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1.  Susceptibility of neuron-like cells derived from bovine Wharton’s jelly to bovine herpesvirus type 5 infections 
Background
Bovine herpesvirus type 5 (BoHV-5), frequently lethal in cattle, is associated with significant agricultural economic losses due to neurological disease. Cattle and rabbits are frequently used as models to study the biology and pathogenesis of BoHV-5 infection. In particular, neural invasion and proliferation are two of the factors important in BoHV-5 infection. The present study investigated the potential of bovine Wharton’s jelly mesenchymal stromal cells (bWJ-MSCs) to differentiate into a neuronal phenotype and support robust BoHV-5 replication.
Results
Upon inducing differentiation within a defined neuronal specific medium, most bWJ-MSCs acquired the distinctive neuronal morphological features and stained positively for the neuronal/glial markers MAP2 (neuronal microtubule associated protein 2), N200 (neurofilament 200), NT3 (neutrophin 3), tau and GFAP (glial fibrillary acidic protein). Expression of nestin, N200, β-tubulin III (TuJI) and GFAP was further demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR). Following BoHV-5 inoculation, there were low rates of cell detachment, good cell viability at 96 h post-infection (p.i.), and small vesicles developed along neuronal branches. Levels of BoHV-5 antigens and DNA were associated with the peak in viral titres at 72 h p.i. BoHV-5 glycoprotein C mRNA expression was significantly correlated with production of progeny virus at 72 h p.i. (p < 0.05).
Conclusion
The results demonstrated the ability of bWJ-MSCs to differentiate into a neuronal phenotype in vitro and support productive BoHV-5 replication. These findings constitute a remarkable contribution to the in vitro study of neurotropic viruses. This work may pave the way for bWJ-MSCs to be used as an alternative to animal models in the study of BoHV-5 biology.
doi:10.1186/1746-6148-8-242
PMCID: PMC3528423  PMID: 23227933
BoHV-5; in vitro replication; Neuronal culture
2.  Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5 
Background
Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5.
Methods
For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis.
Results
The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/− 6.5; 87,7%) were cleaved and 195 (+/− 3.2; 54,6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/− 8.9, 84,5%), and these embryos produced 193 (+/− 3.2, 49,7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/− 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity.
Conclusion
This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.
doi:10.1186/1477-7827-10-53
PMCID: PMC3447700  PMID: 22823939
BoHV-5; Infection; Bovine embryos; Apoptosis
3.  Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system 
BMC Biotechnology  2012;12:18.
Background
The possibility for isolating bovine mesenchymal multipotent cells (MSCs) from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D) environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM). The three-dimensional (3D) cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ) cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking.
Results
Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when bovine-derived UC-WJ cells was included in the culture which demonstrated the immunossupression profile typically observed among isolated mesenchymal cells from other species. After classified the UC-WJ cells as mesenchymal stromal phenotype the in vitro 3D cultures was performed using the AlgiMatrix® protocol. Based on the size of spheroids (283,07 μm ± 43,10 μm) we found that three weeks of culture was the best period to growth the UC-WJ cells on 3D dimension. The initial cell density was measured and the best value was 1.5 × 106 cells/well.
Conclusions
We described for the first time the isolation and characterization of UC-WJ cells in a serum-free condition and maintenance of primitive mesenchymal phenotype. The culture was stable under 60 consecutive passages with no genetic abnormalities and proliferating ratios. Taken together all results, it was possible to demonstrate an easy way to isolate and culture of bovine-derived UC-WJ cells under 2D and 3D serum-free condition, from fetal adnexa with a great potential in cell therapy and biotechnology.
doi:10.1186/1472-6750-12-18
PMCID: PMC3443425  PMID: 22559872
Wharton´s jelly stem cells; Mesenchymal stromal cells; Differentiation; Immunomodulation
4.  Immunohistochemical approach to the pathogenesis of clinical cases of Bovine Herpesvirus type 5 infections 
Diagnostic Pathology  2010;5:57.
Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in cattle has some features that are similar to those of herpetic encephalitis in humans and other animal species. Human Herpesvirus 3 (commonly known as Varicella-zoster virus 1), herpes simplex viruses (HSV), and equid Herpesvirus 1 (EHV-1) induce an intense inflammatory, vascular and cellular response. In spite of the many reports describing the histological lesions associated with natural and experimental infections, the immunopathological mechanisms for the development of neurological disorder have not been established. A total of twenty calf brains were selected from the Veterinary School, University of São Paulo State, Araçatuba, Brazil, after confirmation of BoHV-5 infection by virus isolation as well as by a molecular approach. The first part of the study characterized the microscopic lesions associated with the brain areas in the central nervous system (CNS) that tested positive in a viral US9 gene hybridization assay. The frontal cortex (Fc), parietal cortex (Pc), thalamus (T) and mesencephalon (M) were studied. Secondly, distinct pathogenesis mechanisms that take place in acute cases were investigated by an immunohistochemistry assay. This study found the frontal cortex to be the main region where intense oxidative stress phenomena (AOP-1) and synaptic protein expression (SNAP-25) were closely related to inflammatory cuffs, satellitosis and gliosis, which represent the most frequently observed neurological lesions. Moreover, MMP-9 expression was shown to be localized in the leptomeninges, in the parenchyma and around mononuclear infiltrates (p < 0.0001). These data open a new perspective in understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating BoHV-5 pathogenesis and the strategies of host-virus interaction in order to invade de CNS.
doi:10.1186/1746-1596-5-57
PMCID: PMC2945982  PMID: 20831786

Results 1-4 (4)