Bovine Herpesvirus 1 Invasion of Sensory Neurons by Retrograde Axonal Transport Is Dependent on the pUL37 Region 2 Effector.

Following exposure and replication at mucosal surfaces, most alphaherpesviruses invade the peripheral nervous system by retrograde axonal transport and establish lifelong latent infections in the peripheral ganglia. Reactivation of ganglionic infections is followed by anterograde axonal transport of virions back to body surfaces where viral replication results in disease that can range from moderate to severe in presentation. In the case of bovine herpesvirus 1 (BoHV-1), replication in the epithelial mucosa presents as infectious bovine rhinotracheitis (IBR), a respiratory disease of significant economic impact. In this study, we provide a live-cell analysis of BoHV-1 retrograde axonal transport relative to the model alphaherpesvirus pathogen pseudorabies virus (PRV) and demonstrate that this critical neuroinvasive step is conserved between the two viruses. In addition, we report that the BoHV-1 pUL37 tegument protein supports processive retrograde motion in infected axons and invasion of the calf peripheral nervous system. IMPORTANCE A molecular and cellular understanding of the retrograde axonal transport process that underlies the neuroinvasive properties of the alphaherpesviruses is established from studies of herpes simplex virus and pseudorabies virus. The degree to which this phenotype is conserved in other related viruses has largely not been examined. We provide a time-lapse analysis of the retrograde axonal transport kinetics of bovine herpesvirus 1 and demonstrate that mutation of the pUL37 region 2 effector affords a strategy to produce live-attenuated vaccines for enhanced protection of cattle.

ß-Catenin-Specific Inhibitor, iCRT14, Promotes BoHV-1 Infection-Induced DNA Damage in Human A549 Lung Adenocarcinoma Cells by Enhancing Viral Protein Expression.

Oncolytic bovine herpesvirus type 1 (BoHV-1) infection induces DNA damage in human lung adenocarcinoma cell line A549. However, the underlying mechanisms are not fully understood. We found that BoHV-1 infection decreased the steady-state protein levels of p53-binding protein 1 (53BP1), which plays a central role in dictating DNA damage repair and maintaining genomic stability. Furthermore, BoHV-1 impaired the formation of 53BP1 foci, suggesting that BoHV-1 inhibits 53BP1-mediated DNA damage repair. Interestingly, BoHV-1 infection redistributed intracellular ß-catenin, and iCRT14 (5-[[2,5-Dimethyl-1-(3-pyridinyl)-1H-pyrrol-3-yl]methylene]-3-phenyl-2,4-thiazolidinedione), a ß-catenin-specific inhibitor, enhanced certain viral protein expression, such as the envelope glycoproteins gC and gD, and enhanced virus infection-induced DNA damage. Therefore, for the first time, we provide evidence showing that BoHV-1 infection disrupts 53BP1-mediated DNA damage repair and suggest ß-catenin as a potential host factor restricting both virus replication and DNA damage in A549 cells.

TLR activation, immune response and viral protection elicited in cattle by a commercial vaccine against Bovine Herpesvirus-1.

The innate and acquired immune response induced by a commercial inactivated vaccine against Bovine Herpesvirus-1 (BoHV-1) and protection conferred against the virus were analyzed in cattle. Vaccination induced high levels of BoHV-1 antibodies at 30, 60, and 90 days post-vaccination (dpv). IgG1 and IgG2 isotypes were detected at 90 dpv, as well as virus-neutralizing antibodies. An increase of anti-BoHV-1 IgG1 in nasal swabs was detected 6 days post-challenge in vaccinated animals. After viral challenge, lower virus excretion and lower clinical score were observed in vaccinated as compared to unvaccinated animals, as well as BoHV-1-specific proliferation of lymphocytes and production of IFNγ, TNFα, and IL-4. Downregulation of the expression of endosome Toll-like receptors 8-9 was detected after booster vaccination. This is the first thorough study of the immunity generated by a commercial vaccine against BoHV-1 in cattle.

Assessment of Cidofovir for Treatment of Ocular Bovine Herpesvirus-1 Infection in Cattle Using an Ex-Vivo Model.

