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.

Rapid Resolution of Non-Effusive Feline Infectious Peritonitis Uveitis with an Oral Adenosine Nucleoside Analogue and Feline Interferon Omega.

This is the first report of a successful treatment of a non-effusive feline infectious peritonitis (FIP) uveitis case using an oral adenosine nucleoside analogue drug and feline interferon omega, and alpha-1 acid glycoprotein (AGP) as an indicator of recovery. A 2-year-old male neutered Norwegian Forest Cat presented with uveitis, keratic precipitates, mesenteric lymphadenopathy and weight loss. The cat was hypergammaglobulinaemic and had a non-regenerative anaemia. Feline coronavirus (FCoV) RNA was detected in a mesenteric lymph node fine-needle aspirate by a reverse-transcriptase polymerase chain reaction-non-effusive FIP was diagnosed. Prednisolone acetate eye drops were administered three times daily for 2 weeks. Oral adenosine nucleoside analogue (Mutian) treatment started. Within 50 days of Mutian treatment, the cat had gained over one kilogram in weight, his globulin level reduced from 77 to 51 g/L and his haematocrit increased from 22 to 35%; his uveitis resolved and his sight improved. Serum AGP level reduced from 3100 to 400 µg/mL (within normal limits). Symmetric dimethylarginine (SDMA) was above normal at 28 µg/dL, reducing to 14 µg/dL on the cessation of treatment; whether the SDMA increase was due to FIP lesions in the kidney or Mutian is unknown. Mutian treatment stopped and low-dose oral recombinant feline interferon omega begun-the cat's recovery continued.

Feline Infectious Peritonitis: Update on Pathogenesis, Diagnostics, and Treatment.

Feline infectious peritonitis (FIP) is a mysterious and lethal disease of cats. The causative agent, feline coronavirus (FCoV), is ubiquitous in most feline populations, yet the disease is sporadic in nature. Mutations in the infecting virus combined with an inappropriate immune response to the FCoV contribute to the development of FIP. Diagnosis can be challenging because signs may be vague, clinical pathology parameters are nonspecific, and the gold standard for diagnosis is invasive: histopathology of affected tissue. This article discusses the developments in the understanding of this disease as well as the progress in diagnosis and treatment.

High-throughput viral microneutralization method for feline coronavirus using image cytometry.

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.

A Tale of Two Viruses: The Distinct Spike Glycoproteins of Feline Coronaviruses.

Feline coronavirus (FCoV) is a complex viral agent that causes a variety of clinical manifestations in cats, commonly known as feline infectious peritonitis (FIP). It is recognized that FCoV can occur in two different serotypes. However, differences in the S protein are much more than serological or antigenic variants, resulting in the effective presence of two distinct viruses. Here, we review the distinct differences in the S proteins of these viruses, which are likely to translate into distinct biological outcomes. We introduce a new concept related to the non-taxonomical classification and differentiation among FCoVs by analyzing and comparing the genetic, structural, and functional characteristics of FCoV and the FCoV S protein among the two serotypes and FCoV biotypes. Based on our analysis, we suggest that our understanding of FIP needs to consider whether the presence of these two distinct viruses has implications in clinical settings.

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.

Preliminary investigation on feline coronavirus presence in the reproductive tract of the tom cat as a potential route of viral transmission.

OBJECTIVES: Feline infectious peritonitis (FIP) is an immune-mediated disease initiated by feline coronavirus (FCoV) infection. To date, the only proven route of transmission is the faecal-oral route, but a possible localisation of FCoV in the reproductive tract of tom cats is of concern, owing to the involvement of the male reproductive tract during FIP and to the presence of reproduction disorders in FCoV-endemic feline catteries. The aim of the study was to investigate the presence and localisation of FCoV in semen and/or in the reproductive tract of tom cats, and its possible association with seroconversion and viraemic phase. METHODS: Blood, serum, semen and/or testicle samples were obtained from 46 tom cats. Serology was performed on 38 serum samples, nested reverse transcriptase PCR (nRT-PCR) and reverse transcriptase quantitative PCR (RT-qPCR) were performed on 39 blood samples and on 17 semen samples, and histology, immunohistochemistry and nRT-PCR were performed on 39 testicles. RESULTS: Twenty-four of 38 serum samples were positive on serology. Semen samples were negative on RT-PCR and RT-qPCR for FCoV, while all blood samples were negative at both molecular methods, except for one sample positive at RT-qPCR with a very low viral load. All testicles were negative at immunohistochemistry, while six were positive at nRT-PCR for FCoV. Serology and blood PCR results suggest that the virus was present in the environment, stimulating transient seroconversion. FCoV seems not to localise in the semen of tom cats, making the venereal route as a way of transmission unlikely. Although viral RNA was found in some testicles, it could not be correlated with the viraemic phase. CONCLUSIONS AND RELEVANCE: In the light of these preliminary results, artificial insemination appears safer than natural mating as it eliminates the direct contact between animals, thus diminishing the probability of faecal-oral FCoV transmission.