Seroprevalence of and risk factors for feline coronavirus infection in cats from Greece.

Feline coronavirus (FCoV) is a highly contagious and ubiquitous virus of domestic cats and wild felids. Feline infectious peritonitis (FIP) is a fatal, systemic disease caused by FCoV infection when spontaneous mutations of the viral genome take place. The aims of this study were primarily to determine the prevalence of seropositivity for FCoV in different populations of cats in Greece and assess risk factors for seropositivity. A total of 453 cats were prospectively enrolled in the study. A commercially available IFAT kit was used for the detection of FCoV IgG antibodies in serum. Overall, 55 (12.1 %) of the 453 cats were seropositive for FCoV. Based on multivariable analysis, factors associated with FCoV-seropositivity included cats adopted as strays and contact with other cats. This is the first extensive study on the epidemiology of FCoV in cats from Greece and one of the largest worldwide. Feline coronavirus infection is relatively common in Greece. Therefore, it is necessary to establish optimal strategies for the prevention of FCoV infection, considering the high-risk groups of cats identified in this study.

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.

2022 AAFP/EveryCat Feline Infectious Peritonitis Diagnosis Guidelines.

CLINICAL IMPORTANCE: Feline infectious peritonitis (FIP) is one of the most important infectious diseases and causes of death in cats; young cats less than 2 years of age are especially vulnerable. FIP is caused by a feline coronavirus (FCoV). It has been estimated that around 0.3% to 1.4% of feline deaths at veterinary institutions are caused by FIP. SCOPE: This document has been developed by a Task Force of experts in feline clinical medicine as the 2022 AAFP/EveryCat Feline Infectious Peritonitis Diagnosis Guidelines to provide veterinarians with essential information to aid their ability to recognize cats presenting with FIP. TESTING AND INTERPRETATION: Nearly every small animal veterinary practitioner will see cases. FIP can be challenging to diagnose owing to the lack of pathognomonic clinical signs or laboratory changes, especially when no effusion is present. A good understanding of each diagnostic test's sensitivity, specificity, predictive value, likelihood ratio and diagnostic accuracy is important when building a case for FIP. Before proceeding with any diagnostic test or commercial laboratory profile, the clinician should be able to answer the questions of 'why this test?' and 'what do the results mean?' Ultimately, the approach to diagnosing FIP must be tailored to the specific presentation of the individual cat. RELEVANCE: Given that the disease is fatal when untreated, the ability to obtain a correct diagnosis is critical. The clinician must consider the individual patient's history, signalment and comprehensive physical examination findings when selecting diagnostic tests and sample types in order to build the index of suspicion 'brick by brick'. Research has demonstrated efficacy of new antivirals in FIP treatment, but these products are not legally available in many countries at this time. The Task Force encourages veterinarians to review the literature and stay informed on clinical trials and new drug approvals.

Molecular characterization of Feline coronavirus in Shandong from 2017 to 2019, China (preprint)

Feline coronavirus (FCoV) is the causative agent of feline infectious peritonitis and diarrhoea in kittens worldwide. In this study, a total of 73 feline diarrhoeal faecal samples were collected from veterinary hospitals in ShanDong province from 2017 to 2019. FCoV was detected in 58.23% (46/79) of the samples using the RT-nPCR method. The results showed that the detection rate of FCoV in healthy cats and sick cats were 41.7% (10/24) and 81.6% (40/49), respectively. Full gene amplification and sequencing of N, M and S2 of positive FCoV isolates were performed. There was an amino acid mutation site (M1058L) in the S gene, which could be predicted to be used as a marker for Feline enteric coronavirus (FECV) and Feline infectious peritonitis virus (FIPV). These results enriched the epidemiological information of FCoV and provided an important reference for the prevention of FCoV in Shandong.

An Optimized Bioassay for Screening Combined Anticoronaviral Compounds for Efficacy against Feline Infectious Peritonitis Virus with Pharmacokinetic Analyses of GS-441524, Remdesivir, and Molnupiravir in Cats.

