Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40 % of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40 % in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80 % of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy.

Bovine herpesvirus 5 (BHV5) infection of young cattle is frequently associated with fatal neurological disease and, as such, represents an attractive model for studying the pathogenesis of viral-induced meningoencephalitis. Following replication in the nasal mucosa, BHV5 invades the central nervous system (CNS) mainly through the olfactory pathway. The innate immune response triggered by the host face to virus replication through the olfactory route is poorly understood. Recently, an upregulation of conserved pathogen-associated molecular pattern, as Toll-like receptors (TLRs), has been demonstrated in the CNS of BHV5 experimentally infected cows. A new perspective to understand host-pathogen interactions has emerged elucidating microRNAs (miRNAs) network that interact with innate immune response during neurotropic viral infections. In this study, we demonstrated a link between the expression of TLRs 3, 7, and 9 and miR-155 transcription in the olfactory bulbs (OB) of 16 cows suffering from acute BHV5-induced neurological disease. The OBs were analyzed for viral antigens and genome, miR-155 and TLR 3, 7, and 9 expression considering three major regions: olfactory receptor neurons (ORNs), glomerular layer (GL), and mitral cell layer (ML). BHV5 antigens and viral genomes, corresponding to glycol-C gene, were detected in all OBs regions by fluorescent antibody assay (FA) and PCR, respectively. TLR 3, 7, and 9 transcripts were upregulated in ORNs and ML, yet only ORN layers revealed a positive correlation between TLR3 and miR-155 transcription. In ML, miR-155 correlated positively with all TLRs studied. Herein, our results evidence miR-155 transcription in BHV5 infected OB tissue associated to TLRs expression specifically ORNs which may be a new window for further studies.

Despite of the role of domestic dogs as reservoirs for threatening viral diseases for wild carnivores, few studies have focused to identify circulation of viruses among dogs living in human/wildlife interfaces. To identify canine parvovirus (CPV) types circulating in dogs living in an Atlantic forest biome, faecal samples (n = 100) were collected at the same period (one week) corresponding to each of four areas, during 2014 to 2016 and corresponded to 100 different individuals. CPV was isolated in cell culture from 67 out 100 (67%) samples from healthy dogs. Cytopathic effects were characterized by total or partial cell culture lysis. Genome sequences of CPV-2a (10%), CPV-2b (7%) and CPV-2c (50%) were concomitantly detected by PCR and nucleotide sequencing. The current study addresses the importance of monitoring CPV circulation among dogs presenting potential contact with wildlife species.

Brazil represents the greatest beef producer among tropical countries, and the major obstacle for meat international trade is sanitary problems especially closely related to viral encephalitis. The goal of this study was to estimate the accuracy of the glycol and US9 gene-based polymerase chain reactions (PCRs) for the detection of bovine Herpesvirus type 5 (BoHV-5) from decomposed brain samples (n = 95). For this purpose, a latent-class (bayesian) approach was used. Sensitivity (Se) was estimated to be 70% (95% probability interval, 40-80) and specificity (Sp) 100% in the statistical analysis for both PCRs used. Accordingly, a minimum of ≥40% of the calves was estimated to harbor BoHV-5 DNA even after 72 h of decomposition at room temperature. It was concluded that US9 gene-based PCR could also be considered a cost-effective alternative in sanitary programmers. However, given the importance of veterinary diagnoses, PCR-positive samples should be further confirmed through in vitro isolation and/or sequencing.

Emerging coronavirus infections are a major threat to global public health. In this respect, a novel recombination of canine coronavirus (CCoV) and feline coronavirus (FCoV) was described among human biological samples, giving rise to a potential zoonosis. Despite all efforts, the host‒virus immune response related to CCoV is missing. In this study, pantropic CCoV infection of canine macrophages derived from peripheral blood monocytes was performed. After infection, macrophages were first polarized to the M1 and/or M2 phenotype. Moreover, infection kinetics, cell viability, apoptosis, mitochondrial dysfunction associated with reactive oxygen species and oxide nitric production were measured. Our results demonstrated that virus infection mainly polarized host macrophages to the classically activated (M1) phenotype, as demonstrated by amoeboid morphology with numerous fibrillary cytoplasmic processes followed by classical phenotypes. Viral infection released new particles at 18 h post-infection associated with a decrease in viable cells. Furthermore, upon CCoV infection, M1 cells exhibited reduced phagocytosis properties, as evidenced by a neutral red uptake assay. This in vitro method open an avenue for further studies on host-virus interaction.

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two-dimensional (2D) and three-dimensional (3D) conditions. The 3D was performed in six-well AlgiMatrix™ (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50-125 µm for CC and 175-200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases-1, -2, -9 and -13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E-cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)-derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model.

Canine coronavirus (CCoV) is an important pathogen that affect dogs. Here we assessed a digital real-time based PCR (dPCR) for the detection of CCoV from infected AF-72 cells and directly from faeces from 100 symptomatic dogs. The results obtained from dPCR were compared to real-time TaqMan® based PCR assay (qPCR) and positive samples were submitted to phylogenetic analysis. Thus, dPCR had an equal sensitivity, 101 copies/μl of partial M CCoV gene, when compared to qPCR results with a good agreement in all analysis. These results indicated that the dPCR could be an alternative technique for the diagnosis of CCoV from clinical samples with advantage of simplicity and sensitivity.