Bovine herpesvirus-1 (BoHV-1) infection contributes to keratoconjunctivitis, respiratory disease, and reproductive losses in cattle. The objective of this study was to determine the most appropriate ophthalmic antiviral agent for BoHV-1 inhibition using in-vitro culture and novel ex-vivo bovine corneal modeling. Half-maximal inhibitory concentrations of BoHV-1 were determined for cidofovir, ganciclovir, idoxuridine, and trifluridine via in-vitro plaque reduction assays. In-vitro cytotoxicity was compared amongst these compounds via luciferase assays. Trifluridine and cidofovir were the most potent BoHV-1 inhibitors in vitro, while trifluridine and idoxuridine were the most cytotoxic agents. Therefore, cidofovir was the most potent non-cytotoxic agent and was employed in the ex-vivo corneal assay. Corneoscleral rings (n = 36) from fresh cadaver bovine globes were harvested and equally divided into an uninfected, untreated control group; a BoHV-1-infected, untreated group; and a BoHV-1-infected, cidofovir-treated group. Virus isolation for BoHV-1 titers was performed from corneal tissue and liquid media. Histologic measurements of corneal thickness, epithelial cell density, and tissue organization were compared between groups. Substantial BoHV-1 replication was observed in infected, untreated corneas, but BoHV-1 titer was significantly reduced in cidofovir-treated (1.69 ± 0.08 × 103 PFU/mL) versus untreated (8.25 ± 0.25 × 105 PFU/mL, p < 0.0001) tissues by day 2 of culture. No significant differences in histologic criteria were observed between groups. In conclusion, cidofovir warrants further investigation as treatment for BoHV-1 keratoconjunctivitis, with future studies needed to assess in-vivo tolerability and efficacy.

Natto extract, a Japanese fermented soybean food, directly inhibits viral infections including SARS-CoV-2 in vitro.

Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.

MG-132 interferes with iron cellular homeostasis and alters virulence of bovine herpesvirus 1.

Bovine herpesvirus 1 (BoHV-1) requires an iron-replete cell host to replicate efficiently. BoHV-1 infection provokes an increase in ferritin levels and a decrease of transferrin receptor 1 (TfR-1) expression, ultimately lowering iron pool extent. Thus, cells try to limit iron availability for virus spread. It has been demonstrated that MG-132, a proteasome inhibitor, reduces BoHV-1 release. Since ferritin, the major iron storage protein in mammalian cells, undergoes proteasome-mediated degradation, herein, the influence of MG-132 on iron metabolism during BoHV-1 infection was examined. Following infection in bovine cells (MDBK), MG-132 reduced cell death and viral yield. Western blot analysis showed a significant ferritin accumulation, likely due to the inhibition of its proteasome-mediated degradation pathway. In addition, the concomitant down-regulation of TfR-1 expression, observed during infection, was counteracted by proteasome inhibitor. This trend may be explained by enhanced acidic vesicular organelles, detected by acridine orange staining, determining a reduction of intracellular pH, that promotes new synthesis of TfR-1 degraded in a recycling pathway. In addition, MG-132 influences cellular iron distribution during BoHV-1 infection, as revealed by Perls' Prussian blue staining. However, cellular iron content, evaluated by Atomic Absorption Spectrophotometry, resulted essentially unaltered. These findings reveal that MG-132 may contribute to limit cellular iron availability for virus replication thereby enhancing cell survival.

Inhibition of Stress-Induced Viral Promoters by a Bovine Herpesvirus 1 Non-Coding RNA and the Cellular Transcription Factor, ß-Catenin.

The ability to establish, maintain, and reactivate from latency in sensory neurons within trigeminal ganglia (TG) is crucial for bovine herpesvirus 1 (BoHV-1) transmission. In contrast to lytic infection, the only viral gene abundantly expressed during latency is the latency-related (LR) gene. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency, in part because the glucocorticoid receptor (GR) transactivates viral promoters that drive expression of key viral transcriptional regulator proteins (bICP0 and bICP4). Within hours after dexamethasone treatment of latently infected calves, LR gene products and ß-catenin are not readily detected in TG neurons. Hence, we hypothesized that LR gene products and/or ß-catenin restrict GR-mediated transcriptional activation. A plasmid expressing LR RNA sequences that span open reading frame 2 (ORF2-Stop) inhibited GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) and mouse mammary tumor virus (MMTV) promoter activity in mouse neuroblastoma cells (Neuro-2A). ORF2-Stop also reduced productive infection and GR steady-state protein levels in transfected Neuro-2A cells. Additional studies revealed that the constitutively active ß-catenin mutant reduced the transactivation of the IEtu1 promoter by GR and dexamethasone. Collectively, these studies suggest ORF2 RNA sequences and Wnt/ß-catenin signaling pathway actively promote maintenance of latency, in part, by impairing GR-mediated gene expression.

