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.

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.

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.

Chronic diarrhea in cats is a frequent clinical problem, defined by gastrointestinal signs persisting for more than three weeks. Causes are diverse: intestinal dysbiosis, parasites, viral agents, or inflammation. Among protozoa, Tritrichomonas foetus and Cryptosporidium spp. are major contributors, while Feline Coronavirus (FCoV), though often subclinical, can worsen inflammation and dysbiosis, sometimes progressing to infectious peritonitis. This study reports a clinical case with multiple etiologies: T. foetus, Cryptosporidium spp., FCoV, and severe dysbiosis. Diagnostic workup included clinical examination, coproparasitological testing, PCR, and microbiome analysis. Treatment combined symptomatic measures with antiparasitic and antibiotic therapy. Co-infection caused persistent diarrhea and delayed therapeutic response, but PCR confirmation enabled a tailored protocol, gradually improving clinical signs. The case highlights the need for a comprehensive diagnostic approach, as concurrent infections and dysbiosis significantly affect prognosis and therapeutic strategies.

Feline infectious peritonitis (FIP) is a fatal but now treatable disease in cats caused by feline coronavirus (FCoV). This study prospectively investigated viral coinfections in 100 cats diagnosed with FIP and subsequently treated with oral GS-441524 (Bova UK) and their influence on outcome, focusing on viruses potentially associated with feline chronic gingivostomatitis (FCGS). Cats were tested for feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline calicivirus (FCV), feline herpesvirus (FHV), feline foamy virus (FFV), and feline gammaherpesvirus (FcaGHV1). Coinfections were identified at the following frequencies: FCV (27), FFV (22), FHV (6), FIV (4), FcaGHV1 (2), and FeLV (2, both progressive infections). FFV infection was significantly associated with FIV (pF = 0.0021) and FHV (pF = 0.0226) infection. FCGS was present in 25/97 cats with FCV infection being associated with FCGS (pF = 0.0032); no significant associa-tions were found for the other viruses and FCGS. The 42-day oral GS-441524 treatment’s success rate was 94% (five cats died, one relapsed). Coinfections did not significantly influence disease severity or treatment outcome, although the low number of cases for some pathogens warrants further investigation. However, advanced age was associated with treatment failure, potentially due to delayed diagnosis as FIP is considered to be less common in older individuals, or to age-related changes in immune function. In summary, viral coinfections, particularly with FCV, were common and should be considered in the clinical and hygienic management of cats with FIP.

Identification and evaluation of Nordihydroguaiaretic acid (NDGA) as an active traditional Chinese medicine compound inhibiting the 3C-like protease of feline infectious peritonitis virus.

ObjectivesFeline bocavirus (FBoV) is a single-stranded DNA virus of the genus Bocaparvovirus, family Parvoviridae. First identified in 2012, it comprises three species – FBoV-1, FBoV-2 and FBoV-3 – and is globally distributed. Although associated with gastrointestinal disease in cats, its pathogenesis and shedding patterns remain unclear. This study investigated the shedding dynamics of FBoV in naturally infected cats with gastrointestinal signs.MethodsA longitudinal sampling approach was employed in three separate multi-cat households, involving seven symptomatic cats across multiple time points. Initial FBoV screening was performed using conventional PCR and three singleplex TaqMan-based quantitative PCR (qPCR) assays were developed to detect and quantify FBoV-1, FBoV-2 and FBoV-3. The established singleplex qPCR assays were used for subsequent monitoring. Coinfection with other enteric viruses, particularly feline coronavirus (FCoV), was also evaluated.ResultsFBoV-1 and FBoV-2 were detected in multiple cats from house A, with coinfection observed in 5/9 (55.6%) cats and FBoV-1 alone in 1/9 cats. In contrast, only FBoV-1 was identified in cats from houses B and C. FCoV was frequently codetected in all households. qPCR revealed significant variation in viral load over time and across sample types. Positive viral detection persisted for 10–14 days after the resolution of clinical signs in most cases. Notably, one hospital-resident cat continued to present FBoV-1 for up to 65 days.Conclusions and relevanceThis is the first study to characterise FBoV load, and possibly shedding dynamics, in naturally infected cats using route-specific sampling and species-specific quantification. Findings demonstrate that FBoV can be present well beyond the clinical phase of illness, highlighting the possible risk of prolonged transmission or shedding in multi-cat environments. These insights are important for understanding FBoV pathogenesis and developing effective feline disease control strategies.

