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Feline infectious peritonitis virus (FIPV: virulent feline coronavirus) causes a fatal disease called feline infectious peritonitis (FIP) in wild and domestic cat species. Recent studies identified several antiviral drugs that are effective against FIPV. Drug combination is one of the important strategies in the development of novel treatments for viral infections. GS-441524, a nucleoside analog, and itraconazole, a triazole antifungal drug, have been reported that have antiviral effect against FIPV. This study aims to investigate whether the combination of GS-441524 and itraconazole has synergic antiviral effect against FIPV. The antiviral effect was measured by plaque reduction assay using felis catus whole fatus-4 cell. The plaque reduction of GS-441524 against type I FIPVs increased as the concentration of itraconazole increased. The similar result was obtained for type II FIPV. In addition, the calculated combination index (CI) demonstrated that there was a strong synergy between GS-441524 and itraconazole. It is concluded that the combination of GS-441524 and itraconazole may enhance the individual effect of each drug against replication of type I FIPVs and may contribute to development more effective treatment strategy for FIP.

Feline infectious peritonitis (FIP) is a fatal systemic disease of felids caused by a Coronavirus (CoV) (FIPV). In spite of its clinical relevance and impact on feline health, currently the therapeutic possibilities for treatment of FIP in cats are limited. The emergence of the pandemic Severe Respiratory Syndrome (SARS) coronavirus (CoV) type 2 (SARS-CoV-2), etiological agent of the 2019 Coronavirus Disease (COVID-19), able to infect a broad spectrum of animal species including cats, triggered the interest for the development of novel molecules with antiviral activity for treatment of CoV infections in humans and animals. Essential oils (EOs) have raised significant attention for their antiviral properties integrating and, in some cases, replacing conventional drugs. Thymus vulgaris EO (TEO) has been previously shown to be effective against several RNA viruses including CoVs. In the present study the antiviral efficacy of TEO against FIPV was evaluated in vitro. TEO at 27 μg/ml was able to inhibit virus replication with a significant reduction of 2 log10 TCID50/50 μl. Moreover, virucidal activity was tested using TEO at 27 and 270 μg/ml, over the cytotoxic threshold, determining a reduction of viral titre as high as 3.25 log10 TCID50/50 μl up to 1 h of time contact. These results open several perspectives in terms of future applications and therapeutic possibilities for coronaviruses considering that FIPV infection in cats could be a potential model for the study of antivirals against CoVs.

Feline hypertrophic cardiomyopathy (HCM) is characterized by macrophage-driven myocardial remodeling processes in a pro-inflammatory environment. To further investigate the mechanisms behind these processes, the myocardial transcription of cytokines and remodeling enzymes was comparatively assessed in cats with HCM and cats without cardiac diseases. Sixty-seven cats were included, 17 cats with HCM (including 5 with atrial thrombus; AT), and 50 cats without cardiac diseases. The latter comprised 10 control cats (no cardiac or relevant systemic disease), 34 cats with diseases suspected to be associated with a systemic inflammatory state of which 18 suffered from feline infectious peritonitis (FIP), and 6 cats with multicentric lymphoma. Samples from atria, ventricular free walls and interventricular septum were examined using quantitative reverse transcriptase PCR. The overall highest myocardial marker transcriptions were observed in cats with multicentric lymphoma, FIP and HCM, followed by diseases likely associated with a systemic inflammatory state, and control cats. Inflammatory marker transcription predominated in the myocardium of cats with systemic inflammatory diseases, whereas in HCM the transcription of remodeling enzymes prevailed. Sex significantly influenced the myocardial transcription of several remodeling enzymes. These results suggest a versatile myocardial response depending on the disease and illustrates the relevance of sex for the cardiac response to cardiac and systemic disease in cats. A systemic inflammatory state appears to elicit an inflammatory phenotype in the myocardium, whereas in HCM, the myocardium mediates its own remodeling. In HCM, the identified markers might be involved in the ongoing remodeling processes causing structural and functional changes.