Bovine herpesvirus-1 infection in mouse neuroblastoma (Neuro-2A) cells.

Bovine herpesvirus 1 (BoHV-1) is an important cattle pathogen, that may cause rhinotracheitis, abortions and shipping fever. Virus establishes latency in sensory neurons, but periodically could reactivate. Recent studies identified mouse neuroblastoma (Neuro-2A) cells as a novel cell culture model to study factors that regulate BoHV-1 productive infection in neuronal cells. Herein, following BoHV-1 infection in Neuro-2A, a reduced cell viability occurred. Membrane damage and death morphological alterations, features of apoptosis and necrosis, were distinguished in infected cells. In addition, biochemical signs of apoptosis (caspase 3 activation and PARP cleavage) were observed. These results were accompanied by incomplete autophagy due to enhanced amounts of autophagic markers (LC3-II, ATG5 and Beclin 1), in the presence of increased levels of p62. Interestingly, protein expression of viral infected cell protein 0 (bICP0) was detected in Neuro-2A cells, although BoHV-1 inefficiently replicates in these cells, because just low levels of viral yield were found. Taken together, our results suggest that BoHV-1 may exert its potential neurotoxicity through a combined mechanism of necrosis and apoptosis. Moreover, incomplete autophagy occurred during BoHV-1 replication in Neuro-2A cells, which were favourable for viral persistence.

Proteomic and phosphoproteomic analyses reveal several events involved in the early stages of bovine herpesvirus 1 infection.

Herpesviruses are predicted to express more than 80 proteins during their infection cycle. The proteins synthesized by the immediate early genes and early genes target signaling pathways in host cells that are essential for the successful initiation of a productive infection and for latency. In this study, proteomic and phosphoproteomic tools showed the occurrence of changes in Madin-Darby bovine kidney cells at the early stage of the infection by bovine herpesvirus 1 (BoHV-1). Proteins that had already been described in the early stage of infection for other herpesviruses but not for BoHV-1 were found. For example, stathmin phosphorylation at the initial stage of infection is described for the first time. In addition, two proteins that had not been described yet in the early stages of herpesvirus infections in general were ribonuclease/angiogenin inhibitor and Rab GDP dissociation inhibitor beta. The biological processes involved in these cellular responses were repair and replication of DNA, splicing, microtubule dynamics, and inflammatory responses. These results reveal pathways that might be used as targets for designing antiviral molecules against BoHV-1 infection.

Distinctive features of bovine alphaherpesvirus types 1 and 5 and the virus-host interactions that might influence clinical outcomes.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are two closely related alphaherpesviruses. BoHV-1 causes several syndromes in cattle, including respiratory disease and sporadic cases of encephalitis, whereas BoHV-5 is responsible for meningoencephalitis in calves. Although both viruses are neurotropic, they differ in their neuropathogenic potential. This review summarizes the findings on the specific mechanisms and pathways known to modulate the pathogenesis of BoHV-1 and BoHV-5, particularly in relation to respiratory and neurological syndromes, which characterize BoHV-1 and BoHV-5 infections, respectively.

Two Pioneer Transcription Factors, Krüppel-Like Transcription Factor 4 and Glucocorticoid Receptor, Cooperatively Transactivate the Bovine Herpesvirus 1 ICP0 Early Promoter and Stimulate Productive Infection.

