Establishment and application of triple PCR method for CPV, CCoV and CRV

Canine parvovirus (CPV), canine coronavirus (CCoV) and canine rotavirus (CRV) are the three main causative viruses of diarrhea in dogs with similar clinical symptoms;thereby it is necessary to establish a high effective molecular detection method for differentiating the above pathogens. By optimizing the primer concentration and annealing temperature, a triple PCR method was established for simultaneous detection of CPV, CCoV and CRV, and then the specificity, sensitivity and repeatability of the method were tested. The results showed that the target fragments of CPV VP2 gene (253 bp), CCoV ORF-1b gene (379 bp) and CRV VP6 gene (852 bp) could be accurately amplified by the triple PCR method with high specificity, the detection limits of CPV, CCOV and CRV were 6.44x10-1 pg/L, 8.72x10-1 pg/L and 8.35x10-1 pg/L respectively with high sensitivity, and the method had good stability. Using this triple PCR method, 135 canine diarrhea fecal samples collected in Chengdu region from 2019 to 2020 were detected, and compared with those of single PCR method. The detection rates of CPV, CCoV and CRV were 16.30%, 20.74% and 4.44%, respectively, and the total infection rate was 51.11% (65/135) with 20.00% (13/65) co-infection rate. The detection results were consistent with three single PCR methods. In conclusion, CPV/CCoV/CRV triple PCR method successfully established in this paper can be applied as an effective molecular method to detection of related pathogens and to the epidemiological investigation.

Feline Coronavirus Infections

• First Described: 1963 (Holzworth, 1963);a viral etiology was not identified until the 1970s. • Cause: Feline coronavirus (family Coronaviridae, genus Coronavirus). • Affected Hosts: Cats and wild felids, especially cheetahs. • Modes of Transmission: Fecal-oral (FECV), internal mutation (FIPV) • Geographic Distribution: Worldwide. • Major Clinical Signs: Fever, lethargy, inappetence, vomiting, diarrhea, dehydration, icterus, tachypnea, uveitis, neurologic signs, abdominal distention due to ascites. • Differential Diagnoses: Toxoplasmosis, congestive heart failure, carcinomatosis, lymphoma, pancreatitis, rabies, cryptococcosis, bacterial peritonitis, pyothorax, bacterial meningitis, chronic stomatitis, multiple myeloma, FeLV or FIV infection. • Human Health Significance: Feline coronaviruses do not infect humans. © 2021 Elsevier Inc. All rights reserved.

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.

The first study on clinicopathological changes in cats with feline infectious peritonitis with and without retrovirus coinfection.

Background and Aim: Feline infectious peritonitis (FIP) is an infectious, immune-mediated, and fatal disease in cats caused by a mutant feline coronavirus (FCoV) infection. Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two common retroviruses that play a role in reducing feline immune function with opportunistic retrovirus infection being a predisposing factor for the development of FIP. This study aimed to evaluate the clinicopathological parameters of FIP in cats with and without retrovirus coinfection. Materials and Methods: In total, 62 cats presenting with pleural and/or peritoneal effusion at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand, were selected for the study. Effusion samples were collected and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed on all samples using the 3' untranslated region primer. All FCoV-positive cats were tested for retrovirus infection using a commercial kit (Witness FeLV-FIV [Zoetis]; United States). Clinical signs, hematological, and biochemical parameters of these cats were investigated and grouped. Results: Of the 62 cats with pleural and/or peritoneal effusion, FCoV was detected in 32, of which 21 were highly suspicious for FIP. The cats suspected of FIP were divided into three subgroups following viral detection. A total of 14 had only FCoV infection (Group A), four had FCoV and FeLV infection (Group B), and three had FCoV, FeLV, and FIV infection (Group C). Of the rest, 11 had definitive diagnoses, which included three being FCoV and FeLV-positive (Group D), and eight were retrovirus-negative (Group E). Mild anemia and lymphopenia were found in cats infected with these three viruses. An albumin-to-globulin ratio lower than 0.5 was found in FIP cats with only FCoV infection. Conclusion: Typically, cats with clinical effusion and FIP, with and without retrovirus coinfection, had similar hematological findings. Clinical signs, blood parameters, fluid analysis with cytological assessment, and RT-PCR assays could identify better criteria to diagnose FIP with and without retrovirus coinfection.

