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The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that interacts with substrates, including microbial metabolites. Recent advances reveal that AhR is involved in the host response to coronaviruses (CoVs) infection. Particularly, AhR antagonists decrease the expression of angiotensin-converting enzyme 2 (ACE2) via AhR up-regulation, resulting in suppression of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in mammalian cells. Herein, we report that AhR is expressed in canine fibrosarcoma (A72) cells, where it is considerably activated by infection with genotype II of canine coronavirus (CCoV-II). The pharmacological inhibition of AhR, by CH223191, suppressed cell death signs and increased cell viability. Furthermore, the AhR antagonist induced a meaningful decline in virus yield, accompanied by the inhibition of the expression of viral nuclear protein (NP). Fascinatingly, during CCoV infection, a novel co-expression of NP and AhR expression was found. Taken together, our preliminary findings show that infection with CCoV activates AhR, and pharmacologic AhR inhibition reduces CCoV replication, identifying AhR as a possible candidate target for CCoV antiviral therapy.

Recent studies have demonstrated that 3-O-methylfunicone (OMF), a fungal secondary metabolite from Talaromyces pinophilus belonging to the class of funicone-like compounds, has antiviral activity against canine coronaviruses (CCoV), which causes enteritis in dogs. Herein, we selected two additional funicone-like compounds named vermistatin (VER) and penisimplicissin (PS) and investigated their inhibitory activity towards CCoV infection. Thus, both compounds have been tested for their cytotoxicity and for antiviral activity against CCoV in A72 cells, a fibrosarcoma cell line suitable for investigating CCoV. Our findings showed an increase in cell viability, with an improvement of morphological features in CCoV-infected cells at the non-toxic doses of 1 μM for VER and 0.5 μM for PS. In addition, we observed that these compounds caused a strong inhibition in the expression of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which is activated during CCoV infection. Our results also showed the alkalinization of lysosomes in the presence of VER or PS, which may be involved in the observed antiviral activities.

Canine coronavirus (CCoV), an alphacoronavirus, may cause self-limiting enteric disease in dogs, especially in puppies. The noteworthy plasticity of coronaviruses (CoVs) occurs through mutation and recombination processes, which sometimes generate new dangerous variants. The ongoing SARS-CoV-2 pandemic and the isolation of a novel canine–feline recombinant alphacoronavirus from humans emphasizes the cross-species transmission ability of CoVs. In this context, exploring antiviral compounds is essential to find new tools for fighting against CoVs infections. Fungi produce secondary metabolites, which are often developed as antibiotics, fungicides, hormones, and plant growth regulators. Previous examinations of benzo-γ-pyrone 3-O-methylfunicone (OMF), obtained from Talaromyces pinophilus, showed that it reduces the infectivity of hepatitis C virus and bovine herpesvirus 1. Based on this evidence, this study evaluated the antiviral ability of OMF against CCoV infection in a canine fibrosarcoma (A72) cell line. During CCoV infection, a non-toxic dose of OMF markedly increased features of cell viability. Moreover, OMF induced a significant reduction in virus yield in the presence of an intense downregulation of the viral nucleocapsid protein (NP). These findings occurred in the presence of a marked reduction in the aryl hydrocarbon receptor (AhR) expression. Taken together, preliminary findings suggest that OMF inhibiting AhR shows promising activity against CCoV infection.

Background The synergic action of KHCO 3 and D-ribose is tested on A72 and HTB-126 cell lines proliferation using K:D-Rib solution. Altered Na + /K + ATPase expression and activity were shown in patients with cancer. Studies in human epithelial-derived malignancies indicate that K + depletion also occurs, contributing to the increased intracellular Na + /K + ratio [ 1 ]. D-ribose transformed to piruvate, enters into the Krebs's cycle and has a key role on energetic metabolism. The up-regulation of glycolysis in tumor cells is already well known and it is the rationale of F 18 -FDG PET diagnostic technique. D-ribose is synthesized by the non-oxidative transketolase PPP reaction. Results Results with different K:D-Rib concentrations show that MTT salt interferes with K:D-Rib solution and therefore this method is not reliable. The UV/VIS measurements show that K:D-Rib solutions reduce MTT salt to formazan in absence of cells. Cell proliferation has then been evaluated analysing the digital photos of the Giemsa stained cells with MCID™ software. At 5 mM K:D-Rib concentration, the cell growth arrests between 48 h and 72 h; in fact the cell number after 48 h is around the same with respect to the control after 72 h. In case of HTB-126 human cancer cells, the growth rate was valuated counting the splitting times during 48 days: control cells were split sixteen times while 5 mM treated cells eleven times. Most relevant, the clonogenic assay shows that nine colonies are formed in the control cells while only one is formed in the 5 mM and none in 10 mM treated cells. Conclusions The K:D-Rib solution has an antioxidant behaviour also at low concentrations. Incubation with 5 mM K:D-Rib solution on A72 cells shows a cytostatic effect at 5 mM, but it needs more than 24 h of incubation time to evidence this effect on cell proliferation. At the same concentration on human HTB-126 cells, K:D-Rib solution shows a clear replication slowing but the cytostatic effect at 10 mM K:D-Rib solution only. Results on A72 cells indicate the K + uptake could be determinant either to arrest or to slow down cell growth.

Canine coronavirus type II (CCoV-II), an alphacoronavirus, is responsible for mild enteritis, especially in puppies, but due to its plasticity, it can also cause serious diseases in humans. Formyl peptide receptors (FPRs) play an important role in modulating immune responses, and their expression is variably regulated by cell type and by viral infections. In this study, the role of FPR2 during CCoV infection in a canine fibrosarcoma (A72 cells) cell line as well as in a feline Crandell-Rees Feline Kidney (CRFK) cell line was investigated by in vitro and in silico approaches. During infection, in the presence of WRW4, a specific FPR2 inhibitor, a reduction in gene and protein levels of FPR2 in CCoV infected cells was observed. These results were accompanied by worsened changes in cell viability and morphology in the treated-infected groups, which exhibited substantial growth in virus yield and a significant increase in both gene and protein expression of viral nucleocapsid protein (NP). Interestingly, an opposite trend in the above assays was observed following infection with HP2-20, an agonist of FPR2. The 3D model of canine FPR2 ( c FPR2) showed that WRW4 was confined to the c FPR2 core and did not interact with the extramembrane loops of the receptor, whereas HP2-20 contacted both the core and the second extracellular loop of c FPR2 (ECL2). In addition, the complex c FPR2/HP2-20 exhibited a marked increase in the number of H-bonds, hydrophobic interactions and electrostatic charges compared to the complex c FPR2/WRW4. In conclusion, these results showed that CCoV replication is related to FPR2, suggesting it as an interesting target to develop new drugs to fight CoVs infection.