Crandell-Reese Feline Kidney Cells Research Articles

Comparative Evaluation of GS-441524, Teriflunomide, Ruxolitinib, Molnupiravir, Ritonavir, and Nirmatrelvir for In Vitro Antiviral Activity against Feline Infectious Peritonitis Virus.

Feline infectious peritonitis (FIP), caused by feline coronavirus (FcoV), is considered one of the most enigmatic diseases in cats. Developing effective drugs for FIP is crucial due to its global prevalence and severity. In this study, six antiviral drugs were tested for their cytotoxicity, cell viability, and antiviral efficacies in Crandell-Reese feline kidney cells. A cytotoxicity assay demonstrated that these drugs were safe to be used with essentially no cytotoxicity with concentrations as high as 250 µM for ruxolitinib; 125 µM for GS441524; 63 µM for teriflunomide, molnupiravir, and nirmatrelvir; and 16 µM for ritonavir. GS441524 and nirmatrelvir exhibited the least detrimental effects on the CRFK cells, with 50% cytotoxic concentration (CC50) values of 260.0 µM and 279.1 µM, respectively, while ritonavir showed high toxicity (CC50 = 39.9 µM). In the dose-response analysis, GS441524, nirmatrelvir, and molnupiravir demonstrated promising results with selectivity index values of 165.54, 113.67, and 29.27, respectively, against FIPV. Our study suggests that nirmatrelvir and molnupiravir hold potential for FIPV treatment and could serve as alternatives to GS441524. Continued research and development of antiviral drugs are essential to ensure the well-being of companion animals and improve our preparedness for future outbreaks of coronaviruses affecting animals and humans alike.

Antiviral Effects of Adipose Tissue-Derived Mesenchymal Stem Cells Secretome against Feline Calicivirus and Feline Herpesvirus Type 1.

Mesenchymal stem cells (MSCs) have excellent anti-inflammatory and immunomodulatory capabilities and therapeutic effects in some viral diseases. The therapeutic impact of MSCs mainly relies on the paracrine effects of various secreted substances. Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV1) are common and highly prevalent pathogens causing upper respiratory diseases, and FCV is associated with gingivostomatitis in cats. Recently, feline MSC treatment has been reported to improve the clinical symptoms of feline chronic gingivostomatitis, but the antiviral effects of feline MSCs on FCV and FHV1 are not known. In this study, we evaluated the antiviral efficacy of using feline MSC secretome as a conditioned medium on FCV and FHV1 viral replication in Crandell–Reese feline kidney (CRFK) cells, and RNA sequencing was used to analyze how the CRFK cells were altered by the MSC secretomes. The feline MSC secretome did not inhibit FCV or FHV1 viral entry into the CRFK cells but had antiviral effects on the replication of both FCV and FHV1 in a dose-dependent manner.

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

BackgroundThe role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood.ObjectivesThis study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-β, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes.MethodsCRFK 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-β, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence.ResultsFIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-β 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-β 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.ConclusionTLR7 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-β needs to be explored further.

781. Antiviral Effectiveness of Different Disinfectants on Surfaces Contaminated with Surrogate Non-enveloped Viruses

Abstract Background Contamination of surfaces with virions remains an important medical issues because they can serve as fomites for viral transmission. Long acting disinfectants are important because viruses can remain viable for extended durations. Here we assessed the anti-viral efficacy of various environmental spray disinfectants against F specific coliphage MS2, and Feline Calicivirus (FCV) as surrogate non-enveloped viruses. Methods MS2 was grown and titrated according to Environmental Protection Agency method 1601 and counted as plague forming unit per milliliter (PFU/mL.). Strain F9 of FCV was propagated and titrated in monolayers of Crandell-Reese Feline Kidney cells. Commercially available and an experimental disinfectant based on a biguanide combination were tested. The disinfectants, both wet and as dried residues, were compared on silicone surfaces for reduction of viral loads following 1-minute exposure to high viral load challenges (106-107 PFU/ml). Sterilized deionized water was used as a control. Results Results (as log10 reduction relative to sterilized deionized water control) are tabulated below for the disinfectants in wet and dry states following 1-minute viral challenges. Log10 reductions of MS2 and FCV viral loads following 1-minute exposures to disinfectants in wet and dry residue states. Conclusion The disinfectants were generally much less effective as dried residues than wet. The biguanide-combination and hydrogen peroxide-combination disinfectants were the most effective disinfectants tested in both wet and dry residue states. Both warrant further study for extended eradication of highly infectious viruses on surfaces. Disclosures All Authors: No reported disclosures

