Development and characterization of reverse genetics systems of feline infectious peritonitis virus for antiviral research.

Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome.

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.

Use of selected samples to diagnose a tricky feline viral disease in a cat with uveitis and neurological signs.

This case involved a 2-year-old neutered male domestic mixed-breed cat that was rescued from the street eight months earlier. The animal presented with weakness, hyporexia, progressive weight loss, fatigue, uveitis, pale mucous membranes, dehydration (7%), and pelvic limb paresis. Aqueous humor was collected for molecular analysis for the differential diagnosis of potential etiological agents [Feline coronavirus (FCoV), Feline leukemia virus (FeLV), Feline immunodeficiency virus (FIV), Toxoplasma gondii, Cryptococcus spp., Felid herpesvirus-1 (FHV-1) and Bartonella spp.] of feline uveitis. The sample was positive by real-time reverse transcription-polymerase chain reaction (RT-qPCR) for FCoV and RT-qPCR and real-time polymerase chain reaction (qPCR) for FeLV and qPCR FIV. The cat was euthanized due to poor clinical outcomes and prognosis. A cerebrospinal fluid (CSF) sample was collected and tested, and the same pathogens were found in the aqueous humor. Small-cell follicular multicenter lymphoma and multifocal pyogranulomatous meningoencephalitis were observed upon histopathological analysis. In this study, aqueous humor and cerebrospinal fluid samples were efficient for the detection of coinfection with FIV, FeLV, and FCoV.

O caso refere-se a um gato de dois anos de idade, sem raça definida, resgatado da rua há oito meses. O animal apresentava fraqueza, hiporexia, emagrecimento progressivo, cansaço fácil, uveíte, mucosas pálidas, desidratação (7%) e paresia de membros pélvicos. O humor aquoso foi coletado para o diagnóstico molecular diferencial de potenciais agentes etiológicos [coronavírus felino (FCoV), vírus da leucemia felina (FeLV), vírus da imunodeficiência felina (FIV), Toxoplasma gondii, Cryptococcus spp., herpesvírus felino tipo 1 (FHV-1) and Bartonella spp.] causadores de uveíte felina. A amostra foi positiva na reação em cadeia da polimerase precedida por transcrição reversa em tempo real (RT-qPCR) para FCoV, RT-qPCR e reação em cadeia da polimerase em tempo real (qPCR) para FeLV e qPCR para FIV. O animal foi submetido à eutanásia - devido ao quadro clínico e prognóstico desfavorável. Amostra de líquido cefalorraquidiano (LCR) foi coletada e testada, confirmando a identificação dos mesmos patógenos encontrados no humor aquoso. Linfoma multicêntrico folicular de pequenas células e meningoencefalite piogranulomatosa multifocal foram observados na análise histopatológica. Neste relato, as amostras de humor aquoso e líquido cefalorraquidiano foram eficientes para a detecção de coinfecção por FIV, FeLV e FCoV.

Better therapeutic effect of oral administration of GS441524 compared with GC376.

FIP is a fatal feline disease caused by FIPV. Two drugs (GS441524 and GC376) target FIPV and have good therapeutic effect when administered by subcutaneous injection. However, subcutaneous injection has limitations compared with oral administration. Additionally, the oral efficacy of the two drugs has not been determined. Here, GS441524 and GC376 were shown to efficiently inhibit FIPV-rQS79 (recombination virus with a full-length field type I FIPV and the spike gene replaced with type II FIPV) and FIPV II (commercially available type II FIPV 79-1146) at a noncytotoxic concentration in CRFK cells. Moreover, the effective oral dose was determined via the in vivo pharmacokinetics of GS441524 and GC376. We conducted animal trials in three dosing groups and found that while GS441524 can effectively reducing the mortality of FIP subjects at a range of doses, GC376 only reducing the mortality rate at high doses. Additionally, compared with GC376, oral GS441524 has better absorption, slower clearance and a slower rate of metabolism. Furthermore, there was no significant difference between the oral and subcutaneous pharmacokinetic parameters. Collectively, our study is the first to evaluate the efficacy of oral GS441524 and GC376 using a relevant animal model. We also verified the reliability of oral GS441524 and the potential of oral GC376 as a reference for rational clinical drug use. Furthermore, the pharmacokinetic data provide insights into and potential directions for the optimization of these drugs.

