Viral cis-regulatory elements as sensors of cellular states and environmental cues.

To withstand a hostile cellular environment and replicate, viruses must sense, interpret, and respond to many internal and external cues. Retroviruses and DNA viruses can intercept these cues impinging on host transcription factors via cis-regulatory elements (CREs) in viral genomes, allowing them to sense and coordinate context-specific responses to varied signals. Here, we explore the characteristics of viral CREs, the classes of signals and host transcription factors that regulate them, and how this informs outcomes of viral replication, immune evasion, and latency. We propose that viral CREs constitute central hubs for signal integration from multiple pathways and that sequence variation between viral isolates can rapidly rewire sensing mechanisms, contributing to the variability observed in patient outcomes.

Modulation of Apoptosis by Bovine Gammaherpesvirus 4 Infection in Bovine Endometrial Cells and the Possible Role of LPS in This Process.

The prevalent pathogens associated with bovine uterine infections are bacteria that appear to increase the host's susceptibility to secondary infections with other bacteria or viruses, among which BoGHV4 is the most frequently found. In this work, the study of the pathways of apoptosis induction was carried out on an experimental model of primary culture of endometrial cells, in order to know the implication of BoGHV4 and the presence of bacterial LPS in the pathogenesis of the bovine reproductive tract. For this, different staining techniques and molecular analysis by RT-PCR were used. The results obtained allowed us to conclude that the level of cell death observed in the proposed primary culture is directly related to the time of viral infection and the presence of LPS in BoGHV4 infection. The apoptosis indices in cells infected with BoGHV4 and BoGHV4 + LPS revealed a maximum that correlated with the appearance of cytopathic effects and the maximum viral titers in the model studied. However, morphological, biochemical, and molecular changes were evident during both early and late stages of apoptosis. These findings provide information on the factors that may influence the pathogenesis of BoGHV4 and help to better understand the mechanisms involved in virus infection.

Effect of heat stress on TNF-α, TNFRI and TNFRII expression in BLV infected dairy cattle.

High temperatures for extended periods, which do not allow animals to recover from heat stress, affect in particular those BLV-infected animals that carry a high proviral load. For this study, animals were discriminated between BLV (+) and BLV (-), and those belonging to the first group, were classified based on their proviral load. The expression of the inflammatory cytokine TNF-α and its receptors, which play an important role in disease progression, were quantified by qPCR in two different seasons. During the summer, average temperature was 19.8 °C, maximums higher than 30 °C were frequent. Instead, during the autumn, the average temperature was 12.63 °C, and temperatures never exceeded 27 °C. During this season, almost no periods of temperatures exceeded the comfort limit. Our results revealed that the expression levels of TNF-α and its receptors were downregulated in animals with high proviral load. This fact could affect their antiviral response and predispose to viral dissemination; over time, animals with a poorer immune system are prone to acquiring opportunistic diseases. Conversely, animals with LPL maintained their expression profile, with behavior comparable to non-infected animals. These findings should be considered by producers and researchers, given the problems that global warming is causing lately to the planet.

Expression of apoptosis-related genes at different stages of BoHV-1 and 5 infection of bovine neural tissue.

Bovine alphaherpesviruses (BoHV) 1 and 5 share many genetic and structural characteristics; however, they differ in their ability to cause encephalitis. Previous research suggests that this difference might be caused by a differential modulation of apoptosis. In this study, we analyzed the mRNA expression of Bax, Bcl-2, Fas and caspases 3 and 8 in neural tissue sections of BoHV-1 and BoHV-5 experimentally-infected cattle. Overall, Fas and caspase 3 mRNA was up-regulated during BoHV-5 acute infection, latency, and reactivation. Conversely, caspase 3 mRNA levels increased only in the olfactory cortex during BoHV-1 acute infection, and it was down-regulated during reactivation, while Fas was only up-regulated during BoHV-1 acute infection and latency. Moreover, Bax/Bcl-2 ratio was lower than 1 during BoHV-1 acute infection except in the trigeminal ganglion, whereas many brain regions exhibit a ratio higher than 1 during BoHV-5 acute infection and reactivation. In summary, our findings suggest that during acute infection and reactivation, BoHV-5 induces a pro-apoptotic condition that could partially justify its increased ability to cause neurological damage.

Cytokine TNF-α and its receptors TNFRI and TNFRII play a key role in the in vitro proliferative response of BLV infected animals.