Incidence and sero-surveillance of feline viruses in Korean cats residing in Gyeonggi-do

Incidences of major feline viral diseases provide basic information for preventing viral disease in cats. Despite the growing interest in feline viral diseases, sero-surveillances have been lacking. In this study, we analyzed the diagnoses of feline viral diseases and conducted a sero surveillance of feline panleukopenia virus (FPV), feline calicivirus (FCV), feline herpesvirus-1 (FHV-1), and feline infectious peritonitis virus (FIPV) in Korean cats. Of the 204 confirmed cases since 2015, the numbers of diagnoses for FPV, FIPV, FCV, feline influenza virus, and FHV-1 were 156, 32, 12, 3, and 1 case, respectively. In total, 200 sera, collected between 2019 and 2021, were screened for the presence of antibodies against FPV, 2 FCVs, FHV-1, and FIPV using a hemagglutination inhibition test and a virus-neutralizing assay (VNA). The overall seropositive rates in cats tested for FPV, the 2 FCVs, FHV-1, and FIPV were 92.5%. 42.0%, 37.0%, 52.0%, and 14.0%, respectively. A low correlation (r = 0.466) was detected between the VNA titers of 2 FCV strains. The highest incidence and seropositive rate of FPV reveal that FPV is circulating in Korean cats. The low r-value between 2 FCVs suggests that a new feline vaccine containing the 2 kinds of FCVs is required.

Low Concentration Ozone for Surface Disinfection against Sars-Cov-2 and Feline Coronavirus

Intro: Surface and environment disinfection is an important part of infection control strategies, especially in the ongoing COVID-19 pandemic. Ozone, a highly reactive oxidant, is a widely used disinfectant in many industries including food, healthcare and water treatment. It has a broad-spectrum activity and leaves no harmful residues. However, most demonstrated efficacy has been at high ozone levels (>1ppm) which can be harmful to humans in case of exposure. Here, we undertook a study to evaluate if exposure to ozone is effective in inactivating SARS-CoV-2 and feline coronavirus (FCoV) even at low concentrations. Method(s): Ozone at 0.07, 0.1 and 1.2 ppm were evaluated for its virucidal activity against SARS-CoV-2 and FCoV. An ozone gas generator (Medklinn Air + Surface Sterilizer (CerafusionTM Technology), Medklinn, Malaysia) supplied controlled levels of ozone to a custom-built chamber of 1.5 ft3 (1.5ft x 1ft x 1ft) where dry virus films containing 1 x 104 PFU of test virus were exposed to ozone gas for 0.5h, 1h, 3h, 5h, and 8h. The experiment was performed at ambient temperature (23-24oC) and relative humidity (RH) of 55% (FCoV only) and 85% (SARS-CoV-2 and FCoV). Finding(s): At low level of ozone of 0.1ppm, >90% reduction of both viruses was achieved after 3h exposure at 85% and 55% humidity. At 1.2ppm, >90% reduction of both viruses was achieved after 0.5h exposure at 85% humidity. Ozone at 0.07ppm, however, did not show good efficacy as reduction not exceeding 90% was achieved only after 8h exposure at 85% and 55% humidity. Conclusion(s): The study demonstrated that low concentration of ozone of at least 0.1 ppm reduced SARS-CoV-2 and FCoV by >90% when used at 85% humidity. The use of low level ozone presents a safer alternative for disinfecting enclosed spaces and greatly reduces any potential harmful health effects in case of accidental exposure.Copyright © 2023

Virucidal activity in vitro of mouthwashes against a feline coronavirus type II.