First study on in vitro antiviral and virucidal effects of flavonoids against feline infectious peritonitis virus at the early stage of infection.

Background and Aim: Feline infectious peritonitis (FIP), one of the most important infectious diseases in cats is caused by FIP virus (FIPV), a mutated variant of feline coronavirus. Feline infectious peritonitis has a negative impact on feline health, with extremely high mortality in clinical FIP-infected cats, particularly young cats. There are no approved drugs for FIP treatment, and therapeutic possibilities for FIP treatment are limited. This study aimed to utilize nature-derived bioactive flavonoids with antiviral properties to inhibit FIPV infection in Crandell-Rees feline kidney (CRFK) cells. Materials and Methods: The cytotoxicity of 16 flavonoids was evaluated on CRFK cells using a colorimetric method (MTS) assay. Viral kinetics of FIPV at 50 tissue culture infectious dose (TCID50)/well was determined during the first 24-h post-infection (HPI). Antiviral activity was evaluated based on the replication steps of the virus life cycle, including pre-compound, attachment, penetration, post-viral entry, and virucidal assays. The antiviral efficacy of flavonoids against FIPV was determined based on positive FIPV-infected cells with the immunoperoxidase monolayer assay and viral load quantification using reverse transcription-quantitative polymerase chain reaction. Results: Two flavonoids, namely, isoginkgetin and luteolin, inhibited FIPV replication during post-viral entry in a dose-dependent manner, with 50% maximal effective concentrations = 4.77 ± 0.09 and 36.28 ± 0.03 µM, respectively. Based on viral kinetics, both flavonoids could inhibit FIPV replication at the early stage of infection at 0-6-HPI for isoginkgetin and 2-6-HPI for luteolin using a time-of-addition assay. Isoginkgetin exerted a direct virucidal effect that reduced the viral titers by 2 and 1.89 log10 TCID50/mL at 60 and 120 min, respectively. Conclusion: Isoginkgetin interfered with FIPV replication during both post-viral infection and virucidal experiments on CRFK cells, whereas luteolin inhibited the virus after infection. These results demonstrate the potential of herbal medicine for treating FIP.

Phylogenetic analysis of feline infectious peritonitis virus, feline enteric coronavirus, and severe acute respiratory syndrome coronavirus 2 of cats in Surabaya, Indonesia.

