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Background: Bovine herpesvirus 1 (BoHV-1) is a major pathogen that affects the global bovine population, primarily inducing respiratory and reproductive disorders. Its ability to establish latent infections in neuronal cells and to reactivate under certain conditions poses a continual threat to uninfected hosts. In this study, we aimed to analyze the replication characteristics of BoHV-1 in neuronal cells, as well as the effects of viral replication on host cell immunity and physiology., Methods: Using the Neuro-2a neuronal-origin cell line as a model, we explored the dynamics of BoHV-1 replication and analyzed differential gene expression profiles post-BoHV-1 infection using high-throughput RNA sequencing., Results: BoHV-1 demonstrated restricted replication in Neuro-2a cells. BoHV-1 induced apoptotic pathways and enhanced the transcription of interferon-stimulated genes and interferon regulatory factors while suppressing the complement cascade in Neuro-2a cells., Conclusions: Different from BoHV-1 infection in other non-highly differentiated somatic cells result in viral dominance, BoHV-1 regulated the innate immune response in neuronal cells formed a "virus-nerve cell" relative equilibrium state, which may account for the restricted replication of BoHV-1 in neuronal cells, leading to a latent infection. These findings provide a foundation for further research into the mechanism underlying BoHV-1-induced latent infection in nerve cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bovine herpesvirus type 1 (BoHV-1) establishes lifelong latency in trigeminal ganglionic (TG) neurons following intranasal and ocular infection in cattle. Periodically, the latent virus reactivates in the TG due to stress and is transported anterogradely to nerve endings in the nasal epithelium, where the virus replicates and sheds. Consequently, BoHV-1 is transmitted to susceptible animals and maintained in the cattle population. Modified live BoHV-1 vaccine strains (BoHV-1 MLV) also have a similar latency reactivation. Therefore, they circulate and are maintained in cattle herds. Additionally, they can regain virulence and cause vaccine outbreaks because they mutate and recombine with other circulating field wild-type (wt) strains. Recently, we constructed a BoHV-1 quadruple mutant virus (BoHV-1qmv) that lacks immune evasive properties due to UL49.5 and glycoprotein G (gG) deletions. In addition, it also lacks the gE cytoplasmic tail (gE CT) and Us9 gene sequences designed to make it safe, increase its vaccine efficacy against BoHV-1, and restrict its anterograde neuronal transport noted above. Further, we engineered the BoHV-1qmv-vector to serve as a subunit vaccine against the Rift Valley fever virus (BoHV-1qmv Sub-RVFV) (doi: 10.3390/v15112183). In this study, we determined the latency reactivation and nasal virus shedding properties of BoHV-1qmv (vector) and BoHV-1qmv-vectored subunit RVFV (BoHV-1qmv sub-RVFV) vaccine virus in calves in comparison to the BoHV-1 wild-type (wt) following intranasal inoculation. The real-time PCR results showed that BoHV-1 wt- but not the BoHV-1qmv vector- and BoHV-1qmv Sub-RVFV-inoculated calves shed virus in the nose following dexamethasone-induced latency reactivation; however, like the BoHV-1 wt, both the BoHV-1qmv vector and BoHV-1qmv Sub-RVFV viruses established latency, were reactivated, and replicated in the TG neurons. These results are consistent with the anterograde neurotransport function of the gE CT and Us9 sequences, which are deleted in the BoHV-1qmv and BoHV-1qmv Sub-RVFV.

