SPF) cats against intranasal challenge with FIP virus serotype 2 (FIPV2). Methods: In Study 1, we vaccinated two FIV-infected cats (one negative and another positive for FCoV1 coinfection) intramuscularly twice with CTL epitopes-LNP vaccine and later twice with pan-CoV vaccine-1. Controls included two unvaccinated FIV-infected cats with or without FCoV1 coinfection. Study 2 assessed the sequential vaccinations of three pan-CoV vaccines in four SPF cats. The first two vaccinations were with pan-CoV vaccine-2, followed by pan-CoV vaccine-3 (twice), and lastly with pan-CoV vaccine-1 (once). Three SPF controls included two cats immunized with LNP and one lacking any immunization. Pan-CoV vaccine-2 contained pDNAs with modified FCoV1/SCoV2 B-cell epitopes plus CTL epitopes in LNP. Pan-CoV vaccine-3 contained only pDNAs with FCoV1 B-cell epitopes plus CTL epitopes in LNP. Results: Study 1 demonstrated no adverse effect with 25 μg and 50 μg CTL epitopes-LNP vaccine and 50 μg pan-CoV vaccine-1. However, 100 μg pan-CoV vaccine-1 caused fever 24 h later, which was resolved by a single Meloxicam treatment. Both vaccinees developed cross-FCoV2 neutralizing antibodies (XNAbs), immunoblot binding antibodies (bAbs) to FCoV1 receptor-binding domain (RBD), and T-cell responses to FCoV1 RBD, whereas one vaccinee also developed bAbs to SCoV2 RBD. Study 2 demonstrated no adverse effects after each vaccination. Three vaccinees developed low-titer XNAbs and bAbs to FCoV2 spike-2 by the fourth vaccination. Upon challenge, all cats developed FCoV2 NAbs and bAbs to FCoV2 nucleocapsid and RBD. High vaccine-induced T-cell responses to FCoV1 RBD and T-cell mitogen responses declined with an increase in responses to FCoV2 RBD at three weeks post-challenge. Two of the three controls died from FIP, whereas one vaccinee, with the lowest vaccine-induced immunity, died from skin vasculitis lesions and detection of FIPV2 infection by semi-nested RT-snPCR in feces. Conclusions: In Pilot Study 1, the pan-CoV vaccine-LNP dose of 50 μg had no adverse effects, but adverse effects were observed at 100 μg dose. In Pilot Study 2, the FCoV1-based B-cell vaccine(s) induced low levels of XNAbs against FIPV2 and delayed challenge infection against high-dose FIPV2. The high-dose FIPV2 infections in the vaccinated and control cats started to clear, by single housing at 23–26 weeks post-challenge, whereas two cats in Pilot Study 1 cleared natural FCoV1 transmission by 26 weeks post-infection.

Coronaviruses include various strains that reside in natural animal reservoirs, with zoonotic transmission posing risks to both domesticated animals and human health. Recent efforts to address coronavirus infections have focused on developing inhibitors targeting the main protease (Mpro), some of which exhibit potential broad-spectrum efficacy. This study presents crystal structures of four clinically relevant inhibitors—GC376, PF-00835231, nirmatrelvir, and ibuzatrelvir—bound to Mpro from the feline coronavirus strain FECV-UU23. Structural analysis identified distinct FECV-specific features within the active site where these inhibitors bind and revealed S4 loop as a susceptible structural region essential for the enhanced binding of inhibitors in UU23 Mpro. We therefore propose to incorporate sterically constrained, functionally tailored heterocyclic moieties at the P3 site of known inhibitors which can optimally engage Q187, P188, and S189 residues of the S4 loop. The findings presented enhance understanding of inhibitor specificity and reinforce the promise of these inhibitor scaffolds for developing antivirals against feline coronavirus strains, with possible applications in broad-spectrum coronavirus therapy.