Background and Aim: Questions about the origin of coronavirus and its introduction to human beings have persisted. The detection of a variety of coronavirus related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in bats and pangolins led to the widespread belief that SARS-CoV-2 originated from wild ani-mals and was introduced to humans through an inter-mediate animal. Thus, coronaviruses from animals, especially those in close contact with humans, have attracted particular attention. This study aimed to phylogenetically analyze feline enteric coronavirus (FECV), feline infectious peritonitis virus (FIPV), and SARS-CoV-2 found in cats in Surabaya amid the COVID-19 pandemic. The results will provide a basis for developing basic preventive and pet healthcare strategies. Materials and Methods: Samples were collected on physical examinations of domestic and Persian cats (males and females) from March 2020 to March 2022. Samples were collected if there were clinical signs of FECV and FIP based on a veterinarian's diagnosis in several clinics in Surabaya. Laboratory examinations in this study were performed by reverse-transcription-polymerase chain reaction (RT-PCR) with primers for conserved regions of FIP and FECV, DNA sequencing was performed with Applied Biosystem Genetic Analyzer protocol, homology analysis was performed using Basic Local Alignment Search Tool NCBI, phylogenetic analysis was carried out with BioEdit 7.2 software, and sequences were compared with references from GenBank. Results: Samples were collected from ten cats showing clinical signs of FECV and FIP, based on a veterinarian's diagnosis. On RT-PCR examinations performed with specifically designed primers for detecting FIPV in blood, peritoneal fluid, and feces, only one sample showed positivity for FIPV (1/10), namely, a peritoneal sample from a domestic cat in Surabaya. Homology analysis of the FIPV Surabaya isolate showed 98% similarity with FECV and FIPV reported in GenBank (MT444152 and DQ010921, respectively). In phylogenetic analysis, the FIPV Surabaya isolate was clustered together with SARS-CoV-2 of Clade A (MT198653) from Spain, SARS-CoV-2 Clade A (MT192765) from the USA, SARS-CoV-2 Clade D (039888) from the USA, and SARS-CoV-2 Clade F (MT020781) from Finland. Conclusion: This study revealed a relationship between the SARS-CoV-2 viruses that infect humans and cats (FECV), which is an important finding for those keeping cats at home. However, this finding requires further comprehensive support from laboratory studies.

An updated review of feline coronavirus: mind the two biotypes.

Feline coronavirus (FCoV) includes two biotypes: feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). Although both biotypes can infect cats, their pathogenicities differ. The FIPV biotype is more virulent than the FECV biotype and can cause peritonitis or even death in cats, while most FECV biotypes do not cause lesions. Even pathogenic strains of the FECV biotype can cause only mild enteritis because of their very low virulence. This article reviews recent progress in FCoV research with regard to FCoV etiological characteristics; epidemiology; clinical symptoms and pathological changes; pathogenesis; and current diagnosis, prevention and treatment methods. It is hoped that this review will provide a reference for further research on FCoV and other coronaviruses.

Detection of anti-feline infectious peritonitis virus activity of a Chinese herb extract using geneLEAD VIII, a fully automated nucleic acid extraction/quantitative PCR testing system.

The geneLEAD VIII is a fully-automated nucleic acid extraction/quantitative PCR equipment developed by Precision System Science Co., Ltd., (PSS). To take advantage of its capability, we developed a quantitative assay system to measure growth of animal viruses. The system was used to assay one of the Chinese herbal extracts whose anti-malarial activities were previously reported and demonstrated its dose-dependent anti-viral activity against feline infectious peritonitis virus (FIPV), a feline coronavirus causing the fatal diseases in cats, and relatively low cell toxicity. The assay developed in this study is useful to screen antiviral drugs and the anti-FIPV activity of the herbal extract identified have a potential to lead to development of new drugs against FIPV and other coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

In vitro antiviral effects of GS-441524 and itraconazole combination against feline infectious peritonitis virus.

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.

Isolation and Molecular Detection of Feline Infectious Peritonitis Virus.

Feline coronavirus (FCoV) is an enveloped single-stranded RNA virus, affecting wild and domestic cats. Feline infectious peritonitis viruses (FIPV) variants of FCoV cause fatal peritonitis affecting approximately 5% of FCoV infected animals. The present study aimed to detect and isolate the feline infectious peritonitis virus for the first time in Iraq. In this study, 50 samples (fecal swab and peritoneal fluid) were collected from suspected pet cats from different areas of Baghdad, Iraq. The very suitable age was under two years old.  Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) was used to detect Feline infectious peritonitis in infected collected samples by the amplification of spike protein (S). The result of real-time RT-PCR revealed that out of 50 samples from suspected cats, 10 samples were positive for FIPV. Moreover, 10 positive samples by real-time RT-PCR were used for the isolation of the virus in chicken embryo fibroblast cell culture. Subsequently, the isolated virus was detected by real-time RT-PCR and then by conventional RT-PCR, followed by electrophoresis.

Establishment of Full-Length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats.

Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant feline coronavirus (FCoV) 79-1146_CA (r79-1146_CA). In animal experiments with 1- to 2-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study pathogenesis, antiviral drugs, and vaccines for FCoV. IMPORTANCE Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

Virucidal Efficacy of Blue LED and Far-UVC Light Disinfection against Feline Infectious Peritonitis Virus as a Model for SARS-CoV-2.

Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs through respiratory droplets passed directly from person to person or indirectly through fomites, such as common use surfaces or objects. The aim of this study was to determine the virucidal efficacy of blue LED (405 nm) and far-UVC (222 nm) light in comparison to standard UVC (254 nm) irradiation for the inactivation of feline infectious peritonitis virus (FIPV) on different matrices as a model for SARS-CoV-2. Wet or dried FIPV on stainless steel, plastic, or paper discs, in the presence or absence of artificial saliva, were exposed to various wavelengths of light for different time periods (1-90 min). Dual activity of blue LED and far-UVC lights were virucidal for most wet and dried FIPV within 4 to 16 min on all matrices. Individual action of blue LED and far-UVC lights were virucidal for wet FIPV but required longer irradiation times (8-90 min) to reach a 4-log reduction. In comparison, LED (265 nm) and germicidal UVC (254 nm) were virucidal on almost all matrices for both wet and dried FIPV within 1 min exposure. UVC was more effective for the disinfection of surfaces as compared to blue LED and far-UVC individually or together. However, dual action of blue LED and far-UVC was virucidal. This combination of lights could be used as a safer alternative to traditional UVC.

Two Inhibitors Against the 3C-Like Proteases of Swine Coronavirus and Feline Coronavirus.

Coronaviruses (CoVs) are important human and animal pathogens that cause respiratory and gastrointestinal diseases. Porcine epidemic diarrhoea (PED), characterized by severe diarrhoea and vomiting in pigs, is a highly lethal disease caused by porcine epidemic diarrhoea virus (PEDV) and causes substantial losses in the swine industry worldwide. However, currently available commercial drugs have not shown great therapeutic effects. In this study, a fluorescence resonance energy transfer (FRET)-based assay was applied to screen a library containing 1,590 compounds and identified two compounds, 3-(aminocarbonyl)-1-phenylpyridinium and 2,3-dichloronaphthoquinone, that target the 3C-like protease (3CLpro) of PEDV. These compounds are of low molecular weight (MW) and greatly inhibited the activity of this enzyme (IC50 values were obtained in this study). Furthermore, these compounds exhibited antiviral capacity against another member of the CoV family, feline infectious peritonitis virus (FIPV). Here, the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified (with EC50 values) and cell viability assays were performed. The results of putative molecular docking models indicate that these compounds, labeled compound 1 and compound 2, contact the conserved active sites (Cys144, Glu165, Gln191) of 3CLpro via hydrogen bonds. These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.

Current concepts in the development of therapeutics against human and animal coronavirus diseases by targeting NP.

The coronavirus (CoV) infects a broad range of hosts including humans as well as a variety of animals. It has gained overwhelming concerns since the emergence of deadly human coronaviruses (HCoVs), severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, followed by Middle East respiratory syndrome coronavirus (MERS-CoV) in 2015. Very recently, special attention has been paid to the novel coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 due to its high mobility and mortality. As the COVID-19 pandemic continues, despite vast research efforts, the effective pharmaceutical interventions are still not available for clinical uses. Both expanded knowledge on structure insights and the essential function of viral nucleocapsid (N) protein are key basis for the development of novel, and potentially, a broad-spectrum inhibitor against coronavirus diseases. This review aimed to delineate the current research from the perspective of biochemical and structural study in cell-based assays as well as virtual screen approaches to identify N protein antagonists targeting not only HCoVs but also animal CoVs.

Mutation analysis of the spike protein in Italian feline infectious peritonitis virus and feline enteric coronavirus sequences.