Expression profiles of immune mediators in feline Coronavirus-infected cells and clinical samples of feline Coronavirus-positive cats

BackgroundThere are two biotypes of feline coronavirus (FCoV): the self-limiting feline enteric coronavirus (FECV) and the feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP), a fatal disease associated with cats living in multi-cat environments. This study provides an insight on the various immune mediators detected in FCoV-positive cats which may be responsible for the development of FIP.ResultsIn this study, using real-time PCR and multiplex bead-based immunoassay, the expression profiles of several immune mediators were examined in Crandell-Reese feline kidney (CRFK) cells infected with the feline coronavirus (FCoV) strain FIPV 79–1146 and in samples obtained from FCoV-positive cats. CRFK cells infected with FIPV 79–1146 showed an increase in the expression of interferon-related genes and pro-inflammatory cytokines such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, and IL8. In addition, an increase in the expression of the above cytokines as well as GM-CSF and IFNγ was also detected in the PBMC, serum, and peritoneal effusions of FCoV-positive cats. Although the expression of MX1 and viperin genes was variable between cats, the expression of these two genes was relatively higher in cats having peritoneal effusion compared to cats without clinically obvious effusion. Higher viral load was also detected in the supernatant of peritoneal effusions compared to in the plasma of FCoV-positive cats. As expected, the secretion of IL1β, IL6 and TNFα was readily detected in the supernatant of peritoneal effusions of the FCoV-positive cats.ConclusionsThis study has identified various pro-inflammatory cytokines and interferon-related genes such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, IL8, GM-CSF and IFNγ in FCoV-positive cats. With the exception of MX1 and viperin, no distinct pattern of immune mediators was observed that distinguished between FCoV-positive cats with and without peritoneal effusion. Further studies based on definitive diagnosis of FIP need to be performed to confirm the clinical importance of this study.

Expression of antiviral cytokines in Crandell-Reese feline kidney cells pretreated with Korean red ginseng extract or ginsenosides

The antiviral activity and protective mechanism of Korean red ginseng (KRG) is not well understood. The aim of this study was to investigate the protective mechanism of KRG extract and ginsenosides against feline calicivirus (FCV), a human norovirus surrogate. CRFK cells that were pretreated for 48h with 10μg/mL of KRG extract or purified ginsenoside Rb1 or Rg1, were inoculated with FCV. RNA extracted from each treated group was examined for the expression of antiviral cytokines, including interferon-α (IFN-α), interferon-β (IFN-β), interferon-ω (IFN-ω), Mx, and zinc finger antiviral protein shorter isoform (ZAPS), by relative real-time reverse transcription-polymerase chain reaction. mRNA expression of IFN-α, IFN-β, IFN-ω, Mx, and ZAPS was significantly induced in the FCV-challenged group pretreated with the KRG extract or ginsenosides, and it was higher than the group treated with FCV alone. Mx protein expression was confirmed by western blotting of CRFK cells pretreated with the ginsenoside Rb1 or with Rg1. Induction of antiviral cytokines contributes to the reduction of the viral titer in CRFK cells pretreated with the KRG extract and purified ginsenosides. In future studies, the antiviral protective mechanism of KRG should be demonstrated using other viruses such as human norovirus.