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can occur through saliva and aerosol droplets deriving from the upper aerodigestive tract during coughing, sneezing, talking, and even during oral inspection or dental procedures. The aim of this study was to assess in vitro virucidal activity of commercial and experimental mouthwashes against a feline coronavirus (FCoV) strain. Commercial and experimental (commercial-based products with addition of either sodium dodecyl sulfate (SDS) or thymus vulgaris essential oil (TEO) at different concentrations) mouthwashes were placed in contact with FCoV for different time intervals, that is, 30 s (T30), 60 s (T60), and 180 s (T180); subsequently, the virus was titrated on Crandell Reese Feline Kidney cells. An SDS-based commercial mouthwash reduced the viral load by 5 log10 tissue culture infectious dose (TCID)50 /50 µl at T30 while a cetylpyridinium (CPC)-based commercial mouthwash was able to reduce the viral titer of 4.75 log10 at T60. Furthermore, five experimental mouthwashes supplemented with SDS reduced the viral titer by 4.75-5 log10 according to a dose- (up to 4 mM) and time-dependent fashion.

Coinfection in the cases of feline panleukopenia: a literature review

Coinfection caused by bacteria, parasites, or viruses complicates almost all feline panleukopenia virus (FPV) infections. Pathogens that colonize the gastrointestinal tract, Clostridium perfingens, Clostridium piliforme, Cryptosporidium spp, Giardia spp, Tritrichomonas fetus, canine parvovirus type 2,Salmonella sp., feline coronavirus, feline bocavirus, and feline astrovirus were isolated in the presence of FPV infection. Complex mechanisms between viruses, bacteria, protozoa, and hosts contribute to the pathogenesis and severity of coinfection. Prompt and accurate diagnosis, vaccination precautions, and appropriate treatment play important roles in reducing morbidity and mortality. This article outlines the etiology, pathogenesis, diagnosis, prevention, and treatment that can help veterinarians and pet owners improve their knowledge of managing the diseases.

Restrospective study of hemogram profile in effusive feline infectious peritonitis

Feline Infectious Peritonitis (FIP) is highly mortality disease in cats. The reliable and fast diagnosis is crucial to the best prognosis. The aim of this study to evaluate the hemogram profile in cats infected with effusive FIP. Twenty cats had been diagnosed effusive FIP at Animal Clinic Department of Internal Medicine, Faculty Veterinary Medicine, Universitas Gadjah Mada were used in the study. The diagnosis were based on clinical examination, ultrasound, x-ray, rivalta test, and rapid test. The hemogram profile were analyzed include routine hematology and serum biochemistry. Hemogram profile in effusive FIP showed the decreased hematocrit, hyperproteinemia, and leukocytosis with an average 22.9+or-7.4%;9.0+or-2.2 g/dL;22425+or-4116 cells/mm3 respectively. Erythrocyte, hemoglobin and fibrinogen levels were still in the normal range. The results of differential leukocytes revealed that 90% cats had neutrophilia and 75% lymphopenia with an average 20066+or-3337 cells/mm3 and 1861+or-1818 cells/mm3 respectively. The blood chemistry profile showed 60% of cats experienced increase in SGPT and SGOT with an average 138.4+or-72.3 IU/L and 101+or-60.5 IU/L respectively. Hyperglobulinemia was found in 90% samples with an average 6.7+or-0.8 g/dL. All cats have a low albumin:globulin ratio with an average 0.3+or-0.1. The hemogram profile of effusive FIP were: leukocytosis, neutrophilia, lymphopenia, hyperglobulinemia, and decreased albumin-globulin ratio..

Development of PCR diagnostic method for Trichomonas foetus infection based on 18S rRNA.

To establish a PCR detection method for Trichomonas foetus, the primers were designed and synthesized according to the 18S rRNA gene sequence of T. foetus published by GenBank. The positive recombinant plasmid pUCm-T-TF18S of T. foetus was used as the template, and the genomic DNA of Giardia felis, Coccidia +e-lis, feline parvovirus and cDNA of feline coronavirus were used as the control for PCR detection to analyze the specificity of this method. The positive T. foetus recombinant plasmid was serial to 8 different concentrations with a gap of 10 folds, and PCR was performed to analyze the sensitivity of this method. The pUCm-T-TF18S plasmids stored at -20 " for 3, 6, 9 and 12 months were detected by PCR to analyze the stability of the method. Twenty cat fecal samples were tested using this established PCR assay and compared with those of microscopic examination. The results showed that the recombinant plasmid pUCm-T- TF18S gave specific bands after PCR amplification. The sequencing results showed that the length of the product sequence was 1 264 bp, and the BLAST sequence comparison analysis showed 99.53% sequence identity, which is consistent with that of T. foetus from cats (GenBank registration number M81842.1). The PCR method for detection of T. foetus had no cross-reactivities with C. felis, G. felis, feline coronavirus and feline parvovirus;the minimum detectable template concentration is 4.52 X 105 copies/xl;The target band of T. foetus DNA can still be detected after being stored in the refrigerator at -20 " for 12 months. This method detected 16 positive samples of T. foetus nucleic acid from 20 cat fecal samples, which is more accurate and sensitive than the results from traditional microscopy (13 samples). It is suggested that the PCR method for the detection of T. foetus is highly specific, sensitive and stable, and can be used for clinical detection and epidemiological investigation of T. foetus.Copyright © 2022, National Institute of Parasitic Diseases. All rights reserved.