An important site for bovine herpesvirus 1 (BoHV-1) latency is sensory neurons within trigeminal ganglia (TG). The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. Expression of four Krüppel-like transcription factors (KLF), i.e., KLF4, KLF6, PLZF (promyelocytic leukemia zinc finger), and KLF15, are induced in TG neurons early during dexamethasone-induced reactivation. The glucocorticoid receptor (GR) and KLF15 form a feed-forward transcription loop that cooperatively transactivates the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter that drives bovine infected cell protein 0 (bICP0) and bICP4 expression. Since the bICP0 gene also contains a separate early (E) promoter, we tested the hypothesis that GR and KLF family members transactivate the bICP0 E promoter. GR and KLF4, both pioneer transcription factors, cooperated to stimulate bICP0 E promoter activity in a ligand-independent manner in mouse neuroblastoma cells (Neuro-2A). Furthermore, GR and KLF4 stimulated productive infection. Mutating both half GR binding sites did not significantly reduce GR- and KLF4-mediated transactivation of the bICP0 E promoter, suggesting that a novel mechanism exists for transactivation. GR and KLF15 cooperatively stimulated bICP0 activity less efficiently than GR and KL4: however, KLF6, PLZF, and GR had little effect on the bICP0 E promoter. GR, KLF4, and KLF15 occupied bICP0 E promoter sequences in transfected Neuro-2A cells. GR and KLF15, but not KLF4, occupied the bICP0 E promoter at late times during productive infection of bovine cells. Collectively, these studies suggest that cooperative transactivation of the bICP0 E promoter by two pioneer transcription factors (GR and KLF4) correlates with stimulating lytic cycle viral gene expression following stressful stimuli.IMPORTANCE Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone. We predict that increased corticosteroid levels activate the glucocorticoid receptor (GR). Consequently, viral gene expression is stimulated by the activated GR. The immediate early transcription unit 1 promoter (IEtu1) drives expression of two viral transcriptional regulatory proteins, bovine infected cell protein 0 (bICP0) and bICP4. Interestingly, a separate early promoter also drives bICP0 expression. Two pioneer transcription factors, GR and Krüppel-like transcription factor 4 (KLF4), cooperatively transactivate the bICP0 early (E) promoter. GR and KLF15 cooperate to stimulate bICP0 E promoter activity but significantly less than GR and KLF4. The bICP0 E promoter contains enhancer-like domains necessary for GR- and KLF4-mediated transactivation that are distinct from those for GR and KLF15. Stress-induced pioneer transcription factors are proposed to activate key viral promoters, including the bICP0 E promoter, during early stages of reactivation from latency.

Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter.

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

Immune memory induced by intranasal vaccination with a modified-live viral vaccine delivered to colostrum fed neonatal calves.

Bovine respiratory disease (BRD) remains a major health problem despite extensive use of vaccines during the post-weaning period. Apparent vaccine failure is attributed, in part, to primary vaccination during the period of greatest risk for BRD, providing inadequate time for onset of protective immunity. The current study investigated whether intranasal (IN) vaccination of 3-6 week old calves with a modified-live viral (MLV) vaccine induced sufficient immune memory to prevent respiratory disease and accelerate onset of protective immunity 5 months later. Vaccine groups included naïve controls, a single IN vaccination at 3-6 weeks of age, primary IN vaccination at 6 months, and either an IN or subcutaneous (SC) booster vaccination at 6 months (n = 10/group). All calves were challenged with BHV-1 four days after vaccination at 6 months of age. Primary IN vaccination at 6 months did not significantly reduce clinical disease but significantly (P < 0.01) reduced virus shedding. A single IN vaccination at 3-6 weeks of age significantly (P < 0.05) reduced weight loss but did not reduce fever or virus shedding. Both IN and SC booster vaccinations, significantly (P < 0.01) reduced clinical disease but virus shedding was significantly (P < 0.001) reduced only by IN booster vaccination. Reduction in virus shedding was significantly (P < 0.01) greater following booster versus primary IN vaccination at 6 months. All vaccination regimes significantly (P < 0.01) reduced secondary bacterial pneumonia and altered interferon responses relative to naïve controls. Only IN booster vaccination significantly (P < 0.05) increased BHV-1 specific IgA in nasal secretions. These results confirm primary MLV IN vaccination at 3 to 6 weeks of age, when virus neutralizing maternal antibody was present, induced immune memory with a 5 month duration. This immune memory supported rapid onset of protective immunity four days after an IN booster vaccination.

Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The oocytes recovered were divided into two groups. Group I comprised COCs (n = 312) and denuded oocytes (n = 296), which were experimentally infected with BHV1 and incubated for 1 h at 38.5°C and 5% CO2. Group II comprised COCs (n = 425) and denuded oocytes (n = 405), which were co-incubated with BHV1 under the same conditions for 24 h. The negative control of these two groups was respectively subjected to the same protocol, except for exposure to BHV1. To our knowledge, this study provides the first evidence of BHV1 detection within COCs and denuded oocytes exhibiting intact zona pellucida when co-incubated with the virus for 24 h. Immunolocalization also confirmed the presence of BHV1 in the cytoplasm of the cumulus cells of all COCs exposed to the virus after both incubation periods. In conclusion, detection of BHV1 inside oocytes has a great meaning for the field of animal reproduction. The detection of BHV1 in different layers of cumulus cells also demonstrates that these cells are sources of viral infection.

Cellular microRNA bta-miR-2361 inhibits bovine herpesvirus 1 replication by directly targeting EGR1 gene.

Bovine herpesvirus 1 (BHV-1) is an economically important pathogen of cattle and has led to significant consequences on the cattle industry worldwide. MicroRNAs (miRNAs) are a class of regulators that play critical roles in virus and host interaction. However, the roles of host miRNAs in BHV-1 infection remain largely unclear. In this study, a set of differentially expressed miRNAs by small RNA deep sequencing were analyzed in the Madin-Darby Bovine Kidney Cells (MDBK) infected with BHV-1 after 12 h, 24 h and 48 h post-infection compared to mock infection, and it was confirmed that bta-miR-2361 was significantly down-regulated. Moreover, bta-miR-2361 mimics transfection could inhibit BHV-1 replication. Combined with up-regulated genes from BHV-1-infected MDBK cells by deep RNA-sequencing and predicted by bioinformatics tools, early growth response 1 (EGR1) was putative target of bta-miR-2361. Furthermore, EGR1 was up-regulated during BHV-1 infection, and overexpression of EGR1 promoted BHV-1 replication whereas knockdown of EGR1 had the opposite effects. Subsequently, the target association between bta-miR-2361 and 3'UTR of EGR1 was further validated using a dual-luciferase reporter assay. In addition, overexpression of bta-miR-2361 resulted in decreased EGR1 mRNA and protein levels. Further mechanistic study showed that EGR1 stimulated BHV-1 UL46 promoter activity, but overexpression of bta-miR-2361 suppressed the production of UL46 gene. Collectively, this is the first study to reveal that bta-miR-2361 as a novel host factor regulates BHV-1 replication via directly targeting the EGR1 gene, which is a transcription factor that regulates viral UL46 gene of BHV-1. These results provide further insight into the study of BHV-1 pathogenesis.

The Differential Expression of Mitochondrial Function-Associated Proteins and Antioxidant Enzymes during Bovine Herpesvirus 1 Infection: A Potential Mechanism for Virus Infection-Induced Oxidative Mitochondrial Dysfunction.

Reactive oxidative species (ROS) are important inflammatory mediators. Electrons escaping from the mitochondrial electron transport chain (ETC) during oxidative phosphorylation (OXPHOS) in the mitochondrial respiratory chain (RC) complexes contribute to ROS production. The cellular antioxidant enzymes are important for maintaining ROS release at the physiological levels. It has been reported that BoHV-1 infection induces overproduction of ROS and oxidative mitochondrial dysfunction in cell cultures. In this study, we found that chemical interruption of RC complexes by TTFA (an inhibitor of RC complex II), NaN3 (an inhibitor of RC complex IV), and oligomycin A (an inhibitor of ATP synthase) consistently decreased virus productive infection, suggesting that the integral processes of RC complexes are important for the virus replication. The virus infection significantly increased the expression of subunit SDHB (succinate dehydrogenase) and MTCO1 (cytochrome c oxidase subunit I), critical components of RC complexes II and IV, respectively. The expression of antioxidant enzymes including superoxide dismutase 1 (SOD1), SOD2, catalase (CAT), and glutathione peroxidase 4 (GPX4) was differentially affected following the virus infection. The protein TFAM (transcription factor A, mitochondrial) stimulated by either nuclear respiratory factor 1 (NRF1) or NRF2 is a key regulator of mitochondrial biogenesis. Interestingly, the virus infection at the late stage (at 16 h after infection) stimulated TFAM expression but decreased the levels of both NRF1 and NRF2, indicating that virus infection activated TFAM signaling independent of either NRF1 or NRF2. Overall, this study provided evidence that BoHV-1 infection altered the expression of molecules associated with RC complexes, antioxidant enzymes, and mitochondrial biogenesis-related signaling NRF1/NRF2/TFAM, which correlated with the previous report that virus infection induces ROS overproduction and mitochondrial dysfunction.