Feline coronavirus (FCoV) exists as two different genotypes, FCoV type I and II, each including two biotypes, feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), the latter being a virulent variant originating from the former virus. Recently, two amino acid substitutions, M1058L and S1060A, within the spike protein have been associated to the FECV/FIPV virulence change. In this study, we have analysed the frequency of detection of such mutations in FIPV and FECV strains circulating in Italian cats and obtained information about their evolutionary relationships with reference isolates. A total of 40 FCoV strains, including 19 strains from effusions or tissue samples of FIP cats and 21 strains from faecal samples of non-FIP cats, were analysed. Mutation M1058L was detected in 16/18 FCoV-I and 1/1 FCoV-II strains associated with FIP, while change S1060A was presented by two FIPV strains. By phylogenetic analysis, FCoV sequences clustered according to the genotype but not according to the biotype, with FECV/FIPV strains recovered from the same animal being closely related. Further studies are needed to better define the genetic signatures associated with the FECV/FIPV virulence shift.

Infection with feline coronavirus (FCoV) is a major problem in multiple-cat households, where many cats are kept together in a small space such as catteries and shelters. Sixty cats from 19 breeding catteries included in the study were evaluated for their shedding persistency and intensity patterns using qPCR identification of FCoV in feces. Cats were identified based on shedding persistency as non-shedders (NS) if all four samples negative, intermittent shedders (IS) when at least one positive and one negative sampling followed by another positive sampling, persistent shedders (PS) if all four samples positive and shedders with unclear status (US) if the shedding patterns could not be determined based on only 4 samples. There were 11 NS (18%), 15 IS (25%) and 15 PS (25%) and in 19/60 cats (32%), the shedding patterns could not be determined based only on four samplings. The intensity of shedding was evaluated based on the total number of FCoV particles shed during the 12 months of the study. There were 11 non-shedders (18%), 2 very low intensity shedders (3%), 9 low intensity shedders (15%), 25 medium intensity shedders (42%) and 13 high intensity shedders (22%). Intermittent shedders were shedding significantly lower FCoV particles compared to the persistent shedders (p = 0.0082). Permanent shedders represent the most important source of FCoV infection in multi-cat households and identifying permanent shedders in is the key to minimize the viral load in the environment to control FCoV in a shelters and breeding catteries.

Feline coronavirus (FCoV) is common among cats living indoors in groups. In about 10% of infected cats, a potentially lethal disease, feline infectious peritonitis (FIP) occurs. Virus transmission is faecal-oral. Mutian® Xraphconn (Mutian X) is a product marketed to treat cats with FIP but is also being used to stop virus shedding, although no clear guidelines exist for its use for this purpose. The aim of this study was to establish the minimum dose and treatment duration required to ensure viral clearance from the faeces of asymptomatic virus-shedding cats. In five multicat households, 29 cats naturally infected with FCoV and actively shedding virus in the faeces were given Mutian X pills. Virus shedding was monitored using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) controlled for faecal inhibitors to ensure sensitivity. Mutian X given orally cleared the virus in 29 cats; although four cats required a repeated course to finally stop virus shedding. A dose of 4 mg/kg q24 h for four days was found to be the optimal treatment protocol: 2 mg/kg cleared only 80% of cats. Post-treatment using a sensitive RT-qPCR test was essential to ensure that virus clearance had been achieved, since failure to clear even one cat can result in re-infection of the others. Records of virus shedding by cats before treatment provided a retrospective control: significantly more cats stopped shedding virus after Mutian X than recovered from infection during the control period (p, Highlights • Feline infectious peritonitis (FIP) is caused by feline coronavirus infection • The source of feline coronavirus (FCoV) infection is faeces of virus shedding cats • Treatment of 29 cats for 4 days with Mutian X pills stopped FCoV shedding • Treated cats tested for up to 157 days following treatment remained virus-free

Specific pathogen free kittens were vaccinated with an unattenuated field isolate of canine coronavirus (CCV) either by aerosol or subcutaneously, and received boosting vaccinations four weeks later. Aerosolisation elicited a homologous virus-neutralising (VN) antibody response that increased steadily over a four-week period and levelled off one to two weeks after revaccination. The initial aerosolised dose produced an asymptomatic infection with excretion of CCV from the oropharynx up to eight days after vaccination; virus shedding was not detected, however, after the second inoculation. Cats vaccinated subcutaneously developed low VN antibody titres after the first CCV dose and experienced a strong anamnestic response after the second dose. Neutralising antibody titres then levelled off one to two weeks after revaccination at mean values somewhat lower than in cats vaccinated by aerosol. CCV was not isolated from the oropharynx after either subcutaneous dose. Four weeks after CCV boosting inoculations, vaccinated cats and sham-vaccinated control cats were divided into three subgroups and challenged by aerosol with the virulent UCD1 strain of feline infectious peritonitis virus (FIPV UCD1) at three different dosage levels. Five of six cats (including sham-vaccinated controls) given the lowest challenge dose showed no signs of disease, while all other cats developed lesions typical of feline infectious peritonitis (FIP). The five surviving cats developed FIP after subsequent challenge with a fivefold higher dose of FIPV. Thus heterotypic vaccination of cats with CCV did not provide effective protection against FIPV challenge.