Bovine leukemia virus (BLV) main host cells are B lymphocytes. Infected animals can be classified into high or low proviral load (HPL or LPL respectively), regarding the number of proviral copies infected lymphocytes they carry. After infection, there is an overexpression of several cytokines, particularly TNF-α, which has a delicate regulation mediated by receptors TNFRI and TNFRII; the first one involved with apoptosis, while the other stimulates cell proliferation. The study aimed to quantify TNF-α and its receptors mRNA expression, and in which extent in vitro proliferation was affected, in peripheral blood mononuclear cells (PBMC) from BLV-infected animals with different proviral loads, after the addition or not of synthetic TNF-α (rTNF-α) for 48 h. PBMC from BLV-infected animals showed spontaneous proliferation after 48 h in culture but did not show changes in proliferation rates after 48 h incubation in the presence of the rTNF-α. TNF-α mRNA expression after 48 h culture without exogenous stimulation was significantly lower, regardless of the proviral load of the donor, compared to non-infected animals. In the LPL animals, the expression of TNF-α mRNA was significantly lower with respect to the control group while the expression of TNFRI mRNA was significantly increased. The HPL animals showed a significant decrease in the expression of TNF-α and TNFRII mRNA respect to the control group. After 48 h incubation with rTNF-α, PBMC from infected animals had different responses: TNF-α and TNFRI mRNA expression was reduced in PBMC from the LPL group compared to the BLV negative group, but no differences were observed in PBMC from the HPL group. TNFRII mRNA expression showed no differences between HPL, LPL, and BLV negative groups, though HPL animals expressed 10.35 times more TNFRI mRNA than LPL. These results support the hypothesis that LPL animals, when faced with viral reactivation, present a pro-apoptotic and anti-proliferative state. However, complementary studies are needed to explain the influence of TNFRII on the development of the HLP profile. On the other hand, exogenous stimulation studies reinforce the hypothesis that BLV infection compromises the immune response of the animals.

Alterations in TNF-α and its receptors expression in cows undergoing heat stress.

Heat stress is one of the environmental factors that most severely affects milk industry, as it has impact on production, immune responses and reproductive performance. The present study was conducted with high-performance Holando-Argentino cows. Our objective was to study TNF-α and its receptors pattern expression in cows from a region characterized by extreme climatic seasonality. Animals were evaluated in three periods: spring (n = 15), summer (n = 14) and autumn (n = 11). Meteorological records from a local station were used to estimate the temperature and humidity index (THI) by means of an equation previously defined. A THI higher than 68 is indicative of stressing conditions. During the summer period, the animals were exposed to 8.5 ±â€¯1.09 h of heat stress, or THI > 68. In spring, stress hours were reduced to 1.4 ±â€¯0.5 every day, while during the autumn, there were no recorded heat stress events. Expression of TNF-α, and its receptors was determined by qPCR. During the summer, TNF-α and its receptors expression diminished drastically compared to the rest of the year, when stressful conditions were infrequent. We conclude that animals that are not physiologically prepared to resist high temperatures might have a less efficient immune response, reinforcing the need to develop new strategies to improve animal welfare.

Perforin and granzymes in neurological infections: From humans to cattle.

Perforin and granzymes are essential components of the cytotoxic granules present in cytotoxic T lymphocytes and natural killer cells. These proteins play a crucial role in a variety of conditions, including viral infections, tumor immune surveillance, and tissue rejection. Besides their beneficial effect in most of these situations, perforin and granzymes have also been associated with tissue damage and immune diseases. Moreover, it has been reported that perforin and granzymes released during viral infections could contribute to the pathogenesis of diseases. In this review, we summarize the information available on human perforin and granzymes and their relationship with neurological infections and immune disorders. Furthermore, we compare this information with that available for bovine and present data on perforin and granzymes expression in cattle infected with bovine alphaherpesvirus types1 and -5. To our knowledge, this is the first review analyzing the impact of perforin and granzymes on neurological infections caused by bovine herpesviruses.

Effect of bovine leukemia virus on bovine mammary epithelial cells.

Bovine leukemia virus (BLV) is a retrovirus that infects cattle and is associated with an increase in secondary infections. The objective of this study was to analyze the effect of BLV infection on cell viability, apoptosis and morphology of a bovine mammary epithelial cell line (MAC-T), as well as Toll like receptors (TLR) and cytokine mRNA expression. Our findings show that BLV infection causes late syncytium formation, a decrease in cell viability, downregulation of the anti-apoptotic gene Bcl-2, and an increase in TLR9 mRNA expression. Moreover, we analyzed how this stably infected cell line respond to the exposure to Staphylococcus aureus (S. aureus), a pathogen known to cause chronic mastitis. In the presence of S. aureus, MAC-T BLV cells had decreased viability and decreased Bcl-2 and TLR2 mRNA expression. The results suggest that mammary epithelial cells infected with BLV have altered the apoptotic and immune pathways, probably affecting their response to bacteria and favoring the development of mastitis.