Synergistic Activation of Bovine Herpesvirus 1 Productive Infection and Viral Regulatory Promoters by the Progesterone Receptor and Krüppel-Like Transcription Factor 15.

Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, readily infects the fetus and ovaries, which can lead to reproductive failure. The BoHV-1 latency reactivation cycle in sensory neurons may further complicate reproductive failure in pregnant cows. The immediate early transcription unit 1 (IEtu1) promoter drives expression of important viral transcriptional regulators (bICP0 and bICP4). This promoter contains two functional glucocorticoid receptor (GR) response elements (GREs) that have the potential to stimulate productive infection following stressful stimuli. Since progesterone and the progesterone receptor (PR) can activate many GREs, we hypothesized that the PR and/or progesterone regulates productive infection and viral transcription. New studies demonstrated that progesterone stimulated productive infection. Additional studies revealed the PR and Krüppel-like transcription factor 15 (KLF15) cooperated to stimulate productive infection and IEtu1 promoter activity. IEtu1 promoter activation required both GREs, which correlated with the ability of the PR to interact with wild-type (wt) GREs but not mutant GREs. KLF15 also cooperated with the PR to transactivate the bICP0 early promoter, a promoter that maintains bICP0 protein expression during productive infection. Intergenic viral DNA fragments (less than 400 bp) containing two GREs and putative KLF binding sites present within genes encoding unique long 52 (UL-52; component of DNA primase/helicase complex), Circ, bICP4, and IEtu2 were stimulated by KLF15 and the PR more than 10-fold, suggesting that additional viral promoters are activated by these transcription factors. Collectively, these studies suggest progesterone and the PR promote BoHV-1 spread to reproductive tissues, thus increasing the incidence of reproductive failure.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is the most frequently diagnosed cause of abortions in pregnant cows and can cause "abortion storms" in susceptible herds. Virulent field strains and even commercially available modified live vaccines can induce abortion, in part because BoHV-1 replicates efficiently in the ovary and corpus luteum. We now demonstrate that progesterone and the progesterone receptor (PR) stimulate productive infection. The BoHV-1 genome contains approximately 100 glucocorticoid receptor (GR) response elements (GREs). Interestingly, the PR can bind and activate many promoters that contain GREs. The PR and Krüppel-like transcription factor 15 (KLF15), which regulate key steps during embryo implantation, cooperate to stimulate productive infection and two viral promoters that drive expression of key viral transcriptional regulators. These studies suggest that the ability of progesterone and the PR to stimulate productive infection has the potential to promote virus spread in reproductive tissue and induce reproductive failure.

Modulation of bovine herpesvirus 1 infection by virally encoded microRNAs.

Bovine herpesvirus 1 (BoHV-1), is a member of the subfamily Alphaherpesvirinae in the order Herpesviridae and is a ubiquitous pathogen of cattle responsible for significant economic loss worldwide. The BoHV-1 genome encodes at least 10 BoHV-1 microRNA (miRNA) genes, whose functions remain poorly understood. This study sought to understand the role of three BoHV-1 miRNA genes, Bhv1-miR-B6, Bhv1-miR-B8 and Bhv1-miR-B9, which are located proximal to the BoHV-1 origins of replication (OriS). Therefore, plasmids expressing the precursor miRNA hairpins for the Bhv1-miR-B6, Bhv1-miR-B8, and Bhv1-miR-B9 genes were constructed and transfected into Madin-Darby bovine kidney cells prior to BoHV-1 infection. Interestingly, transient expression of either Bhv1-miR-B8 or Bhv1-miR-B9 in Madin-Darby bovine kidney cells prior to infection resulted in partial suppression of BoHV-1 replication, quantified through estimating levels of glycoprotein C mRNA and protein levels. Putative interactions between the mature miRNA bhv1-miR-B8-3p and bhv1-miR-B9 and BoHV-1 transcripts were identified providing plausible pathways for these molecules to affect virus replication. Therefore, these two miRNAs are implicated in the post-transcriptional regulation of BoHV-1 transcripts important for virus replication and could be used to limit BoHV-1 replication.

