Following the COVID-19 epidemic outbreak in Ariano Irpino, Campania region (Italy), we tested lactating cows for the presence of SARS-CoV-2 on a cattle farm at which, prior to the investigation, 13 of the 20 farmworkers showed COVID-19-like symptoms, and one of them died. Twenty-four lactating cows were sampled to detect SARS-CoV-2. All nasal and rectal swabs and milk samples were negative for SARS-CoV-2 RNA. Of the 24 collected serum samples, 11 showed antibodies against SARS-CoV-2 nucleocapsid protein, 14 showed antibodies against SARS-CoV-2 spike protein, and 13 developed neutralising antibodies for SARS-COV-2; all samples were negative for Bovine Coronavirus (BCoV), another betacoronavirus. To our knowledge, this is the first report of natural serological evidence of SARS-CoV-2 infection in lactating cows. We hypothesise that this may be a case of reverse zoonosis. However, the role of cattle in SARS-CoV-2 infection and transmission seems to be negligible.

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.

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.

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.

Feline coronavirus (FCoV) is an alphacoronavirus (αCoV) that causes moderate or chronic asymptomatic infection in cats. However, in a single infected cat, FCoV can modify its cellular tropism by acquiring the ability to infect macrophages, resulting in the development of feline infectious peritonitis (FIP). In this context, to restrain the impact of FCoV infection, scientific research has focused attention on the development of antiviral therapies involving novel mechanisms of action. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) signaling regulates the host response to different human and animal CoVs. Hence, the mechanism of action of AhR was evaluated upon FCoV infection in Crandell Feline Kidney (CRFK) and in canine fibrosarcoma (A72) cells. Following infection with feline enteric CoV (FECV), strain “München”, a significant activation of AhR and of its target CYP1A1, was observed. The selective AhR antagonist CH223191 provoked a reduction in FCoV replication and in the levels of viral nucleocapsid protein (NP). Furthermore, the effect of the AhR inhibitor on the acidity of lysosomes in infected cells was observed. Our findings indicate that FCoV acts on viral replication that upregulates AhR. CH223191 repressed virus yield through the inhibition of AhR. In this respect, for counteracting FCoV, AhR represents a new target useful for identifying antiviral drugs. Moreover, in the presence of CH223191, the alkalinization of lysosomes in FCoV-infected CRFK cells was detected, outlining their involvement in antiviral activity.