Purpose: Infectious bovine rhinotracheitis (IBR) is one of the most important diseases affecting production and productivity., Methodology: Cross-sectional study was aimed at to determine the seroprevalence of IBR and associated risk factors, and animal owners' knowledge, attitude and practice towards the disease from April 2021 to June 2022. Accordingly, a total of 384 serum samples were collected from both crossbreed (70) and local breed (314) cattle from purposively selected districts of East Wollega zone of Western Ethiopia. Competitive enzyme-linked immunosorbent assay (ELISA) was used for testing glycoprotein antibodies (anti-gB) for bovine herpes virus-1 (BoHV-1) virus in collected serum, and the obtained data were analysed by multiple logistic regressions by using R software 3.62 version. However, questionnaire data were analysed for descriptive statistics by SPSS version 20.0 (IBM. Corp, 2011)., Result: The total prevalence of IBR in the study area was found to be 70.54% at herd and 80.47% at individual cattle level. The significant association (p  < 0.05 $ &lt; \ 0.05$ ) was found for breed, age, body condition and herd size but not for district and sex as risk factors. The BoHV-1 virus seropositivity in adult animals increased significantly, with an odds ratio of 1.65 (95% CI 0.705-3.85) compared to young. Local breed cattle were 2.055 times more likely to test positive for IBR with an odds ratio of 0.77 (95% CI 0.23-2.22) compared to crossbreed cattle. The chances of cattle in medium herds testing positive for the BoHV-1 virus with an odds ratio of (1.78 95% CI 1.303-7.50) are greater than the chances of cattle in smaller herds testing positive. The survey results showed that 70% of animal owners identified IBR as a major challenge in animal production, whereas 35% mentioned long calving intervals. However, 92% of the participants were not informed about the level of knowledge and attitude regarding particular diseases such as IBR., Conclusion: This study showed that there is a high prevalence of IBR in cattle in the study area, and that owners have low awareness of the disease. Therefore, it is necessary to develop an immediate control system and conduct additional research on molecular detection to evaluate its effects on reproductive performance., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Bovine alphaherpesvirus 1 (BoHV-1) infection causes respiratory tract disorders and immune suppression and may induce bacterial pneumonia. BoHV-1 establishes lifelong latency in sensory neurons after acute infection. Reactivation from latency consistently occurs following stress or intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators necessary for productive infection and reactivation from latency. The IEtu1 promoter contains two glucocorticoid receptor (GR) responsive elements (GREs) that are transactivated by activated GR. GC-rich motifs, including consensus binding sites for specificity protein 1 (Sp1), are in the IEtu1 promoter sequences. E2F family members bind a consensus sequence (TTTCCCGC) and certain specificity protein 1 (Sp1) sites. Consequently, we hypothesized that certain E2F family members activate IEtu1 promoter activity. DEX treatment of latently infected calves increased the number of E2F2 + TG neurons. GR and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivate a 436-bp cis -regulatory module in the IEtu1 promoter that contains both GREs. A luciferase reporter construct containing a 222-bp fragment downstream of the GREs was transactivated by E2F2 unless two adjacent Sp1 binding sites were mutated. Chromatin immunoprecipitation studies revealed that E2F2 occupied IEtu1 promoter sequences when the BoHV-1 genome was transfected into mouse neuroblastoma (Neuro-2A) or monkey kidney (CV-1) cells. In summary, these findings revealed that GR and E2F2 cooperatively transactivate IEtu1 promoter activity, which is predicted to influence the early stages of BoHV-1 reactivation from latency., Importance: Bovine alpha-herpesvirus 1 (BoHV-1) acute infection in cattle leads to establishment of latency in sensory neurons in the trigeminal ganglia (TG). A synthetic corticosteroid dexamethasone consistently initiates BoHV-1 reactivation in latently infected calves. The BoHV-1 immediate early transcription unit 1 (IEtu1) promoter regulates expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators. Hence, the IEtu1 promoter must be activated for the reactivation to occur. The number of TG neurons expressing E2F2, a transcription factor and cell cycle regulator, increased during early stages of reactivation from latency. The glucocorticoid receptor (GR) and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivated a 436-bp cis -regulatory module (CRM) in the IEtu1 promoter that contains two GR responsive elements. Chromatin immunoprecipitation studies revealed that E2F2 occupies IEtu1 promoter sequences in cultured cells. GR and E2F2 mediate cooperative transactivation of IEtu1 promoter activity, which is predicted to stimulate viral replication following stressful stimuli., Competing Interests: The authors declare no conflict of interest.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1 + TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli., Competing Interests: The authors declare no conflict of interest.

Bubaline alphaherpesvirus 1 (BuHV-1) is a pathogen of water buffaloes responsible for economic loss worldwide. MicroRNAs (miRNAs) regulate gene expression produced by alphaherpesviruses and hosts. This study aimed at (a) unravelling the ability of BuHV-1 to produce miRNAs, including hv1-miR-B6, hv1-miR-B8, hv1-miR-B9; (b) measuring the host immune-related miRNAs associated to herpesvirus infection, including miR-210-3p, miR-490-3p, miR-17-5p, miR-148a-3p, miR-338-3p, miR-370-3p, by RT-qPCR; (c) identifying candidate markers of infection by receiver-operating characteristic (ROC) curves; (d) exploiting the biological functions by pathway enrichment analyses. Five water buffaloes BuHV-1 and Bovine alphaherpesvirus 1 (BoHV-1) free were immunized against Infectious Bovine Rhinotracheitis (IBR). Five additional water buffaloes served as negative controls. All animals were challenged with a virulent wild-type (wt) BuHV-1 via the intranasal route 120 days after the first vaccination. Nasal swabs were obtained at days (d) 0, 2, 4, 7, 10, 15, 30, and 63 post-challenge (pc). The animals of both groups shed wt BuHV-1 up to d7 pc. Results demonstrated that (a) miRNAs produced by the host and BuHV-1 could be efficiently quantified in the nasal secretion up to d63 and d15 pc, respectively; b) the levels of host and BuHV-1 miRNAs are different between vaccinated and control buffaloes; c) miR-370-3p discriminated vaccinated and control animals; d) host immune-related miRNAs may modulate genes involved in the cell adhesion pathway of the neuronal and immune system. Overall, the present study provides evidence that miRNAs can be detected in nasal secretions of water buffaloes and that their expression is modulated by BuHV-1., (© 2023. The Author(s).)