Neurological diseases of cattle in the state of Goiás, Brazil (2010-2017) / Doenças neurológicas de bovinos no estado de Goiás (2010-2017)

A retrospective study was conducted on neurological diseases of cattle in the state of Goiás, Brazil, from March 2010 to August 2017. Samples of three veterinary diagnostic laboratories were analyzed. Diagnosis was established in 170 out of 407 cattle with neurological signs. Epidemiological, clinical, and anatomic pathology features of each case were researched in the files. Main disorders included diseases caused by viruses (rabies 29.41%, meningoencephalitis by bovine herpesvirus 15.88%, and malignant catarrhal fever 1.76%), by bacteria (botulism 5.88%, suppurative meningitis 3.53%, encephalic abscesses 2.94%, listeriosis 1.76%, and thrombotic meningoencephalitis 1.76%), of metabolic origin (polioencephalomalacia 17.06%), of indefinite cause (lymphoplasmacytic meningoencephalitis 11.18%, traumatic hemorrhages 3.53%, and multifocal malacia with gliosis 1.18%), congenital (hydrocephaly 1.18% and multiple malformations 0.59%), toxic (urea poisoning 1.18% and insecticide poisoning 0.59%), and parasitic (meningoencephalitis associated with infection by Trypanosoma sp. 0.59%).(AU)

Foi realizado um estudo retrospectivo de doenças neurológicas de bovinos no estado de Goiás durante o período de março de 2010 a agosto de 2017, analisando amostras de três laboratórios de diagnóstico veterinário. De 407 bovinos que apresentaram sinais clínicos neurológicos, o diagnóstico foi estabelecido em 170 casos. Desses casos, foram pesquisadas nas fichas as características epidemiológicas, clínicas e anatomopatológicas. As principais doenças diagnosticadas foram causadas por vírus (raiva 29,41%, meningoencefalite por herpesvírus bovino 15,88% e febre catarral maligna 1,76%), de origem metabólica (polioencefalomalacia 17,06%), por bactérias (botulismo 5,88%, meningite supurativa 3,53%, abscessos encefálicos 2,94%, listeriose 1,76% e meningoencefalite trombótica 1,76%), sem causa definida (meningoencefalite linfoplasmocítica 11,18%, hemorragias traumáticas 3,53% e malacia multifocal com gliose 1,18%), congênitas (hidrocefalia 1,18% e malformações múltiplas 0,59%), tóxicas (intoxicação por ureia 1,18% e intoxicação por inseticida 0,59%), e parasitária (meningoencefalite associada à infecção por Trypanosoma sp. 0,59%).(AU)

Bovine herpesvirus type 5 replication and induction of apoptosis in vitro and in the trigeminal ganglion of experimentally-infected cattle.

Bovine herpesvirus (BoHV) types 1 and 5 are neuroinvasive. Cases of BoHV-1-induced encephalitis are not as frequent as those caused by BoHV-5. In this study, the capability of BoHV-5 to induce apoptosis in cell cultures and in the trigeminal ganglion during acute infection of experimentally-infected cattle was analyzed. Apoptotic changes in cell cultures agree with the ability of the viral strains to replicate in each cell line. Marked differences were observed between the in vitro induction of apoptosis by BoHV-1Cooper and BoHV-5 97/613 strains. Apoptotic neurons were clearly evident in the trigeminal ganglion of BoHV-1-infected calves. For BoHV-5 a fewer number of positive neurons was observed. There is an association between the magnitude of bovine herpesviruses replication and the induction of apoptosis in trigeminal ganglion. These findings suggest that the induction of apoptosis and the innate immune response orchestrate the final outcome of alpha herpesviruses infection of the bovine nervous system.