Feline infectious peritonitis virus (FIPV) replication in macrophages/monocytes induced tumor necrosis factor (TNF)-alpha production, and that the TNF-alpha produced was involved in aggravating the pathology of FIP. We previously reported the preparation of a feline TNF-alpha (fTNF-alpha)-neutralizing mouse monoclonal antibody (anti-fTNF-alpha mAb). This anti-fTNF-alpha mAb 2–4 was confirmed to inhibit the following fTNF-alpha-induced conditions in vitro. In the present study, we investigated whether mAb 2–4 improved the FIP symptoms and survival rate of experimentally FIPV-inoculated SPF cats. Progression to FIP was prevented in 2 out of 3 cats treated with mAb 2–4, whereas all 3 cats developed FIP in the placebo control group. Plasma alpha1-glycoprotein and vascular endothelial growth factor levels were improved by the administration of mAb 2–4, and the peripheral lymphocyte count also recovered. These results strongly suggested that the anti-fTNF-alpha antibody is effective for the treatment of FIP., Highlights • Feline infectious peritonitis (FIP) is a coronavirus-induced fatal disease in cats. • We investigated therapeutic effect of anti-fTNF-α mAb for experimental FIP infection. • Anti-fTNF-α mAb improved the FIP symptoms and survival rate in 2 of 3 cats. • Anti-fTNF-α mAb improved plasma AGP and VEGF level and lymphopenia. • The results suggested the anti-fTNF-α mAb may be effective for the treatment of FIP.

Feline infectious peritonitis (FIP) is a feline coronavirus (FCoV)-induced fatal disease in wild and domestic cats. There are two FCoV serotypes. Both type I and II FCoV can replicate in Felis catus whole fetus (fcwf)-4 cells, but the replicability of type I FCoV in feline cell lines is lower than that of type II FCoV, the reason for which is unclear. Inhibition of IFNβ production by non-structural and structural proteins, excluding spike protein has been reported in many coronavirus infections. In this study, we investigated whether IFNβ is involved in the difference in replicability in feline cell lines between types I and II FCoV. When fcwf-4 cells were infected with FCoV, the virus titer of type II FCoV in the culture supernatant was higher than that of type I FIPV. When the IFNβ expression level in FCoV-infected fcwf-4 cells was semi-quantitatively analyzed, infection with type I FIPV, excluding type I FIPV UCD-1, highly induced IFNβ expression. In contrast, induction of IFNβ by type II FCoV infection was significantly lower than that by type I FIPV. In addition, when fcwf-4 cells were adsorbed by FIPV and then stimulated with Poly(I:C), type II FCoV infection inhibited Poly(I:C)-induced IFNβ gene expression. Also, the proliferation of type I FIPV was enhanced by a IFN inhibitor. These findings clarified that, unlike type I FIPV, type II FCoV strongly inhibits IFNβ expression in infected cells. It was also suggested that the IFNβ-inducing ability is different among type I FIPV strains., Highlights • Feline coronavirus is classified into 2 serotypes. • Induction of IFNβ by type II FCoV infection was lower than that by type I FCoV. • Type II FCoV infection inhibited Poly(I:C)-induced IFNβ gene expression. • Type II FCoV strongly inhibits IFNβ expression in infected cells.