Enhanced Antiviral Activity of Soybean β‐Conglycinin‐Derived Peptides by Acylation with Saturated Fatty Acids

Peptide mixtures prepared from soybean β-conglycinin (7S-peptides) were acylated with saturated fatty acids of different chain length (6C-18C) in order to improve their antiviral activity against Feline calicivirus (FCV) strain F9 which is a typical norovirus surrogate. Among the fatty acids varieties, it was revealed that 7S-peptides acylated with myristic and palmitic acids potently inhibited FCV replication. Myristorylation and palmitoylation of 7S-peptides kept host cells viability at 91.51% and 98.90%, respectively. The infectivity of FCV on Crandell-Reese feline kidney cells was further determined after exposure of initial titer of 10(6.47) TCID₅₀/mL. Myristoylated and palmitoylated 7S-peptides significantly (P < 0.006) reduced FCV infectivity as compared to native 7S-peptides. Native 7S-peptides showed 25% FCV inhibitory activity while myristoylated and palmitoylated 7S-peptides exhibited 98.59% and 99.98% reduction in FCV infectivity, respectively. Myristoylated and palmitoylated 7S-peptides demonstrated higher anti-FCV activity in a wide range of concentration with complete reduction at 25 μg/mL. Surface hydrophobicity was significantly (P < 0.05) increased after attachment of long hydrocarbon fatty acids to 7S-peptides as supported by changes in fluorescence intensity. Enzymatic hydrolysis together with acylation will give an insight into surface and physiological functional lipopeptides derived from soy β-conglycinin.

Disinfection of fabrics and carpets artificially contaminated with calicivirus: relevance in institutional and healthcare centres

Fabrics and carpets are used widely as surface coverings or linens in healthcare settings and are prone to contamination with infectious agents such as noroviruses (NoVs). Laundering, water cleaning and vacuuming are considered to be adequate for routine cleaning of these materials, but no standard procedure for their disinfection is available in case of contamination. Testing disinfectants for their efficacy against NoVs is difficult because these viruses cannot be cultivated in vitro. Therefore, feline calicivirus (FCV) has gained acceptance as a surrogate model for NoVs in disinfectant efficacy testing. The present study evaluated five disinfectants against FCV on various fabrics or carpets. FCV was dried on fabrics and carpets, followed by treatment with a given disinfectant for a defined contact time of 1, 5 or 10 min. The surviving virus was then eluted and titrated in Crandell-Reese feline kidney cells to determine virus inactivation. A disinfectant was considered to be effective if it inactivated at least 99% of the applied virus. Metricide, an activated dialdehyde-based product, was found to be the most effective disinfectant on all types of fabric and carpet, inactivating more than 99.99% of the virus in 1-10 min. In general, effectiveness of disinfectants increased with an increase in exposure time from 1 to 10 min. The disinfection of carpets was more difficult than the disinfection of fabrics; 100% polyester was the least amenable to disinfection. Only Metricide and Microbac-II (a phenolic compound) were able to inactivate 99% of FCV on 100% polyester. In summary, activated dialdehyde was found to be uniformly active against FCV on all types of material tested.

Gene-expression changes induced by Feline immunodeficiency virus infection differ in epithelial cells and lymphocytes

Infection of cats with Feline immunodeficiency virus (FIV) is an important model for understanding comparative lentivirus biology. In vivo, FIV infects lymphocytes and monocyte/macrophages, but in vitro infection is commonly investigated in epithelial Crandell-Reese Feline Kidney (CRFK) cells. In this study, the transcriptional responses of CRFK cells and primary lymphocytes to infection with FIV 34TF, a cloned subtype A virus, and FIV USgaB01, a biological subtype B isolate, were determined. Reverse-transcribed mRNA from both cell types was hybridized to microarrays containing 1700 human expressed sequence tags in duplicate and data were analysed with Significance Analysis of Microarrays (sam) software. Results from six experiments assessing homeostatic cross-species hybridization excluded 3.48 % inconsistently detected transcripts. Analysis of data from five time points over 48 h after infection identified 132 and 24 differentially expressed genes in epithelial cells and lymphocytes, respectively. Genes involved in protein synthesis, the cell cycle, structure and metabolism were affected. The magnitude of gene-expression changes ranged from 0.62 to 1.62 and early gene induction was followed by downregulation after 4 h. Transcriptional changes in CRFK cells were distinct from those in lymphocytes, except for heat-shock cognate protein 71, which was induced at multiple time points in both cell types. These findings indicate that FIV infection induces transcriptional changes of a modest magnitude in a wide range of genes, which is probably reflective of the relatively non-cytopathic nature of virus infection.