Bovine herpesvirus-1 (BoHV-1) can infect all breeds of cattle and cause severe respiratory organs and genital tract diseases. However, the mechanism of BoHV-1 entering the cells remains unclear. In this study, we explored the mechanism of BoHV-1 entering MDBK cells. We found that the entry of BoHV-1 was blocked by NH 4 Cl and bafilomycin A1, indicating that BoHV-1 entry is dependent on the acidic environment of endosome. Specific inhibitor dynasore and small interfering RNA (siRNA) knockdown of dynamin-2 inhibited BoHV-1 entry, showing that dynamin is required in BoHV-1 entry. The results of specific inhibitor, siRNA knockdown and co-localization indicating clathrin- and caveolin- mediated endocytosis play a role in BoHV-1 entry. BoHV-1 infection was not affected by EIPA which is a specific inhibitor of macropinocytosis. In addition, we found that BoHV-1 triggered PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways to induce clathrin-mediated and caveolin-mediated endocytosis at the early stage of BoHV-1 infection. BoHV-1 binding was sufficient to activate the endocytic signaling pathways and promote viral entry. These two signaling pathways were activated by transfection of viral gD protein, and were inhibited by deletion of viral gD protein and the siRNA knockdown of cellular receptor nectin-1. The results of co-localization indicating the entered BoHV-1 is traced to late endosomes via early endosomes. Our results suggested the interaction of viral gD protein and cellular receptor nectin-1 triggered the PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways and induced clathrin-mediated and caveolin-mediated endocytosis to promote BoHV-1 entry into MDBK cells at the early stage of BoHV-1 infection., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bovine herpesvirus 1 (BoHV-1) is a significant risk factor for the bovine respiratory disease complex (BRDC), a severe disease causing great economic losses to the cattle industry worldwide. Previous studies have reported that both phospholipase C-γ1 (PLC-γ1) and β-catenin are activated during BoHV-1 infection for efficient replication. However, the interplay between PLC-γ1 and β-catenin as a consequence of virus infection remains to be elucidated. Here, we reported that PLC-γ1 interacted with β-catenin, which was enhanced following virus infection. PLC-γ1-specific inhibitor, U73122, significantly reduced the mRNA levels of β-catenin in BoHV-1-infected cells; however, the steady-state protein levels were not affected due to the virus infection. Interestingly, the treatment of virus-infected cells with U73122 reduced the accumulation of activated β-catenin [p-β-catenin(S552)] in fractions of the cytoplasmic membrane as that observed with the treatment of methyl-β-cyclodextrin (MβCD), which can disrupt cytoplasmic membrane structure via sequestering cholesterol. Nucleus accumulation of p-β-catenin(S552) was increased following U73122 treatment in virus-infected cells. In addition, the association of p-β-catenin(S552) with cytoplasmic membrane induced by the virus infection was significantly disrupted by the treatment of U73122 and MβCD. These data indicated that the PLC-γ1 signaling is potentially involved in the regulation of β-catenin signaling stimulated by BoHV-1 infection partially via affecting the subcellular localization of p-β-catenin(S552)., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, establishes lifelong latency in sensory neurons within trigeminal ganglia (TG) after acute infection. The BoHV-1 latency-reactivation cycle, like other alphaherpesvirinae subfamily members, is essential for viral persistence and transmission. Notably, cells within pharyngeal tonsil (PT) also support a quiescent or latent BoHV-1 infection. The synthetic corticosteroid dexamethasone, which mimics the effects of stress, consistently induces BoHV-1 reactivation from latency allowing early stages of viral reactivation to be examined in the natural host. Based on previous studies, we hypothesized that stress-induced cellular factors trigger expression of key viral transcriptional regulatory genes. To explore this hypothesis, RNA-sequencing studies compared viral gene expression in PT during early stages of dexamethasone-induced reactivation from latency. Strikingly, RNA encoding infected cell protein 4 (bICP4), which is translated into an essential viral transcriptional regulatory protein, was detected 30 min after dexamethasone treatment. Ninety minutes after dexamethasone treatment bICP4 and, to a lesser extent, bICP0 RNA were detected in PT. All lytic cycle viral transcripts were detected within 3 h after dexamethasone treatment. Surprisingly, the latency related (LR) gene, the only viral gene abundantly expressed in latently infected TG neurons, was not detected in PT during latency. In TG neurons, bICP0 and the viral tegument protein VP16 are expressed before bICP4 during reactivation, suggesting distinct viral regulatory genes mediate reactivation from latency in PT versus TG neurons. Finally, these studies confirm PT is a biologically relevant site for BoHV-1 latency, reactivation from latency, and virus transmission. IMPORTANCE BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. RNA-sequencing studies revealed the viral infected cell protein 4 (bICP4) RNA was expressed in PT of latently infected calves within 30 min after dexamethasone was used to initiate reactivation. As expected, bICP4 RNA was not detected during latency. All lytic cycle viral genes were expressed within 3 h after dexamethasone treatment. Conversely, bICP0 and the viral tegument protein VP16 are expressed prior to bICP4 in trigeminal ganglionic neurons during reactivation. The viral latency related gene, which is abundantly expressed in latently infected neurons, was not abundantly expressed in PT during latency. These studies provide new evidence PT is a biologically relevant site for BoHV-1 latency and reactivation. Finally, we predict other alphaherpesvirinae subfamily members utilize PT as a site for latency and reactivation.