Intra-vitam diagnosis of feline infectious peritonitis (FIP) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. With the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive FIP were collected intra-vitam for detection of feline coronavirus (FCoV) antibodies and RNA by means of indirect immunofluorescence (IIF) assay and real-time RT-PCR (qRT-PCR), respectively. In 5 effusions there was no evidence for either FCoV RNA or antibodies, 51 and 52 specimens tested positive by IIF and qRT-PCR, respectively, although antibody titres ≥ 1:1600, which are considered highly suggestive of FIP, were detected only in 37 effusions. Three samples with high antibody levels tested negative by qRT-PCR, whereas 18 qRT-PCR positive effusions contained no or low-titre antibodies. qRT-PCR positive samples with low antibody titres mostly contained low FCoV RNA loads, although the highest antibody titres were detected in effusions with CT values > 30. In conclusion, combining the two methods, i.e., antibody and RNA detection would help improving the intra-vitam diagnosis of effusive FIP., Highlights • Effusions from cats with suspected FIP were analysed for detection of feline coronavirus (FCoV) antibodies and RNA. • Using a cut-off of 1:1600 for FCoV antibodies, only 40/61 samples were in agreement between the two tests. • Most effusions with low FCoV loads were found to contain low specific antibody titres. • Combining serological and molecular methods results in an increase of the diagnostic efficiency.

Highlights • Holy Birman cats (HB) seem to be predisposed to feline infectious peritonitis (FIP). • In this study, indicators of innate and specific immunity were assessed in HB cats. • CD4/CD8, IFN-γ, IL12/IL4 were higher and IL-4 was lower in HB than in other breeds. • The rate of shedders, but not of seropositivity, was lower in HB than in other cats. • Th1 immunity seems to be stronger in HB. This may reduce the rate of shedders., The aim of this study was to assess whether Holy Birman cats (HB) have a peculiar immune profile and a higher rate of infection by feline coronaviruses (FCoV). Leucocyte and lymphocyte subsets, antibody titers, α1-acid glycoprotein (AGP), globulin fractions, IL-4, IL-12 and IFN-γ in blood and fecal FCoV excretion were determined in HB (n = 75) and in cats from other breeds (n = 94). Significantly higher CD4/CD8 ratio, IFN-γ concentration and IL12/IL4 ratio and significantly lower IL-4 concentration and proportion of shedders were found in HB than in other breeds. No other differences were found. In conclusion, this study did not provide evidence of peculiar immune profiles in HB, except for a prevalent Th1 profile, that may explain why in our caseload the rate of shedders was lower in HB than in other breeds.

Highlights • ORF 3c is affected by deletions and stop codons more frequently than ORF 7b. • Deletions in ORF 3c are not a compelling feature of FIPV. • Sequences of different tissue specimens within one cat are mostly identical. • PCR amplification of ORF 7b is more efficiently than that of ORF 3c., Feline infectious peritonitis (FIP) is the most frequent lethal infectious disease in cats. However, understanding of FIP pathogenesis is still incomplete. Mutations in the ORF 3c/ORF 7b genes are proposed to play a role in the occurrence of the fatal FIPV biotype. Here, we investigated 282 tissue specimens from 28 cats that succumbed to FIP. Within one cat, viral sequences from different organs were similar or identical, whereas greater discrepancies were found comparing sequences from various cats. Eleven of the cats exhibited deletions in the 3c gene, resulting in truncated amino acid sequences. The 7b gene was affected by deletions only in one cat. In three of the FIP cats, coronavirus isolates with both intact 3c genes as well as 7b genes of full length could also be detected. Thus, deletions or stop codons in the 3c sequence seem to be a frequent but not compelling feature of FIPVs.

Feline infectious peritonitis (FIP) is a lethal infectious disease affecting domestic and wild cats. Several reports suggested that TNF-alpha is related to the progression of FIP. Thus, the administration of a feline TNF-alpha-neutralizing antibody to cats with FIP may reduce the disease progression. In this study, we have prepared nine monoclonal antibodies (MAbs) that recognize feline TNF-alpha. All MAbs neutralized recombinant TNF-alpha. The 50% inhibitory concentrations (IC50) of the MAbs for the cytotoxicity of recombinant TNF-alpha were 5–684 ng/ml. MAb 2–4 exhibited high neutralizing activity against natural TNF-alpha derived from FIPV-infected macrophages, and was confirmed to inhibit the following feline TNF-alpha-induced conditions in vitro: (i) an increase in the survival rate of neutrophils from cats with FIP, (ii) aminopeptidase N (APN) mRNA expression in macrophages, and (iii) apoptosis of a feline T-lymphocyte cell line.

