Transcriptome analysis of Crandell Rees Feline Kidney (CRFK) cells infected with Feline calicivirus strain 023 (FCV 023).

Endogenous retroviruses (ERVs) are retroviral sequences present in the host genomes. Although most ERVs are inactivated, some are produced as replication-competent viruses from host cells. We previously reported that several live-attenuated vaccines for companion animals prepared using the Crandell-Rees feline kidney (CRFK) cell line were contaminated with a replication-competent feline ERV termed RD-114 virus. We also found that the infectious RD-114 virus can be generated by recombination between multiple RD-114 virus-related proviruses (RDRSs) in CRFK cells. In this study, we knocked out RDRS env genes using the genome-editing tool TAL Effector Nuclease (TALEN) to reduce the risk of contamination by infectious ERVs in vaccine products. As a result, we succeeded in establishing RDRS knockout CRFK cells (RDKO_CRFK cells) that do not produce infectious RD-114 virus. The growth kinetics of feline herpesvirus type 1, calicivirus, and panleukopenia virus in RDKO_CRFK cells differed from those in parental cells, but all of them showed high titers exceeding 107 TCID50/mL. Infectious RD-114 virus was undetectable in the viral stocks propagated in RDKO_CRFK cells. This study suggested that RDRS env gene-knockout CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with no risk of contamination with infectious ERV.

Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype

IntroductionIn addition to standard highly active antiretroviral therapy protocols, complementary therapies using natural compounds are widely used by human immunodeficiency virus (HIV)‐infected human patients. One such compound is the fermented wheat germ extract (FWGE), named Avemar.Materials and methodsIn this study, we investigate the effects of Avemar in a feline‐acquired immunodeficiency syndrome model. MBM lymphoid cells were acutely infected by the American feline immunodeficiency virus (FIV)‐Petaluma (FIV‐Pet) and the European FIV Pisa‐M2 strains. FL‐4 lymphoid cells, continuously producing FIV‐Pet, served as a model for chronic infection. Crandell Rees feline kidney (CRFK) cells were infected by either FIV‐Pet or feline adenovirus (FeAdV) as a model for transactivation and opportunistic viral infection. Cell cultures were treated pre‐ and post‐infection with serial dilutions of spray‐dried FWGE (Avemar pulvis, AP), a standardized active ingredient in commercial Avemar products. Residual FIV and FeAdV infectivity was quantified.ResultsIn a concentration‐dependent manner, AP inhibited replication of FIV strains in MBM and CRFK cells by 3–5 log. Low AP concentration prevented FIV‐Pet release from FL‐4 cells. Higher concentrations destroyed virus‐producing cells with cytopathic effects resembling apoptosis. AP strongly inhibited FeAdV production inside CRFK cells but not in HeLa cells. Adenovirus particles are then released via the disintegration of CRFK cells.DiscussionThis report is the first to describe the antiviral effects of Avemar. Further studies are required to confirm its in vitro and in vivo effects and to investigate the potential for its use as a nutraceutical in FIV‐infected felines or HIV‐infected humans.ConclusionAvemar, as a single nutraceutical, inhibits FIV replication and destroys retrovirus carrier cells. An important conclusion is that prolonged Avemar treatment might reduce the number of retrovirus‐producing cells in the host.

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

Feline panleukopenia virus (FPV) causes fatal leukopenia and severe hemorrhagic diarrhea in cats. Although FPV isolates have been reported worldwide from several animals, the biological and genetic features of South Korean FPVs remain unclear. We characterized molecularly South Korean FPV isolates. Crandell-Rees feline kidney (CRFK) cells were used to isolate FPV from 60 organ homogenates. The isolates were confirmed to be FPVs via analyses of cytopathic effects, immunofluorescence studies, electron microscopy, and polymerase chain reaction. Viral genetic analyses used the full VP2 sequences. Eight isolates propagated in CRFK cells were confirmed to be FPVs. All isolates yielded viral titers ranging from 104.5 to 106.0 TCID50/mL 5 days after inoculation into CRFK cells and exhibited hemagglutination titers ranging from 27 to 212 (using pig erythrocytes). The Korean FPV isolates grew well in cat cells such as CRFK and Fcwf4 cells. The FPV isolates were most similar to the KS42 strain isolated from a Korean cat in 2008. The FPV isolates will serve as useful antigens in future sero-epidemiological studies and will aid in the development of diagnostic tools.

