Modulation of granzymes mRNA expression in neural tissue of BoAHV-1 and BoAHV-5-infected cattle.

Bovine alphaherpesviruses (BoAHV)-1 and 5 are neurotropic viruses of cattle which differ in their neuropathogenic potential. BoAHV-5 is responsible for non-suppurative meningoencephalitis in calves while BoAHV-1 can occasionally cause encephalitis. Granzymes (GZMs) are serine-proteases that participate in CD8+ T cells-mediated killing of virally-infected cells upon release through perforin (PFN)-formed pores in the cell membrane. Recently, six GZMs have been identified in cattle (A, B, K, H, M and O). However, their expression in bovine tissues has not been evaluated. In this study, the mRNA expression levels of PFN and GZMs A, B, K, H and M in the nervous system of calves experimentally-inoculated with BoAHV-1 or BoAHV-5 were analyzed at the three distinct stages of the infectious cycle of alphaherpesviruses: acute infection, latency and reactivation. This is the first report describing the expression of GZMs in bovine neural tissue and the first analysis of GZM expression in the context of bovine alphaherpesviruses neuropathogenesis. The findings revealed that PFN and GZM K are upregulated during BoAHV-1 or BoAHV-5 acute infection. In contrast to BoAHV-1, during BoAHV-5 latency a significant up-regulation of PFN, GZM K and GZM H was detected. PFN and GZM A, K and H expression was also up-regulated during BoAHV-5 reactivation. Therefore, a distinct pattern of PFN and GZM expression is evident along the infectious cycle of each alphaherpesvirus and this might contribute to the differences in BoAHV-1 and BoAHV-5 neuropathogenesis.

Study of the dynamics of in vitro infection with bovine gammaherpesvirus type 4 and apoptosis markers in susceptible cells.

Bovine gammaherpesvirus type 4 (BoHV-4) shows tropism for the endometrium, in which it causes the death of epithelial and stroma cells. Despite having anti-apoptotic genes in its genome, experiments based on immortalized cell lines have shown that BoHV-4 induces cell death by apoptosis. In the present study, we evaluated BoHV-4 replication, pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) mitochondrial genes expression and chromatin condensation in bovine endometrium primary culture cells (BEC) and in the Madin Darby bovine kidney (MDBK) cell line. Results showed that BoHV-4 has a preference for replication in BEC cells over the MDBK cell line, demonstrated by the high viral titer that is consistent with the tropism of the virus. In BEC cells, chromatin condensation was consistent with the values of viral kinetics at the late stage of infection, accompanied with a balance in the mRNA levels of apoptotic mitochondrial proteins. As a consequence, in those cells viral transmission would be enhanced by inhibiting apoptosis in the early stage of virus proliferation, allowing the complete production of viral progeny, and then, the induction of apoptosis in late stages would allow neighboring cells infection. In MDBK cells replication kinetics was coincident with the up-regulation of Bcl-2, which suggests that the productive infection in MDBK is associated with a lytic phase of the virus or another cell death pathway (probably autophagy mechanism) at the late stage of infection. The results agree with the study of nuclear morphology, where a constant chromatin condensation was observed over time. It is clear that the documented BoHV-4 apoptotic responses observed in the cell lines studied above are not valid in cells from primary cultures. The data presented in this study suggest that BoHV-4 could induce apoptosis in BEC cells without a leading role of the mitochondria pathway. Further studies will be necessary to characterize in detail the programmed cell death pathways involved in BoHV-4 infection in the primary cell cultures evaluated.

Effect of bovine leukemia virus (BLV) infection on bovine mammary epithelial cells RNA-seq transcriptome profile.

Bovine leukemia virus (BLV) is a δ-retrovirus responsible for Enzootic Bovine Leukosis (EBL), a lymphoproliferative disease that affects cattle. The virus causes immune system deregulation, favoring the development of secondary infections. In that context, mastitis incidence is believed to be increased in BLV infected cattle. The aim of this study was to analyze the transcriptome profile of a BLV infected mammary epithelial cell line (MAC-T). Our results show that BLV infected MAC-T cells have an altered expression of IFN I signal pathway and genes involved in defense response to virus, as well as a collagen catabolic process and some protooncogenes and tumor suppressor genes. Our results provide evidence to better understand the effect of BLV on bovine mammary epithelial cell's immune response.

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity.

P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor-Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA). Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.