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.

SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.

Current status on treatment options for feline infectious peritonitis and SARS-CoV-2 positive cats.

Feline infectious peritonitis (FIP) is a viral-induced, immune-mediated disease of cats caused by virulent biotypes of feline coronaviruses (FCoV), known as the feline infectious peritonitis virus (FIPV). Historically, three major pharmacological approaches have been employed to treat FIP: (1) immunomodulators to stimulate the patient's immune system non-specifically to reduce the clinical effects of the virus through a robust immune response, (2) immunosuppressive agents to dampen clinical signs temporarily, and (3) re-purposed human antiviral drugs, all of which have been unsuccessful to date in providing reliable efficacious treatment options for FIPV. Recently, antiviral studies investigating the broad-spectrum coronavirus protease inhibitor, GC376, and the adenosine nucleoside analogue GS-441524, have resulted in increased survival rates and clinical cure in many patients. However, prescriber access to these antiviral therapies is currently problematic as they have not yet obtained registration for veterinary use. Consequently, FIP remains challenging to treat. The purpose of this review is to provide an update on the current status of therapeutics for FIP. Additionally, due to interest in coronaviruses resulting from the current human pandemic, this review provides information on domesticated cats identified as SARS-CoV-2 positive.

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses.

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 µM to 25.90 ± 1.40 µM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 µM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 µM and 1.99 ± 0.30 to 4.03 ± 0.60 µM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.

Development of Feline Ileum- and Colon-Derived Organoids and Their Potential Use to Support Feline Coronavirus Infection.

Feline coronaviruses (FCoVs) infect both wild and domestic cat populations world-wide. FCoVs present as two main biotypes: the mild feline enteric coronavirus (FECV) and the fatal feline infectious peritonitis virus (FIPV). FIPV develops through mutations from FECV during a persistence infection. So far, the molecular mechanism of FECV-persistence and contributing factors for FIPV development may not be studied, since field FECV isolates do not grow in available cell culture models. In this work, we aimed at establishing feline ileum and colon organoids that allow the propagation of field FECVs. We have determined the best methods to isolate, culture and passage feline ileum and colon organoids. Importantly, we have demonstrated using GFP-expressing recombinant field FECV that colon organoids are able to support infection of FECV, which were unable to infect traditional feline cell culture models. These organoids in combination with recombinant FECVs can now open the door to unravel the molecular mechanisms by which FECV can persist in the gut for a longer period of time and how transition to FIPV is achieved.

Cellular Metabolic Profiling of CrFK Cells Infected with Feline Infectious Peritonitis Virus Using Phenotype Microarrays.

Feline infectious peritonitis (FIP) is a fatal feline immune-mediated disease caused by feline infectious peritonitis virus (FIPV). Little is known about the biological pathways associated in FIP pathogenesis. This is the first study aiming to determine the phenotypic characteristics on the cellular level in relation to specific metabolic pathways of importance to FIP pathogenesis. METHODS: The internalization of type II FIPV WSU 79-1146 in Crandell-Rees Feline Kidney (CrFK) cells was visualized using a fluorescence microscope, and optimization prior to phenotype microarray (PM) study was performed. Then, four types of Biolog Phenotype MicroArray™ plates (PM-M1 to PM-M4) precoated with different carbon and nitrogen sources were used to determine the metabolic profiles in FIPV-infected cells. RESULTS: The utilization of palatinose was significantly low in FIPV-infected cells; however, there were significant increases in utilizing melibionic acid, L-glutamine, L-glutamic acid and alanyl-glutamine (Ala-Gln) compared to non-infected cells. CONCLUSION: This study has provided the first insights into the metabolic profiling of a feline coronavirus infection in vitro using PMs and deduced that glutamine metabolism is one of the essential metabolic pathways for FIPV infection and replication. Further studies are necessary to develop strategies to target the glutamine metabolic pathway in FIPV infection.