Bovine Herpesvirus Type 1 (BoHV-1) infection causes infectious bovine rhinotracheitis and genital disease in cattle, with significant economic and welfare impacts. However, the role of cellular host factors during viral replication remains poorly characterised. A previously performed genome-wide CRISPR knockout screen identified pro- and antiviral host factors acting during BoHV-1 replication. Herein we validate a pro-viral role for a candidate from this screen: the cellular protein tetracopeptide repeat protein 4 (TTC4). We show that TTC4 transcript production is upregulated during BoHV-1 infection. Depletion of TTC4 protein impairs BoHV-1 protein production but does not reduce production of infectious virions, whereas overexpression of exogenous TTC4 results in a significant increase in production of infectious BoHV-1 virions. TTC4 itself is poorly characterized (especially in the context of virus infection), but is a known co-chaperone of heat shock protein 90 (HSP90). HSP90 has a well-characterized pro-viral role during the replication of diverse herpesviruses, and we therefore hypothesized that HSP90 is also pro-viral for BoHV-1. Drug-mediated inhibition of HSP90 using geldanamycin at sub-cytotoxic concentrations inhibited both BoHV-1 protein production and viral genome replication, indicating a pro-viral role for HSP90 during BoHV-1 infection. Our data demonstrates pro-viral roles for both TTC4 and HSP90 during BoHV-1 replication; possibly, interactions between these two proteins are required for optimal BoHV-1 replication, or the two proteins may have independent pro-viral roles., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Infection of cattle with bovine herpesvirus type 1 (BHV-1) can lead to upper respiratory tract disease, conjunctivitis, or genital disease and cause serious economic losses to the cattle industry worldwide. The role of long noncoding RNAs in BHV-1 infection is not well understood. To explore the role of lncRNA- MSTRG.16919.1 in bovine herpes virus type I (BHV-1) infected MDBK cells, the lncRNA- MSTRG.16919.1 gene was silenced and sequenced transcriptome and sequencing data were analyzed by Edge R software, Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and an interaction network of proteins. Real-time quantitative PCR (RT-qPCR) and Western blotting were used to verify the results of bioinformatic analyses. The results showed that 1151 differential genes were obtained in the siRNA- MSTRG.16919.1 group compared with an NC group. Compared with BHV-33 h, 6586 differentially expressed genes were obtained. A total of 498 differentially expressed genes were screened from the two groups. To verify the accuracy of the sequencing, six genes were randomly selected for RT-qPCR, and the results showed that the expression trend of selected genes was consistent with the sequencing results. GO enrichment analysis showed that the differential genes were related to such biological processes as nucleotide binding, enzyme binding, cell cycle, and glial macromolecule metabolism. KEGG analysis enriched 378 and 2634 signaling pathways, respectively, that were associated with virus infection, ubiquitin-mediated protein hydrolysis, phosphoinositol metabolism, apoptosis, and other metabolic pathways. The STRING protein interaction database was used to analyze the interaction network of proteins encoded by differential genes, and the degree algorithm in Cytoscape was used to screen the top 20 proteins. The results showed that SKIV2L2, JAK2, PIK3CB, and MAPK8 were related to virus infection. Western blot analysis of TNF, NF-κB, MAPK8, MAPK9, and MAPK10 proteins showed that lncRNA- MSTRG.16919.1 was involved in regulating the expression of these functional proteins. The results of this study provide basic information for exploring the function and regulatory mechanism of lncRNA- MSTRG.16919.1 in organisms and help for further studying the interaction between virus and host.