Feline infectious peritonitis (FIP) is a fatal disease of cats that currently lacks licensed and affordable vaccines or antiviral therapeutics. The disease has a spectrum of clinical presentations including an effusive ("wet") form and non-effusive ("dry") form, both of which may be complicated by neurologic or ocular involvement. The feline coronavirus (FCoV) biotype, termed feline infectious peritonitis virus (FIPV), is the etiologic agent of FIP. The objective of this study was to determine and compare the in vitro antiviral efficacies of the viral protease inhibitors GC376 and nirmatrelvir and the nucleoside analogs remdesivir (RDV), GS-441524, molnupiravir (MPV; EIDD-2801), and ß-D-N4-hydroxycytidine (NHC; EIDD-1931). These antiviral agents were functionally evaluated using an optimized in vitro bioassay system. Antivirals were assessed as monotherapies against FIPV serotypes I and II and as combined anticoronaviral therapies (CACT) against FIPV serotype II, which provided evidence for synergy for selected combinations. We also determined the pharmacokinetic properties of MPV, GS-441524, and RDV after oral administration to cats in vivo as well as after intravenous administration of RDV. We established that orally administered MPV at 10 mg/kg, GS-441524 and RDV at 25 mg/kg, and intravenously administered RDV at 7 mg/kg achieves plasma levels greater than the established corresponding EC50 values, which are sustained over 24 h for GS-441514 and RDV.

Clinical Follow-Up and Postmortem Findings in a Cat That Was Cured of Feline Infectious Peritonitis with an Oral Antiviral Drug Containing GS-441524.

This is the first report on a clinical follow-up and postmortem examination of a cat that had been cured of feline infectious peritonitis (FIP) with ocular manifestation by successful treatment with an oral multicomponent drug containing GS-441524. The cat was 6 months old when clinical signs (recurrent fever, lethargy, lack of appetite, and fulminant anterior uveitis) appeared. FIP was diagnosed by ocular tissue immunohistochemistry after enucleation of the affected eye. The cat was a participant in a FIP treatment study, which was published recently. However, 240 days after leaving the clinic healthy, and 164 days after the end of the 84 days of treatment, the cured cat died in a road traffic accident. Upon full postmortem examination, including histopathology and immunohistochemistry, there were no residual FIP lesions observed apart from a generalized lymphadenopathy due to massive lymphoid hyperplasia. Neither feline coronavirus (FCoV) RNA nor FCoV antigen were identified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively, in any tissues or body fluids, including feces. These results prove that oral treatment with GS-441524 leads to the cure of FIP-associated changes and the elimination of FCoV from all tissues.

Shedding persistency and intensity patterns of feline coronavirus (FCoV) in feces of cats living in breeding catteries in the Czech Republic.

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.

Successful treatment of a South African cat with effusive feline infectious peritonitis with remdesivir.

Historically, feline infectious peritonitis (FIP) has been considered almost invariably fatal. The recent COVID-19 pandemic has fuelled research in coronavirus pathophysiology and treatment. An unintended consequence is that we now have an effective treatment accessible for FIP. This paper reports on the successful resolution of immunohistochemistry-confirmed effusive FIP in an adolescent cat in South Africa following monotherapy with remdesivir at 4.9-5.6 mg/kg daily for 80 days.

Ionophore Antibiotics Inhibit Type II Feline Coronavirus Proliferation In Vitro.

Feline coronaviruses (FCoVs) infect cats worldwide and cause severe systemic diseases, such as feline infectious peritonitis (FIP). FIP has a high mortality rate, and drugs approved by the Food and Drug Administration have been ineffective for the treatment of FIP. Investigating host factors and the functions required for FCoV replication is necessary to develop effective drugs for the treatment of FIP. FCoV utilizes an endosomal trafficking system for cellular entry after binding between the viral spike (S) protein and its receptor. The cellular enzymes that cleave the S protein of FCoV to release the viral genome into the cytosol require an acidic pH optimized in the endosomes by regulating cellular ion concentrations. Ionophore antibiotics are compounds that form complexes with alkali ions to alter the endosomal pH conditions. This study shows that ionophore antibiotics, including valinomycin, salinomycin, and nigericin, inhibit FCoV proliferation in vitro in a dose-dependent manner. These results suggest that ionophore antibiotics should be investigated further as potential broad-spectrum anti-FCoV agents.

Detection of Feline Coronavirus Variants in Cats without Feline Infectious Peritonitis.