BackgroundFeline Infectious Peritonitis (FIP) is a lethal systemic disease, caused by the FIP Virus (FIPV); a virulent mutant of Feline Enteric Coronavirus (FECV). Currently, the viruses virulence determinants and host gene expressions during FIPV infection are not fully understood.MethodsRNA sequencing of Crandell Rees Feline Kidney (CRFK) cells, infected with FIPV strain 79–1146 at 3 hours post infection (h.p.i), were sequenced using the Illumina next generation sequencing approach. Bioinformatic’s analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench mapped both control and infected cell reads to 18899 genes out of 19046 annotated genes. Kal’s Z test statistical analysis was used to analyse the differentially expressed genes from the infected CRFK cells. Real time RT-qPCR was developed for further transcriptional profiling of three genes (PD-1, PD-L1 and A3H) in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diseased cats.ResultsBased on Kal’s Z-test, with False Discovery Rate (FDR) <0.05 and >1.99 fold change on gene expressions, a total of 61 genes were differentially expressed by both samples, where 44 genes were up-regulated and the remainder were down-regulated. Most genes were closely clustered together, suggesting a homogeneous expression. The majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and Th1 cell functions, and the regulation of apoptosis. Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data.ConclusionThe possible roles of these genes, and their importance in feline coronaviruses infection, are discussed.

The life cycle of calicivirus is not fully understood because most of the viruses cannot be propagated in tissue culture cells. We studied the mechanism of calicivirus entry into cells using feline calicivirus (FCV), a cultivable calicivirus. From the cDNA library of Crandell-Rees feline kidney (CRFK) cells, feline junctional adhesion molecule 1 (JAM-1), an immunoglobulin-like protein present in tight junctions, was identified as a cellular-binding molecule of the FCV F4 strain, a prototype strain in Japan. Feline JAM-1 expression in nonpermissive hamster lung cells led to binding and infection by F4 and all other strains tested. An anti-feline JAM-1 antibody reduced the binding of FCV to permissive CRFK cells and strongly suppressed the cytopathic effect (CPE) and FCV progeny production in infected cells. Some strains of FCV, such as F4 and F25, have the ability to replicate in Vero cells. We found that regardless of replication ability, FCV bound to Vero and 293T cells via simian and human JAM-1, respectively. In Vero cells, an anti-human JAM-1 antibody inhibited binding, CPE, and progeny production by F4 and F25. In addition, feline JAM-1 expression permitted FCV infection in 293T cells. Taken together, our results demonstrate that feline JAM-1 is a functional receptor for FCV, simian JAM-1 also functions as a receptor for some strains of FCV, and the interaction between FCV and JAM-1 molecules may be a determinant of viral tropism. This is the first report concerning a functional receptor for the viruses in the family Caliciviridae.

Transcriptome analysis of infected Crandell Rees Feline Kidney (CRFK) cells by canine parvovirus type 2c Laotian isolates

Investigation of the antiviral properties of copper iodide nanoparticles against feline calicivirus

Interstitial nephritis in cats inoculated with Crandell Rees feline kidney cell lysates

The JM1/1 strain of fowl adenovirus (FAV) serotype 1 isolated from gizzard erosion was used to investigate the biology of FAV in homologous (susceptible) and heterologous cells. The FAV JM1/1 strain is capable of efficient multiplication in primary chicken kidney (CK) cells, but not in Crandell-Rees feline kidney (CRFK) cells or Vero cells. FAV adsorption in heterologous cells was slightly higher than in CK cells. An early gene encoding a DNA-binding protein and a late gene encoding the hexon protein were expressed in CK cells. Only the early gene was expressed in Vero cells. Neither of these genes was expressed in CRFK cells. These results suggest that the virus was unable to multiply effectively due to suppression of viral gene expression in the heterologous cells used in this study.