Highlights • Serum oxidative stress biomarkers in cats with feline infectious peritonitis (FIP) were studied. • Serum paraoxonase-1 (PON1) with three different substrates and total antioxidant capacity (TAC) were measured. • PON1 decreased in cats with FIP as well as with other inflammatory conditions. • TAC decreased only in cats with feline infectious peritonitis. • The study demonstrated a situation of oxidative stress with changes in both PON1 and TAC in cats with FIP., The purpose of this study was to elucidate the possible presence of oxidative stress in cats naturally affected by feline infectious peritonitis (FIP) by investigating two antioxidant biomarkers in serum: paraoxonase-1 (PON1) and total antioxidant capacity (TAC). PON1 was measured by spectrophotometric assays using three different substrates: p-nitrophenyl acetate (pNA), phenyl acetate (PA) and 5-thiobutil butyrolactone (TBBL), in order to evaluate possible differences between them. The PA and TBBL assays for PON1 and the assay for TAC were validated, providing acceptable precision and linearity although PA and TAC assays showed limit of detection higher than the values found in some cats with FIP. Cats with FIP and other inflammatory conditions showed lower PON1 values compared with a group of healthy cats with the three assays used, and cats with FIP showed significant decreased TAC concentrations. This study demonstrated the existence of oxidative stress in cats with FIP.

In order to identify parameters differentiating exudative from transudative effusions, it was postulated that the activity of adenosine deaminase (AD) (EC 3.5.4.4) might be highly correlated with granulomatous inflammatory processes of the serosa, and the activity of the enzyme in body effusions and serum from cats was examined. The method of Slaats et al (1985) for the determination of the enzyme was evaluated by using an Hitachi 705 autoanalyser, and its activity was measured in body cavity effusions of 174 cats. The activity of AD was high in effusions from cats with infectious serositis and bacterial or feline infectious peritonitis (FIP). In cases of FIP the activity of AD was very significantly different from all other cases of thoracic (P = 0.004) and abdominal (P0.0001) effusions. The determination of AD in the serum of cats did not contribute to the aetiological differentiation of hydrops. The increases in the activity of AD appeared to originate from the body effusion, because the ratio of the activity of the enzyme in the effusion to its activity in serum was relatively high in cases of FIP.

Ribavirin, either free in aqueous solution or incorporated into liposomes, was evaluated in 50 specific-pathogen-free kittens after experimental challenge exposure with feline infectious peritonitis virus (FIPV). Ribavirin was administered daily for 10 to 14 days at 16.5 mg kg-1 bodyweight given per os, intramuscularly or intravenously beginning 18 hours after kittens were challenge-exposed with FIPV. All kittens, including ribavirin-treated and untreated kittens, succumbed to FIP. Clinical signs of disease were more severe in the ribavirin-treated kittens and their mean survival times were shortened. The clinical efficacy of free ribavirin given intravenously at a reduced dosage (5.5 mg kg-1 bodyweight) was compared to that of ribavirin incorporated into lecithin-containing liposomes (5 mg kg-1) intravenously. Drugs were given once daily for three consecutive days of each week for three weeks, beginning 18 hours after virus challenge exposure. There was no significant difference either in survival rate or severity of disease between kittens given free ribavirin, liposomal ribavirin or saline only. Because of its intrinsic toxicity and low therapeutic index against FIPV and its marginal antiviral activities in vivo at maximal doses, ribavirin cannot presently be recommended as primary antiviral chemotherapy against FIP.

Two cals previously challenge-exposed and seropositive to feline infectious peritonitis virus ( fipv ) were evaluated for delayed-type hypersensitivity ( dth ) skin responses to intradermal fipv. Before testing, cat 1 ( fip -resistant) had survived a severe experimental fipv challenge-exposure and had remained asymptomatic, whereas cat 2 ( fip -susceptible) developed acute fulminant fip after a considerably smaller virus challenge-exposure. Cat 1 developed a focal thickened plague at the fipv -injected skin site at 48 hours after injection. Histological examinations of serial punch biopsies from virus-inoculated skin revealed perivascular and diffuse dermal infiltrations of macrophages, lymphocytes and polymorphonuclear leucocytes which were maximal at 48 to 72 hours after injection. In contrast, cat 2 did not react grossly and showed only very mild dermal infiltrates at 72 hours after injection. The present findings of strong dth responses to fipv in a resistant cat and minimal responses in a cat with acute fulminant fip suggest that certain in vivo cellular immune reactions may be associated with disease resistance.