Focus on laryngeal disease in the cat

This is a title only record which contains no .

Ophthalmic and immunopathological characterization of systemic infectious diseases in cats.

Ocular involvement in systemic diseases is frequent in cats; however, without concurrent clinical and ophthalmic examinations with gross and/or histologic analysis of the eye, these findings can be underdiagnosed. This article aims to provide gross, histologic, and immunohistochemical characteristics of ocular lesions from cats submitted to necropsy, focusing on those caused by systemic infectious agents. Cats that died due to a systemic infectious disease were selected based on necropsy diagnosis and presence of ocular lesions. Gross, histologic, and immunohistochemical findings were recorded. From April 2018 to September 2019, 849 eyes of 428 cats were evaluated. Histologic abnormalities were seen in 29% of cases, which were classified as inflammatory (41%), neoplastic (32%), degenerative (19%), and metabolic/vascular (8%). Macroscopic changes were present in one-third of eyes with histologic lesions. Of these, 40% were attributed to inflammatory or neoplastic diseases associated with infectious agents. The most important infectious agents causing ocular disease in this study were feline leukemia virus, feline infectious peritonitis virus, and Cryptococcus sp. The most common ocular abnormalities associated with infectious agents were uveitis (anterior, posterior, or panuveitis), optic neuritis, and meningitis of the optic nerve. Ocular lesions secondary to systemic infections in cats are frequent; however, these are not always diagnosed because gross lesions are less common than histologic lesions. Therefore, both gross and histologic evaluation of the eyes of cats is recommended, mainly for cases in which the clinical suspicion or necropsy diagnosis suggests that an infectious agent might be related to the cause of death.

Incidence of acute haemolysis in cats receiving canine packed red blood cells (xenotransfusions).

OBJECTIVES: The aim of this study was to report the incidence of transfusion reactions in cats, including acute haemolysis (AH), occurring within 24 h of receiving a xenotransfusion. An additional aim was to determine whether cases with AH could be classified as having an acute haemolytic transfusion reaction (AHTR) as per the definition provided by the Association of Veterinary Haematology and Transfusion Medicine's Transfusion Reaction Small Animal Consensus Statement. METHODS: Medical records of cats that received canine packed red blood cells (PRBCs) between July 2018 and September 2020 at a veterinary hospital were reviewed. The incidence of AH, AHTRs, febrile non-haemolytic transfusion reactions (FNHTRs), transfusion-associated circulatory overload and septic transfusion reactions were recorded. RESULTS: The medical records of 53 cats were retrospectively evaluated. Twenty-three (43%) cats had transfusion reactions. Thirteen (25%) cats had AH; however, only four (8%) met the definition of an AHTR. Ten (19%) cats were determined to have FNHTRs. Survival to discharge of cats affected by AH was 50% (25% for cases that met the definition of an AHTR). Survival to discharge of cats not suffering from AHTR was 40%. CONCLUSIONS AND RELEVANCE: This report indicates that a higher proportion of cats undergo AH (25%) when administered canine PRBCs than previously reported, although many could not be classed as having an AHTR due to an apparently adequate packed cell volume rise. Challenges with sourcing feline blood in emergency situations occasionally necessitates the use of xenotransfusion in transfusion medicine. Clinicians should be aware that haemolysis after xenotransfusion can occur within 24 h and that a repeat feline transfusion may be required sooner than anticipated in some cases.