Acute infection of the ocular, oral, or nasal cavity by bovine herpesvirus 1 (BoHV-1) culminates in lifelong latency in sensory neurons within trigeminal ganglia. The BoHV-1 latency reactivation cycle, including calves latently infected with commercially available modified live vaccines, can lead to reproductive complications, including abortions. Recent studies demonstrated progesterone stimulated BoHV-1 productive infection and sporadically induced reactivation from latency in male rabbits. The progesterone receptor (PR) and progesterone transactivate the immediate early transcription unit 1 (IEtu1) promoter and the infected cell protein 0 (bICP0) early promoter. These viral promoters drive expression of two viral transcriptional regulatory proteins (bICP0 and bICP4) that are crucial for productive infection. Based on these observations, we hypothesize that progesterone induces reactivation in a subset of calves latently infected with BoHV-1. These studies demonstrated progesterone was less efficient than dexamethasone at initiating reactivation from latency in female calves. Notably, heat stress correlated with enhancing the ability of progesterone to induce reactivation from latency. Previous studies demonstrated that heat stress activates the glucocorticoid receptor (GR), which suggested GR activation augments progesterone-mediated reactivation from latency. Additional studies revealed GR and PR cooperatively stimulated productive infection and synergistically transactivated the IEtu1 promoter when cultures were treated with dexamethasone. Mutating one or both GR binding sites in the IEtu1 promoter blocked transactivation. Collectively, these studies indicated that progesterone intermittently triggered reactivation from latency, and heat stress augmented reactivation from reactivation. Finally, these studies suggest progesterone enhances virus spread in tissues and cells where PR is abundantly expressed. IMPORTANCE Steroid hormone fluctuations are predicted to enhance or initiate bovine herpesvirus 1 (BoHV-1) replication and virus spread in cattle. For example, stress increases the incidence of BoHV-1 reactivation from latency in cattle, and the synthetic corticosteroid dexamethasone consistently induces reactivation from latency. The glucocorticoid receptor (GR) and dexamethasone stimulate key viral regulatory promoters and productive infection, in part because the viral genome contains numerous consensus GR-responsive elements (GREs). The progesterone receptor (PR) and GR belong to the type I nuclear hormone receptor family. PR and progesterone specifically bind to and transactivate viral promoters that contain GREs and stimulate BoHV-1 productive infection. Although progesterone did not induce reactivation from latency in female calves as efficiently as dexamethasone, heat stress enhanced progesterone-mediated reactivation from latency. Consequently, we predict that low levels of stressful stimuli can cooperate with progesterone to induce reactivation from latency or promote virus spread.

Bovine alphaherpesvirus 1 (BoHV-1) is a pathogen causing respiratory and reproductive clinical signs in cattle. Infected animals may develop rhinotracheitis, vulvovaginitis, balanoposthitis, and abortion. Viral latency is generally established in neuronal ganglia simultaneously to a decrease in both genes or genome expression and viral replication. Under stressful conditions, infection is reactivated leading to viral replication and the manifestation of clinical signs. In this study, we evaluated both viral reactivation and apoptosis in trigeminal ganglia cells as BoHV-1 progressed from the latent to the acute phase of infection after dexamethasone administration in experimentally infected calves. To test ganglia cell death as a consequence of BoHV-1 infection, we stained the BoHV-1 samples with TUNEL after the viral shedding by the calves. RT-qPCR of apoptotic genes was also performed, showing the upregulation of the caspase 8 gene in the trigeminal ganglia from cattle experimentally infected with BoHV-1. These results showed the occurrence of apoptosis in ganglion cells of calves infected by BoHV-1., (© 2021. Sociedade Brasileira de Microbiologia.)

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 × 10 3 PFU/mL) versus untreated (8.25 ± 0.25 × 10 5 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.

Bovine herpesvirus-1 (BoHV-1) is a major cause of rhinotracheitis and vulvovaginitis in cattle. VP8, the major tegument protein of BoHV-1, is essential for viral replication in the host. VP8 is phosphorylated by the viral kinase US3, mediating its translocation to the cytoplasm. VP8 remains nuclear when not phosphorylated. Interestingly, VP8 has a significant presence in mature BoHV-1YmVP8, in which the VP8 phosphorylation sites are mutated. This suggests that VP8 might be packaged during primary envelopment of BoHV-1. This was investigated by mass spectrometry and Western blotting, which showed VP8, as well as VP22, to be constituents of the primary enveloped virions. VP8 and VP22 were shown to interact via co-immunoprecipitation experiments, in both BoHV-1-infected and VP8-transfected cells. VP8 and VP22 also co-localised with one another and with nuclear lamin-associated protein 2 in BoHV-1-infected cells, suggesting an interaction between VP8 and VP22 in the perinuclear region. In cells infected with VP22-deleted BoHV-1 (BoHV-1ΔUL49), VP8 was absent from the primary enveloped virions, implying that VP22 might be critical for the early packaging of VP8. In conclusion, a novel VP22-dependent mechanism for packaging of VP8 was identified, which may be responsible for a significant amount of VP8 in the viral particle.

Bovine herpesvirus 1 (BoHV-1) is a promising oncolytic virus with broad antitumor spectrum; however, its oncolytic effects on human lung adenocarcinoma in vivo have not been reported. In this study, we report that BoHV-1 can be used as an oncolytic virus for human lung adenocarcinoma, and elucidate the underlying mechanism of how BoHV-1 suppresses tumor cell proliferation and growth. First, we examined the oncolytic activities of BoHV-1 in human lung adenocarcinoma A549 cells. BoHV-1 infection reduced the protein levels of histone deacetylases (HDACs), including HDAC1-4 that are promising anti-tumor drug targets. Furthermore, the HDAC inhibitor Trichostatin A (TSA) promoted BoHV-1 infection and exacerbated DNA damage and cytopathology, suggesting a synergy between BoHV-1 and TSA. In the A549 tumor xenograft mouse model, we, for the first time, showed that BoHV-1 can infect tumor and suppressed tumor growth with a similar high efficacy as the treatment of TSA, and HDACs have potential effects on the virus replication. Taken together, our study demonstrates that BoHV-1 has oncolytic effects against human lung adenocarcinoma in vivo.