(1) Background: This study aimed to detect feline coronavirus (FCoV) and characterize spike (S) gene mutation profiles in cats suffering from diseases other than feline infectious peritonitis (FIP) using commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) and reevaluating results by sequencing. (2) Methods: In 87 cats in which FIP was excluded by histopathology and immunohistochemistry, FCoV 7b gene and S gene mutation RT-qPCR was performed prospectively on incisional biopsies and fine-needle aspirates of different organs, body fluids, and feces. Samples positive for S gene mutations or mixed FCoV underwent sequencing. (3) Results: In 21/87 cats, FCoV RNA was detectable. S gene mutations were detected by commercial RT-qPCR (and a diagnostic algorithm that was used at the time of sample submission) in at least one sample in 14/21 cats (66.7%), with only mutated FCoV in 2/21, only mixed in 1/21, and different results in 11/21 cats; in the remaining 7/21 cats, RNA load was too low to differentiate. However, sequencing of 8 tissue samples and 8 fecal samples of 9 cats did not confirm mutated FCoV in any of the FCoV RNA-positive cats without FIP. (4) Conclusions: Sequencing results did not confirm results of the commercial S gene mutation RT-qPCR.

Serotype I and II Feline Coronavirus Replication and Gene Expression Patterns of Feline Cells-Building a Better Understanding of Serotype I FIPV Biology.

Feline infectious peritonitis (FIP) is a disease of domestic cats caused by the genetic variant of the feline coronavirus (FCoV) and feline infectious peritonitis virus (FIPV), currently grouped into two serotypes, I and II. Although serotype I FIPV is more prevalent in cats with FIP, serotype II has been more extensively studied in vitro due to the relative ease in propagating this viral serotype in culture systems. As a result, more is known about serotype II FIPV than the more biologically prevalent serotype I. The primary cell receptor for serotype II has been determined, while it remains unknown for serotype I. The recent development of a culture-adapted feline cell line that more effectively propagates serotype I FIPV, FCWF-4 CU, derived from FCWF-4 cells available through the ATCC, offers the potential for an improved understanding of serotype I FIPV biology. To learn more about FIPV receptor biology, we determined targeted gene expression patterns in feline cells variably permissive to replication of serotype I or II FIPV. We utilized normal feline tissues to determine the immunohistochemical expression patterns of two known coronavirus receptors, ACE2 and DC-SIGN. Lastly, we compared the global transcriptomes of the two closely related FCWF-4 cell lines and identified viral transcripts with potential importance for the differential replication kinetics of serotype I FIPV.

Fecal Feline Coronavirus RNA Shedding and Spike Gene Mutations in Cats with Feline Infectious Peritonitis Treated with GS-441524.

As previously demonstrated by our research group, the oral multicomponent drug Xraphconn® containing GS-441524 was effective at curing otherwise fatal feline infectious peritonitis (FIP) in 18 feline coronavirus (FCoV)-infected cats. The aims of the current study were to investigate, using samples from the same animals as in the previous study, (1) the effect of treatment on fecal viral RNA shedding; (2) the presence of spike gene mutations in different body compartments of these cats; and (3) viral RNA shedding, presence of spike gene mutations, and anti-FCoV antibody titers in samples of 12 companion cats cohabitating with the treated cats. Eleven of the eighteen treated FIP cats (61%) were shedding FCoV RNA in feces within the first three days after treatment initiation, but all of them tested negative by day 6. In one of these cats, fecal shedding reoccurred on day 83. Two cats initially negative in feces were transiently positive 1-4 weeks into the study. The remaining five cats never shed FCoV. Viral RNA loads in feces decreased with time comparable with those in blood and effusion. Specific spike gene mutations linked to systemic FCoV spread were consistently found in blood and effusion from treated FIP cats, but not in feces from treated or companion cats. A new mutation that led to a not yet described amino acid change was identified, indicating that further mutations may be involved in the development of FIP. Eight of the twelve companion cats shed FCoV in feces. All but one of the twelve companion cats had anti-FCoV antibodies. Oral treatment with GS-441524 effectively decreased viral RNA loads in feces, blood, and effusion in cats with FIP. Nonetheless, re-shedding can most likely occur if cats are re-exposed to FCoV by their companion cats.

Role of Feline Coronavirus as Contributor to Diarrhea in Cats from Breeding Catteries.