Food as a transmitter of viruses: A review

Viruses have been present throughout human history, causing diseases due to infections and food poisoning;they have caused frequent public health problems worldwide. These illnesses are usually mild, moderate, or severe in nature. The personal hygiene of food handlers and processing processes should be checked periodically. Virus detection protocols and safety measures should be continually reviewed as viruses change their mode of infection. The objective of this review was to discuss the possible routes of virus transmission to humans through food. Important topics have been reviewed such as: definition of food viruses, presence, and types of viruses in food, enteric viruses, zoonotic viruses, water as a means of transmission, risks of infection, other non-conventional foods as potential transmitters of viruses and food safety, in addition to current and future challenges, research work on viruses more resistant to heat treatments in food should be sought. Also, future work on survival time of active viruses on food surfaces. In addition, studies that determine the mechanisms of virus mutation in relation to the conditions of food handling and processing.

The infectious peritonitis Literature review

The authors summarize the current knowledge about FIP (feline infectious peritonitis) using the latest scientific literature and their own experiences. The feline coronaviruses, both the feline enteric coronavirus (FECV) and the FIP virus (FIPV) belong to the same Alphacoronavirus 1 species, in the Alphacoronavirus genus within the Coronaviridae family, and infect wild and domestic felids. FIPV is the mutated form of the ubiquitous and contagious feline enteric coronavirus, which, in contrast, causes a fatal and non-infectious illness. The lethal disease develops in only a subset of infected cats as a result of complex immunopathological processes. The clinical manifestation of the disease is very diverse. the effusive form ("wet form") has a more rapid course than the non-effusive form ("dry form"). However, these two main manifestations are rather the endpoints of a continuum of diseases. Macroscopically the wet form is characterized by effusions in the serosal cavities, and the dry form by perivascular (pyo)granulomas in the organs. The most characteristic histoogical lesions are granulomatou's to necrotizing vasculitis in the wet form, and vasocentric pyogranulomatous inflammation in the dry form. Ante-mortem diagnosis of the disease is challenging yet extremely important, partially because of recent successes in therapy. The most reliable diagnosis is likely to be made only post-mortem, but a properly constructed diagnostic workflow can be similarly effective. Although the active substances of previous successful therapies are relatively easily available, they are not approved for veterinary use. In the absence of an effective vaccine, prevention is based mainly on epidemiological considerations and the reduction of stressors that unnecessarily affect the cats. Presenting the example of FIP and COVID-19, it is perfectly understandable why the experience of different drugs in the treatment of animal coronaviral infections can be of tremendous value in preparing their use in human experiments.

A Combined Method Based on the FIPV N Monoclonal Antibody Immunofluorescence Assay and RT-nPCR Method for the Rapid Diagnosis of FIP-Suspected Ascites

Feline infectious peritonitis (FIP), which is caused by feline infectious peritonitis virus (FIPV), is a fatal and immunologically mediated infectious disease among cats. At present, due to the atypical clinical symptoms and clinicopathological changes, the clinical diagnosis of FIP is still difficult. The gold standard method for the differential diagnosis of FIP is immunohistochemistry (IHC) which is time-consuming and requires specialized personnel and equipment. Therefore, a rapid and accurate clinical diagnostic method for FIPV infection is still urgently needed. In this study, based on the etiological investigation of FIPV in parts of southern China, we attempted to explore a new rapid and highly sensitive method for clinical diagnosis. The results of the etiological investigation showed that the N gene of the FIPV BS8 strain had the highest homology with other strains. Based on this, a specific FIPV BS8 N protein monoclonal antibody was successfully prepared by expression of the recombinant proteins, immunization of mice, fusion and selection of hybridoma cell lines, and screening and purification of monoclonal antibodies. Furthermore, we carried out a time-saving combination method including indirect immunofluorescence assay (IFA) and nested reverse transcription polymerase chain reaction (RT-nPCR) to examine FIP-suspected clinical samples. These results were 100% consistent with IHC. The results revealed that the combined method could be a rapid and accurate application in the diagnosis of suspected FIPV infection within 24 hours. In conclusion, the combination of IFA and RT-nPCR was shown to be a fast and reliable method for clinical FIPV diagnosis. This study will provide insight into the exploitation of FIPV N antibodies for the clinical diagnosis of FIP-suspected ascites samples.