A stochastic quantitative risk assessment model was developed to estimate the annual probability of introduction of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) on 127 dairy farms through indirect contacts. Vehicles transporting calves, cattle to slaughterhouse, dead animals, and mixture of feed, as well as visits by veterinarians and hoof trimmers, farm workers and contacts with neighbors were considered in the model. Data from biosecurity questionnaires of each farm, scientific literature and expert opinion from field veterinarians, animal vehicle drivers, hoof trimmers and personnel from rendering transport companies were used to estimate values for input parameters. Results showed that the annual probability of introducing BVDV or BoHV-1 through indirect contacts was very heterogeneous. The overall distribution of median values for each farm ranged from 0.5 to 14.6% and from 1.0 to 24.9% for BVDV and BoHV-1, respectively. The model identified that providing protective clothing and boots to visits, not allowing the animal vehicle driver to come into contact with animals present on the farm and ensuring that calf vehicles arrived empty, were the measures with the highest impact on the probability of infection for most farms. This model could be a useful tool to show the impact of the measures to farmers and veterinarians, thus increasing their awareness on biosecurity. In addition, it could support decision making on which measures should be prioritized in dairy cattle herds to reduce the probability of introduction of diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The dairy sector is one of the leading in agricultural production sectors in the world and the bovine herpesvirus 1 (BoHV-1) is an important pathogen that causes great losses in most production systems. Moreover, BoLA DRB3 immunological gene presents different alleles related to protection against many pathogens., Methods: Serological diagnosis was carried out to determine the BoHV-1 infection and through PCR-RFLP 506 Holstein cows from several municipalities of Antioquia were genotyped for BoLA DRB3.2 gene polymorphisms., Results: Alleles 8, 16, 22, and 24 were the most common out of the 42 alleles found. By indirect ELISA technique, a 58.7% prevalence of BoHV-1 infection in this population was diagnosed and Odd ratios for found alleles were calculated by logistic regression; the only significant association was held for allele 37, which showed that it effects confers susceptibility to infection. On the other hand, by using generalized linear models, a significant association between BoLA DRB3.2 gene and milk and fat yield in primiparous and services per conception in multiparous was found, with the most favorable alleles being 11 and 28 in primiparous and 22 and 28 in multiparous; allele 37 was unfavorable only in primiparous., Conclusion: BoLA DRB3.2 gene polymorphisms have shown high variability and significant effects on Holstein cattle and their performance in production systems in Antioquia are at both sanitary or health and productive levels., Competing Interests: The authors declare that there is no conflict of interest.

Many studies report age as a risk factor for BoHV-1 infection or seropositivity. However, it is unclear whether this pattern reflects true epidemiological causation or is a consequence of study design and other issues. Here, we seek to understand the age-related dynamics of BoHV-1 seroprevalence in seasonal calving Irish dairy herds and provide decision support for the design and implementation of effective BoHV-1 testing strategies. We analysed seroprevalence data from dairy herds taken during two Irish seroprevalence surveys conducted between 2010 and 2017. Age-dependent seroprevalence profiles were constructed for herds that were seropositive and unvaccinated. Some of these profiles revealed a sudden increase in seroprevalence between adjacent age-cohorts, from absent or low to close to 100% of seropositive animals. By coupling the outcome of our data analysis with simulation output of an individual-based model at the herd scale, we have shown that these sudden increases are related to extensive virus circulation within a herd for a limited time, which may then subsequently remain latent over the following years. BoHV-1 outbreaks in dairy cattle herds affect animals independent of age and lead to almost 100% seroconversion in all age groups, or at least in all animals within a single epidemiological unit. In the absence of circulating infection, there is a year-on-year increase in the age-cohort at which seroprevalence changes from low to high. The findings of this study inform recommendations regarding testing regimes in the context of contingency planning or an eradication programme in seasonal calving dairy herds.

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs 467 YTSL 470 and 563 YTVV 566 , which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes great economic losses in the cattle industry. Herpesvirus infection generally induces endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) in infected cells. However, it is not clear whether ER stress and UPR can be induced by BoHV-1 infection. Here, we found that ER stress induced by BoHV-1 infection could activate all three UPR sensors (the activating transcription factor 6 (ATF6), the inositol-requiring enzyme 1 (IRE1), and the protein kinase RNA-like ER kinase (PERK)) in MDBK cells. During BoHV-1 infection, the ATF6 pathway of UPR did not affect viral replication. However, both knockdown and specific chemical inhibition of PERK attenuated the BoHV-1 proliferation, and chemical inhibition of PERK significantly reduced the viral replication at the post-entry step of the BoHV-1 life cycle. Furthermore, knockdown of IRE1 inhibits BoHV-1 replication, indicating that the IRE1 pathway may promote viral replication. Further study revealed that BoHV-1 replication was enhanced by IRE1 RNase activity inhibition at the stage of virus post-entry in MDBK cells. Furthermore, IRE1 kinase activity inhibition and RNase activity enhancement decrease BoHV1 replication via affecting the virus post-entry step. Our study revealed that BoHV-1 infection activated all three UPR signaling pathways in MDBK cells, and BoHV-1-induced PERK and IRE1 pathways may promote viral replication. This study provides a new perspective for the interactions of BoHV-1 and UPR, which is helpful to further elucidate the mechanism of BoHV-1 pathogenesis.