(1) Background: Feline coronavirus infection (FCoV) is common in multi-cat environments. A role of FCoV in causing diarrhea is often assumed, but has not been proven. The aim of this study was to evaluate an association of FCoV infection with diarrhea in multi-cat environments. (2) Methods: The study included 234 cats from 37 catteries. Fecal samples were analyzed for FCoV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Potential co-infections were determined by applying a qPCR panel on different potential enteropathogens and fecal flotation. A fecal scoring system was used to categorize feces as diarrheic or non-diarrheic. (3) Results: Of the 234 cats included, 23 had diarrhea. The prevalence of FCoV infection was 87.0% in cats with and 58.8% in cats without diarrhea. FCoV infection was significantly associated with diarrhea (Odds Ratio (OR) 5.01; p = 0.008). In addition, presence of Clostridium perfringens α toxin (OR 6.93; p = 0.032) and feline panleukopenia virus (OR 13.74; p = 0.004) were associated with an increased risk of diarrhea. There was no correlation between FCoV load and fecal score. FCoV-positive cats with co-infections were not more likely to have diarrhea than FCoV-positive cats without co-infections (p = 0.455). (4) Conclusions: FCoV infection is common in cats from catteries and can be associated with diarrhea.

Expression of Toll-like receptors 3, 7, 9 and cytokines in feline infectious peritonitis virus-infected CRFK cells and feline peripheral monocytes.

BACKGROUND: The role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood. OBJECTIVES: This study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-ß, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes. METHODS: CRFK cells and monocytes from feline coronavirus (FCoV)-seronegative cats and FCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours post-infection (hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-ß, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence. RESULTS: FIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-ß expression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showed lower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfected monocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositive cats downregulated TLR7 and TNF-α expression between 4 and 24 hpi, and 4 and 12 hpi, respectively. IFN-ß was upregulated early in FIPV-infected monocytes from FCoV-seropositive cats, with a significant difference observed at 12 hpi compared to FCoV-seronegative cats. The viral load in the CRFK and FIPV-infected monocytes in both cohorts of cats was similar over time. CONCLUSION: TLR7 may be the key TLR involved in evading the innate response against inhibiting TNF-α production. Distinct TLR expression profiles between FCoV-seronegative and FCoV-seropositive cats were observed. The associated TLR that plays a role in the induction of IFN-ß needs to be explored further.

Alpha-1 Acid Glycoprotein Reduction Differentiated Recovery from Remission in a Small Cohort of Cats Treated for Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a systemic immune-mediated inflammatory perivasculitis that occurs in a minority of cats infected with feline coronavirus (FCoV). Various therapies have been employed to treat this condition, which was previously usually fatal, though no parameters for differentiating FIP recovery from remission have been defined to enable clinicians to decide when it is safe to discontinue treatment. This retrospective observational study shows that a consistent reduction of the acute phase protein alpha-1 acid glycoprotein (AGP) to within normal limits (WNL, i.e., 500 µg/mL or below), as opposed to duration of survival, distinguishes recovery from remission. Forty-two cats were diagnosed with FIP: 75% (12/16) of effusive and 54% (14/26) of non-effusive FIP cases recovered. Presenting with the effusive or non-effusive form did not affect whether or not a cat fully recovered (p = 0.2). AGP consistently reduced to WNL in 26 recovered cats but remained elevated in 16 cats in remission, dipping to normal once in two of the latter. Anaemia was present in 77% (23/30) of the cats and resolved more quickly than AGP in six recovered cats. The presence of anaemia did not affect the cat's chances of recovery (p = 0.1). Lymphopenia was observed in 43% (16/37) of the cats and reversed in nine recovered cats but did not reverse in seven lymphopenic cats in the remission group. Fewer recovered cats (9/24: 37%) than remission cats (7/13: 54%) were lymphopenic, but the difference was not statistically different (p = 0.5). Hyperglobulinaemia was slower than AGP to return to WNL in the recovered cats. FCoV antibody titre was high in all 42 cats at the outset. It decreased significantly in 7 recovered cats but too slowly to be a useful parameter to determine discontinuation of antiviral treatments. Conclusion: a sustained return to normal levels of AGP was the most rapid and consistent indicator for differentiating recovery from remission following treatment for FIP. This study provides a useful model for differentiating recovery from chronic coronavirus disease using acute phase protein monitoring.

