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.

The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza), myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P < 0.05) in bfMSC cultured under 100 µM of 5-Aza compared to 1 and 10 µM. Treatment of bfMSC with 10 µM of 5-Aza resulted in down-regulation of MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential down-regulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.

Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50% of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37% of cells being in the early stages of apoptosis; 63.69% of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.

The possibility of isolating bovine mesenchymal multipotent stromal cells (MSCs) from fetal adnexa is an interesting prospect due to the potential use of these cells in biotechnological applications. However, little is known about the properties of these progenitor cells in bovine species. Wharton's jelly (WJ) MSC cells were obtained from the umbilical cord of bovine fetuses at three different stages of pregnancy and divided into groups 1, 2 and 3 according to gestational trimester. Cell morphology, from the three stages of pregnancy, typically appeared fibroblast-like spindle-shaped, presenting the same viability and number. Moreover, the proliferative ability of T-cells in response to a mitogenic stimulus was suppressed when WJMSC cells were added to the culture. Multilineage properties were confirmed by their ability to undergo adipogenic, osteogenic/chondrogenic and neurogenic differentiation. Mesenchymal phenotyping, CD105+, CD29+, CD73+ and CD90+ cell markers were detected in all three cell groups, yet these markers were considered more expressed in MSCs of group 2 (p < 0.005). Expression of cytokines IL2, IL6RR, INFAC, INFB1, IFNG, TNF and LTBR were downregulated, whereas IL1F10 expression was upregulated in all tested WJMSCs. The present study demonstrated that WJMSCs harvested from the bovine umbilical cord at different gestational stages showed proliferative capacity, immune privilege and stemness potential.

The present study reports an investigation on the phenotype of inflammatory and immune cells, cytokine and viral gene expression in the brains of cattle naturally infected with bovine herpesvirus 5 (BHV5). Brain sections of 38 affected animals were analysed for the nature and extent of perivascular cuffs in the Virchow-Robin space and parenchyma. Histopathological changes were severe in the olfactory bulbs (Obs), hippocampus, piriform, frontal, temporal and parietal cortices/lobes and were characterized by inflammatory infiltrates in Virchow-Robin spaces. The histopathological changes correlated positively with the distribution of BHV5 antigens (r = 0.947; P < 0.005). Cells of CD3+ phenotype were predominant in areas with severe perivascular cuffs. Viral antigens and genomic viral DNA were detected in the Obs and piriform lobe, simultaneously (r = 0.987; P < 0.005). Similarly, pro-inflammatory cytokine genes INFG, IL2, TNF and LTBR were expressed in the same brain areas (P < 0.005). These results provide important information on the inflammatory and immunological events accompanying BHV5 neurological infections. Our findings provide the first evidence for increased immune activation followed by inflammatory cytokine expression, positively correlated with viral replication in the cranial areas of the brain. Taken together, these results suggest that the host immune response and inflammation play a crucial role in the pathogenesis of acute encephalitis by BHV5 in cattle.