Evaluation of a norovirus sampling method using sodium dodecyl sulfate/EDTA-pretreated chromatography paper strips

Noroviruses are the most common cause of nonbacterial gastroenteritis outbreaks. A novel method was tested that utilizes sodium dodecyl sulfate (SDS)/EDTA-pretreated chromatography paper strips to collect and transport norovirus-containing fresh stool samples. As confirmation, the strips were infected with different dilutions of human norovirus-containing faeces and stored at different temperatures (−80, −20, 4 °C, room temperature and 37 °C). Reverse transcription (RT)-PCR analysis was carried out after storage of the strips for 1, 7, 14, 60, and 120 days. The presence of amplifiable norovirus RNA on the strips was inversely correlated with the duration of storage and the storage temperature. Norovirus RNA remained detectable for a sufficient amount of time at room temperature or even in warmer climatic conditions to allow transportation of the samples from a field site to a reference laboratory using regular postal mail. To prove that viral infectivity was lost upon contact with the SDS/EDTA-treated strips, feline calicivirus F9 (10 7 TCID 50/ml) was loaded onto the strips. After elution and dialysis, no cytopatic effect could be observed upon incubation on Crandell–Reese feline kidney cells. The SDS/EDTA-pretreated chromatography filter paper strips are an inexpensive, biosafe and adequate method to collect, transport and store norovirus samples.

The pathway of feline calicivirus entry

The requirement for a low pH-dependent step during feline calicivirus (FCV) entry into Crandell-Reese feline kidney cells was investigated. Chloroquine, a lysosomotropic agent that prevents acidification of intracellular vesicles, inhibited the production of infectious virus when present during adsorption and the initial stages of FCV replication, but had little effect when added after 2 h post infection. The effect of chloroquine was reversible, allowing the virus growth curve to proceed when removed from the culture. In the presence of chloroquine small amounts of viral RNA were detected at 4, 6, and 8 h post infection, compared to untreated infected cells. These results suggest that entry of feline calicivirus into cells requires a low pH-dependent step.

Early interaction of feline calicivirus with cells in culture.

The kinetics and biochemical properties of feline calicivirus (FCV) attachment to Crandell-Reese feline kidney cells were determined. Maximum binding was observed at pH 6.5. Cells in suspension at 4 degrees C bound virus more efficiently than cells in monolayers at 4 degrees C or 37 degrees C. High initial binding rate was observed in monolayers or cells in suspension and proceeded to a maximum at 90 min, although half maximal binding was observed as early as 15 min. Binding was specific and competitively blocked by serotypically homologous or heterologous FCV as well as by San Miguel sea lion virus. Treatment of cells with proteases increased FCV binding, whereas phospholipase had no effect on virus attachment. Conversely, cells treated with neuraminidase followed by O-glycanase treatment showed a decreased binding ability. Cells of feline origin bound FCV very efficiently, and non-permissive cells showed a poor binding ability. Following transfection of viral RNA, infectious virus could be recovered from all non-permissive cells, except from Madin-Darby canine kidney cells. These results suggest that FCV binds to a receptor in which carbohydrates may be an important component and that FCV replication in non-permissive cells is primarily restricted by the absence of appropriate receptors on the cell surface.