Antiviral Effects of Hydroxychloroquine and Type I Interferon on In Vitro Fatal Feline Coronavirus Infection.

Feline infectious peritonitis (FIP) is a viral disease with a high morbidity and mortality by the FIP virus (FIPV, virulent feline coronavirus). Several antiviral drugs for FIP have been identified, but many of these are expensive and not available in veterinary medicine. Hydroxychloroquine (HCQ) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., SARS-CoV-2, dengue virus) have been confirmed. We investigated whether HCQ in association with interferon-ω (IFN-ω) is effective for FIPV in vitro. A total of 100 µM of HCQ significantly inhibited the replication of types I and II FIPV. Interestingly, the combination of 100 µM of HCQ and 104 U/mL of recombinant feline IFN-ω (rfIFN-ω, veterinary registered drug) increased its antiviral activity against type I FIPV infection. Our study suggested that HCQ and rfIFN-ω are applicable for treatment of FIP. Further clinical studies are needed to verify the combination of HCQ and rIFN-ω will be effective and safe treatment for cats with FIP.

Clinical and Molecular Relationships between COVID-19 and Feline Infectious Peritonitis (FIP).

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) has led the medical and scientific community to address questions surrounding the pathogenesis and clinical presentation of COVID-19; however, relevant clinical models outside of humans are still lacking. In felines, a ubiquitous coronavirus, described as feline coronavirus (FCoV), can present as feline infectious peritonitis (FIP)-a leading cause of mortality in young cats that is characterized as a severe, systemic inflammation. The diverse extrapulmonary signs of FIP and rapidly progressive disease course, coupled with a closely related etiologic agent, present a degree of overlap with COVID-19. This paper will explore the molecular and clinical relationships between FIP and COVID-19. While key differences between the two syndromes exist, these similarities support further examination of feline coronaviruses as a naturally occurring clinical model for coronavirus disease in humans.

Alpha-1 acid glycoprotein reduction differentiates recovery from remission in feline infectious peritonitis treatment (preprint)

Feline infectious peritonitis (FIP) is a systemic immune-mediated inflammatory perivasculitis which occurs in a minority of cats infected with feline coronavirus (FCoV). A variety of therapies have been employed to treat this condition, which previously was usually fatal, but no parameters for differentiating FIP recovery from remission have been defined to enable clinicians to decide when it is safe to discontinue treatment. This observational study shows that a consistent reduction of the acute phase protein alpha-1 acid glycoprotein (AGP) to within normal limits (WNL i.e. 500μg/ml or below), as opposed to duration of survival, distinguishes recovery from remission. AGP consistently reduced to WNL in 26 recovered cats but remained elevated in 16 cats in remission, dipping to normal once in two of the latter. Anaemia was present in 58% (23/40) of the cats, and resolved more quickly than AGP in six recovered cats. Lymphopenia was observed in 43% (16/37) of the cats and reversed in nine recovered cats, but none of the remission group. Hyperglobulinaemia was slower than AGP to return to WNL in the recovered cats. FCoV antibody titre was high in all 42 cats at the outset and decreased significantly in 7 recovered cats, but too slowly to be a useful parameter to determine discontinuation of anti-viral treatments. Conclusion: a sustained return to normal levels of AGP was the most rapid and consistent indicator for differentiating recovery from remission following treatment for FIP. This study provides a useful model for differentiating acute from chronic coronavirus disease using acute phase protein monitoring.

Potent protease inhibitors of deadly lagoviruses: rabbit hemorrhagic disease virus and European brown hare syndrome virus (preprint)

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family and mainly affect rabbits and hares, respectively. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as Riparian brush rabbits. In the US, only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by RHD type 2 virus (RHDV2) was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several US states infecting wild rabbits and hares, making it highly likely that RHD will become endemic in the US. Vaccines are available for RHD, however, there is no specific treatment for these diseases. RHDV and EBHSV encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses that encode 3CLpro including caliciviruses and coronaviruses. Here we established the enzyme and cell-based assays for these uncultivable viruses to determine the in vitro activity of 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis, and identified potent inhibitors of RHDV1 and 2 and EBHSV. In addition, structure-activity relationship study and homology modelling of the 3CLpros and inhibitors revealed that lagoviruses share similar structural requirements for 3CLpro inhibition with other caliciviruses.