Bovine herpesvirus (BoHV) types 1 and 5 are two closely related alpha-herpesviruses of cattle with neuroinvasive potential. BoHV-5 causes non-suppurative meningoencephalitis in calve whereas encephalitis caused by BoHV-1 has been occasionally reported. As an initial step to understand the biology of both BoHV types in neural cells, undifferentiated SH-SY5Y human neuroblastoma cells were infected with BoHV-1 strains Cooper and Los Angeles (LA), BoHV-5 strain 97/613 and A663, a BoHV-5/BoHV-1 natural recombinant. Cytopathic effect (CPE) in these cells was evident earlier for BoHV-5 strain 97/613 and CPE progression was slower for BoHV-1, particularly for Cooper strain. Virus antigen was detected as early as 8 h post-infection (hpi) for all strains, with the exception of BoHV-1 Cooper for which antigen expression was detectable by 24 hpi. All strains released detectable infectious virus in the extracellular medium by 8 hpi, confirming that undifferentiated SH-SY5Y cells are fully permissive to BoHV infection. Significantly different extracellular virus titers among the different strains were detected by 24 hpi, with BoHV-5 97/613 reaching the maximal virus production. The lowest extracellular titer was recorded for BoHV-1 Cooper at all the evaluated time-points. BoHV-1 Cooper, BoHV-1 LA and BoHV-5 97/613 had a steady increase in intracellular virus production. The evaluation of lysis plaques formation revealed that BoHV-5 A663 produced the largest plaques followed by BoHV-5 97/613. Both BoHV-1 strains produced smaller plaques when compared with BoHV-5. Despite a slower replicative cycle, strain A663 is more efficient in cell to cell dissemination. Thus, it is evident that BoHV-5 strains have growth advantages in undifferentiated neural cells compared with BoHV-1. This in vitro model might be useful to analyze the neuropathogenic potential of bovine alphaherpesviruses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Accumulating studies have shown that the epidermal growth factor receptor (EGFR) signaling pathway plays an essential role in mediating cellular entry of numerous viruses. In this study, we report that bovine herpesvirus 1 (BoHV-1) productive infection in both the human lung carcinoma cell line A549 and bovine kidney (MDBK) cells leads to activation of EGFR, as demonstrated by the increased phosphorylation of EGFR at Tyr1068 (Y1068), which in turn plays important roles in virus infection. A time-of-addition assay supported that virus replication at post-entry stages was affected by the EGFR specific inhibitor Gefitinib. Interestingly, both phospholipase C-γ1 (PLC-γ1) and Akt, canonical downstream effectors of EGFR, were activated following virus infection in A549 cells, while Gefitinib could inhibit the activation of PLC-γ1 but not Akt. In addition, virus titers in A549 cells was inhibited by chemical inhibition of PLC-γ1, but not by the inhibition of Akt. However, the Akt specific inhibitor Ly294002 could significantly reduce the virus titer in MDBK cells. Taken together, our data suggest that PLC-γ1 is stimulated in part through EGFR for efficient replication in A549 cells, whereas Akt can be stimulated by virus infection independent of EGFR, and is not essential for virus productive infection, indicating that Akt modulates BoHV-1 replication in a cell type-dependent manner. This study provides novel insights on how BoHV-1 infection activates EGFR signaling transduction to facilitate virus replication.