Lymphocyte proliferation and apoptosis of lymphocyte subpopulations in bovine leukemia virus-infected dairy cows with high and low proviral load.

Bovine leukemia virus (BLV) is one of the most important virus in dairy cattle. The infection behavior follows what we call the iceberg phenomenon: 60% of infected animals do not show clinical signs; 30% develop persistent lymphocytosis (PL); and the remaining 10%, die due to lymphosarcoma. BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms by which cattle governs the control of viral dissemination will be desirable for designing effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. It is known that BLV affects cellular homeostasis: proliferation and apoptosis. It is also known that the BLV tropism is directed towards B lymphocytes, and that lymphocytotic animals have elevated amounts of these cells. Usually, when an animal is infected by BLV, the B markers that increase are CD21, CD5 and CD11b. This increase could be related to the modulation of apoptosis in these cells. This is the first work in which animals infected with BLV are classified according to their proviral load and the subpopulations of B and T lymphocytes are evaluated in terms of their percentage in peripheral blood and its stage of apoptosis and viability. PBMCs from HPL animals proliferated more than LPL and non-infected animals. CD11b+/CD5+ lymphocytes in LPL animals presented greater early and late apoptosis than HPL animals and cells of HPL animals had increased viability than LPL animals. Our results confirm that BLV alters the mechanism of apoptosis and proliferation of infected cells.

Stable infection of a bovine mammary epithelial cell line (MAC-T) with bovine leukemia virus (BLV).

Bovine leukemia virus (BLV) is a retrovirus that affects cattle causing a lymphoproliferative disease. BLV infection has been associated with misbalance of the immune response causing a higher incidence of other infections. Mastitis is one of the most important conditions that affect milk production in cattle. The aim of this study was to stably infect a bovine mammary epithelial cell line (MAC-T). MAC-T cell line was successfully infected with BLV and the infection was confirmed by nested PCR, qPCR, immunocytochemistry, western blot and transmission electron microscopy. This is the first report of a bovine mammary epithelial cell line stably infected with BLV. This new cell line could be used as an in vitro model to study the effect of BLV on the immune response in the mammary gland and the relationship with other agents causing mastitis.

Can Bovine Leukemia Virus Be Related to Human Breast Cancer? A Review of the Evidence.

The incidence of breast cancer is continuously increasing worldwide, as influenced by many factors that act synergistically. In the last decade there was an increasing interest in the possible viral etiology of human breast cancer. Since then, many viruses have been associated with this disease (murine mammary tumor virus, MMTV; Epstein-Barr virus, EBV; and human papillomavirus, HPV). Recently, BLV has been identified in human breast cancers giving rise to the hypothesis that it could be one of the causative agents of this condition. BLV is a retrovirus distributed worldwide that affects cattle, causing lymphosarcoma in a small proportion of infected animals. Because of its similarity with human retroviruses like HTLV and HIV, BLV was assumed to also be involved in tumor emergence. Based on this assumption, studies were focused on the possible role of BLV in human breast cancer development. We present a compilation of the current knowledge on the subject and some prospective analysis that is required to fully end this controversy.

Toll-like receptors, IFN-γ and IL-12 expression in bovine leukemia virus-infected animals with low or high proviral load.

Bovine leukemia virus (BLV) infection is widespread mainly in dairy cattle and 5-10% of infected animals will die due to lymphosarcoma; most cattle remain asymptomatic but 30% develop persistent lymphocytosis (PL). BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms which govern the control of viral dissemination will be desirable for the design of effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. We aimed to characterize cytokines and toll-like receptors (TLR) expression related to the proviral load profiles. IFN-γ and IL-12 mRNA expression level was significantly higher in PBMC from infected cattle (LPL n=6 and HPL n=7) compared to uninfected animals (n=5). While no significant differences were observed in IL-12 expression between LPL and HPL group, IFN-γ expression was significantly higher in LPL animals. Infected cattle exhibited higher expression levels of TLR3, 7-9. Animals with HPL had significantly higher expression of TLR7/8 than uninfected cattle. TLR8 and TLR9 were up-regulated in HPL group, and TLR3 was up-regulated in LPL group. This is the first report related to TLR gene expression in BLV infected cattle and represents evidence of the involvement of these receptors in BLV recognition. Further studies on different subpopulations of immune cells may help clarify their role in response to BLV and its consequences on viral dissemination.