Both Feline Coronavirus Serotypes 1 and 2 Infected Domestic Cats Develop Cross-Reactive Antibodies to SARS-CoV-2 Receptor Binding Domain: Its Implication to Pan-CoV Vaccine Development.

The current study was initiated when our specific-pathogen-free laboratory toms developed unexpectedly high levels of cross-reactive antibodies to human SARS-CoV-2 (SCoV2) receptor binding domain (RBD) upon mating with feline coronavirus (FCoV)-positive queens. Multi-sequence alignment analyses of SCoV2 Wuhan RBD and four strains each from FCoV serotypes 1 and 2 (FCoV1 and FCoV2) demonstrated an amino acid sequence identity of 11.5% and a similarity of 31.8% with FCoV1 RBD (12.2% identity and 36.5% similarity for FCoV2 RBD). The sera from toms and queens cross-reacted with SCoV2 RBD and reacted with FCoV1 RBD and FCoV2 spike-2, nucleocapsid, and membrane proteins, but not with FCoV2 RBD. Thus, the queens and toms were infected with FCoV1. Additionally, the plasma from six FCoV2-inoculated cats reacted with FCoV2 and SCoV2 RBDs, but not with FCoV1 RBD. Hence, the sera from both FCoV1-infected cats and FCoV2-infected cats developed cross-reactive antibodies to SCoV2 RBD. Furthermore, eight group-housed laboratory cats had a range of serum cross-reactivity to SCoV2 RBD even 15 months later. Such cross-reactivity was also observed in FCoV1-positive group-housed pet cats. The SCoV2 RBD at a high non-toxic dose and FCoV2 RBD at a 60-400-fold lower dose blocked the in vitro FCoV2 infection, demonstrating their close structural conformations essential as vaccine immunogens. Remarkably, such cross-reactivity was also detected by the peripheral blood mononuclear cells of FCoV1-infected cats. The broad cross-reactivity between human and feline RBDs provides essential insights into developing a pan-CoV vaccine.

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.

Stopping Feline Coronavirus Shedding Prevented Feline Infectious Peritonitis.

After an incubation period of weeks to months, up to 14% of cats infected with feline coronavirus (FCoV) develop feline infectious peritonitis (FIP): a potentially lethal pyogranulomatous perivasculitis. The aim of this study was to find out if stopping FCoV faecal shedding with antivirals prevents FIP. Guardians of cats from which FCoV had been eliminated at least 6 months earlier were contacted to find out the outcome of their cats; 27 households were identified containing 147 cats. Thirteen cats were treated for FIP, 109 cats shed FCoV and 25 did not; a 4-7-day course of oral GS-441524 antiviral stopped faecal FCoV shedding. Follow-up was from 6 months to 3.5 years; 11 of 147 cats died, but none developed FIP. A previous field study of 820 FCoV-exposed cats was used as a retrospective control group; 37 of 820 cats developed FIP. The difference was statistically highly significant (p = 0.0062). Cats from eight households recovered from chronic FCoV enteropathy. Conclusions: the early treatment of FCoV-infected cats with oral antivirals prevented FIP. Nevertheless, should FCoV be re-introduced into a household, then FIP can result. Further work is required to establish the role of FCoV in the aetiology of feline inflammatory bowel disease.

Natural selection differences detected in key protein domains between non-pathogenic and pathogenic feline coronavirus phenotypes.

Feline coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed feline enteric coronavirus [FECV]), with around 12 per cent developing into deadly feline infectious peritonitis (FIP; feline infectious peritonitis virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV- and FECV-specific signals of positive selection. We analyzed full-length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site (FCS) and the other within the fusion domain of Spike. We also found fifteen sites subject to positive selection associated with FIPV within Spike, eleven of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were fourteen sites (twelve novel sites) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 FCS and adjacent to C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype and included twenty-four positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV-wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that it is unlikely to be one singular 'switch' mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.