BackgroundFeline herpesvirus 1 (FHV-1) is a common cause of respiratory and ocular disease in cats. Especially in young kittens that have not yet reached the age of vaccination, but already lost maternal immunity, severe disease may occur. Therefore, there is a need for an effective antiviral treatment. In the present study, the efficacy of six antiviral drugs, i.e. acyclovir, ganciclovir, cidofovir, foscarnet, adefovir and 9-(2-phosphonylmethoxyethyl)-2, 6-diaminopurine (PMEDAP), against FHV-1 was compared in Crandell-Rees feline kidney (CRFK) cells using reduction in plaque number and plaque size as parameters.ResultsThe capacity to reduce the number of plaques was most pronounced for ganciclovir, PMEDAP and cidofovir. IC50 (NUMBER) values were 3.2 μg/ml (12.5 μM), 4.8 μg/ml (14.3 μM) and 6 μg/ml (21.5 μM), respectively. Adefovir and foscarnet were intermediately efficient with an IC50 (NUMBER) of 20 μg/ml (73.2 μM) and 27 μg/ml (140.6 μM), respectively. Acyclovir was least efficient (IC50 (NUMBER) of 56 μg/ml or 248.7 μM). All antiviral drugs were able to significantly reduce plaque size when compared with the untreated control. As observed for the reduction in plaque number, ganciclovir, PMEDAP and cidofovir were most potent in reducing plaque size. IC50 (SIZE) values were 0.4 μg/ml (1.7 μM), 0.9 μg/ml (2.7 μM) and 0.2 μg/ml (0.7 μM), respectively. Adefovir and foscarnet were intermediately potent, with an IC50 (SIZE) of 4 μg/ml (14.6 μM) and 7 μg/ml (36.4 μM), respectively. Acyclovir was least potent (IC50 (SIZE) of 15 μg/ml or 66.6 μM). The results demonstrate that the IC50 (SIZE) values were notably lower than the IC50 (NUMBER) values. The most remarkable effect was observed for cidofovir and ganciclovir. None of the products were toxic for CRFK cells at antiviral concentrations.ConclusionIn conclusion, measuring reduction in plaque number and plaque size are two valuable and complementary means of assessing the efficacy of an antiviral drug. By using these parameters for six selected antiviral drugs, we found that ganciclovir, PMEDAP, and cidofovir are the most potent inhibitors of FHV-1 replication in CRFK cells. Therefore, they may be valuable candidates for the treatment of FHV-1 infection in cats.

The steroids 3,6,17-trihydroxy-stigmasta-4,7,24(28)-triene (A) and 14,15,18,20-diepoxyturbinarin (B) were isolated from the cyclohexane extract of brown alga, Turbinaria conoides (J. Agardh) Kutzing, and have been reported for their antimicrobial activity by us. In this study, the isolated compounds were evaluated for comprehensive antihistaminic, antiviral and cytotoxicity screening. The antihistaminic study was performed using in vitro standard animal models. Evaluation of the potency (EC50), affinity (pA2) and the maximal response (E max) of the histamine alone and in the presence of the compounds were determined. Antiviral activity and cytotoxicity were performed in Crandell-Rees feline kidney (CRFK) cells by a colorimetric formazan-based MTS assay. No significant antiviral activity or cytotoxicity were observed for the compounds in the CRFK cells. Compound A inhibited the histamine-induced concentration at 20 µg mL−1(p < 0.05). The most significant inhibition (97%) was observed for compound B (p < 0.01) at the same concentration, which was comparable to that of the positive control chlorpheniramine maleate (10 µg mL−1). This potentiality suggests that 14,15,18,20-diepoxyturbinarin (B) can be developed as a new lead antihistaminic agent.

Detection of RD-114 virus by a reverse transcriptase assay based on product enhancement

Feline calicivirus replication induces apoptosis in cultured cells