Structure elucidation and quantification of the active pharmaceutical ingredient in a non-approved drug and in cat serum using QTRAP-MS/MS and ZenoTOF-MS/MS.

Oxidative stress index levels may be used to determine redox balance and oxidative damage of any systemic disease in animals. Feline coronaviruses are large, enveloped, single-stranded RNA viruses that have a wide range of disease forms, from enteric disease to feline infectious peritonitis. In this study, it was aimed to investigate the redox balance, determine the oxidative stress index, and evaluate the antioxidant supplementation in Feline coronavirus–seropositive cats. A total of 60 cats from different breeds, ages, and genders were classified into two groups based on the rapid feline coronavirus Ab test kit (Bionote®, Anigen, Rapid FCoV Ab) results as the Feline coronavirus–seropositive group (n=41) and the Feline coronavirus– seronegative group (n=19). The free radicals determination system was used to measure the reactive oxygen species value (D-Roms test, Diacron; GROMSseto, Italy), and potential antioxidant was measured using the Oxi-adsorbent test (Diacron, Grosseto, Italy). The oxidative stress index wascalculated as reactive oxygen species/potential antioxidant. The mean reactive oxygen species value in Feline coronavirus seropositive cats was not significantly different compared to Feline coronavirus–seronegative cats (p &gt;.05). However, the mean potential antioxidant and oxidative stress index values were significantly higher in the Feline coronavirus–seronegative group compared to the Feline coronavirus–seropositive group (p &lt; .001). This study showed that higher oxidative stress index values were observed in Feline coronavirus–seropositive cats compared to healthy seronegative cats. Antioxidant supplementation may be Cite this article as: Bilgiç, B., Moscati, L., Dokuzeylül, B., Kayar, A., Marenzoni, M. L., &amp; Or, ME. (2025). Redox balance in feline coronavirus–seropositive cats. Acta Veterinaria Eurasia, 51, 0074, doi:10.5152/actavet.2025.25074.

Simple SummaryStray cats (Felis vaga) can carry and spread diseases that affect both other cats and sometimes people. In this study, we wanted to find out how common certain important viruses are among stray cats in Shenzhen, China. We also checked if the cats had protection against rabies, a serious disease that can be passed to humans. We collected samples from 126 stray cats between June and August 2024. Our tests showed that these viruses were very common overall. Most cats were infected with at least one virus, and many were infected with more than one at the same time. We did not find the rabies virus in any cat. However, very few cats had antibodies against rabies, meaning they were not protected from the disease and could potentially spread it if infected. This study tells us that stray cats in Shenzhen commonly carry several cat viruses and have low immunity to rabies. This information is important for planning strategies to manage the stray cat population and to protect both cat and human health.Stray cats (Felis vaga) are key hosts for feline and zoonotic pathogens. From June to August 2024, we conducted a cross-sectional study across six districts in Shenzhen, China, involving 126 cats sampled from three types of sites. Multiple specimens were tested via quantitative real-time PCR (qPCR) for feline coronavirus type I (FCoV-I), feline calicivirus (FCV), feline herpesvirus type I (FHV-I), feline panleukopenia virus (FPV), and rabies virus (RABV); serum was analyzed for RABV-neutralizing antibodies by the fluorescent antibody virus neutralization (FAVN) assay. The overall pathogen positivity was 89.68%. FPV was most prevalent (61.90%), followed by FCV (57.14%), FCoV-I (46.83%), and FHV-I (23.02%). No RABV nucleic acid was detected. The co-infection rate reached 62.70%, primarily dual infections (33.33%). Geographical variation was observed, with significantly higher FCoV-I in Longgang than Futian (p < 0.05). RABV seropositivity was only 6.00%. FCV and FPV co-occurred most frequently (Jaccard = 0.456). All pathogen pairs had relative risk (RR) > 1, suggesting non-random co-infections, though not significant after correction. In summary, major feline pathogens are widespread with frequent co-infections among Shenzhen stray cats, while low rabies immunity indicates potential public health risk. Targeted control measures are warranted.