Artificial insemination and in vitro embryo production are increasingly used to improve the reproductive efficiency of herds, however success of these techniques depends on the sanitary quality of the semen. Insemination centers commonly use antibiotics in their routine procedure, but they are not able against viruses. In this paper, we demonstrate a new approach for disinfecting virus in bovine semen using photoimmunoinactivation, an adaptation of the photodynamic inactivation (PDI) methodology. The photosensitizers (PSs), hematoporphyrin (HP) and zinc tetracarboxy-phthalocyanine (ZnPc) were conjugated to Immunoglobulin Y (IgY) anti-bovine alphaherpesvirus 1 (BoHV-1) and used for PDI against the BoHV-1 viruses in cell culture and compared to the unconjugated PSs. Both treatments proved to be efficient, but a significant decrease in the irradiation time required to completely eliminate the virus was observed in the samples treated with the immunoconjugates. Photophysical measurements help us to understand the coupling between PSs and IgY and the evaluated production of singlet oxygen. Following the cell culture test, the same approach was applied in semen artificially infected with BoHV-1. The immunoconjugates were also efficient for complete virus inactivation up to 5 min of irradiation and proved to be safe using several parameters of sperm viability, demonstrating the feasibility of our strategy for disinfection viruses in semen., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study aimed to determine the seroprevalence and identify the risk factors associated with Neospora caninum, Bovine herpesvirus type 1 (BHV-1), and Bovine viral diarrhea virus (BVDV) infection on industrial Holstein dairy cattle farms in Isfahan province, Central Iran. Blood samples were taken from 216 apparently healthy cattle from 16 randomly selected Holstein dairy farms in the North, South, East, and West of Isfahan in the summer of 2017. The antibodies to N. caninum, BHV-1, and BVDV were detected using a commercially available ELISA kit. The overall seroprevalence for N. caninum, BHV-1, and BVDV was 19%, 72.2%, and 52.8%, respectively. The significant major risk factors of BHV-1 in cattle were identified as farm direction, age groups, parity, and milk yield by the univariate analysis (p < 0.05). The significant major risk factors of BVDV in cattle were identified as age groups, parity, milk yield, and stage of pregnancy (p < 0.05). The only significant major risk factor of N. caninum was farm direction (p < 0.05). A significant association of concurrent infection with BVDV and BHV-1 has shown in the current study (p < 0.05). This study is the first to report the risk factors for N. caninum, BHV-1, and BVDV infection in the central part of Iran and allows us to conclude that these agents are widely distributed in this region.

MicroRNAs have emerged as important and versatile modulators of immune responses and participate actively in the regulation of host-pathogen interactions. However, the role of miR-2890 in the host response against viral infection is unclear. In this study, we show that bta-miR-2890 facilitates the production of type I interferon (IFN) and IFN-stimulated genes, and inhibits bovine alphaherpesvirus 1 (BoHV-1) replication. Further research showed that bta-miR-2890 actives the type I IFN signal pathway via up-regulating JAK1 and STAT1 by directly targeting BoHV-1 UL41 3'UTR. Here, we found that BoHV-1 UL41 inhibits JAK1 expression. Mechanistically, BoHV-1 UL41 up-regulates E3 ubiquitin ligase SYVN1, which promotes K48-linked polyubiquitination and proteasomal degradation of JAK1. Together, our results suggest that bta-miR-2890 promotes JAK1 and STAT1 expression via targeting BoHV-1 UL41 is an important event for IFN-dependent antiviral immune response, and suggest that bta-miR-2890 has an important role in controlling viral infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, can cause abortions in pregnant cows. Progesterone maintains pregnancy and promotes spermiogenesis and testosterone biosynthesis in males: furthermore, progesterone is a neuro-steroid. Recent published studies demonstrated progesterone stimulated the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, and two glucocorticoid receptor response elements within the promoter were required for progesterone mediated transactivation. In this study, we tested whether progesterone induces reactivation from latency in rabbits. As expected, the synthetic corticosteroid dexamethasone consistently induced reactivation from latency in males and females. While progesterone induced reactivation from latency in approximately one-half of male rabbits, virus shedding was sporadic compared to dexamethasone and less efficient in female rabbits. Progesterone significantly increased productive infection in rabbit skin cells, which correlated with stimulating reactivation. These studies suggest progesterone promotes BoHV-1 spread in cattle, in part, by increasing the frequency of reactivation from latency., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine alphaherpesvirus 1 (BoHV-1) as a cusative agent for some diseases in cattle infects sheep and goat; and it is believed that these animals may be reservoir host for this virus. Thus, BoHV-1 infection in sheep and goat should be considerd when there is a program for control and eradication of this virus in cattle. Therefore, the aim of this study was to determine the seroprevalence of BoHV-1 in sheep, relationship between host and environmental factors with infection, and the role of sheep in the epidemiology of the BoHV-1. Blood samples were randomly collected from 310 healthy sheep in 6 cities of Khuzestan province (Southwest of Iran) including Ahvaz, Hendijan, Shushtar, Dezful, Masjed Soleyman and Behbahan. Sera were analyzed by virus neutralisation (VN) test for detection antibodies to BoHV-1. According to VN test, apparent and true seroprevalence seroprevalence of BoHV-1 infection was 28.4 % (95%CI: 23.4-33.4%) and 28.4 % (95%CI: 23.3-33.4%), respectively. Logistic regression revealed that the odds of infection between the age was 1.06 (95%CI: 0.9-1.25) (P > 0.05), implying that the odds of infection increased 6 % with rising one year of age. Besides, the relative frequency of infection in males was more than females', and the odds of infection in male sheep was identified to be 1.13 (95%CI: 0.47-2.71) (P > 0.05), compared to that in females. Moreover, in comparison to sheep with no history of abortion, the odds of infection in sheep with a history of abortion was 1.28 (95%CI: 0.57-2.87) (P > 0.05). The seroprevalence in Shushtar, Masjed Soleyman, Dezful, Ahvaz, Hendijan, and Behbahan were found to be 48.3, 46.7, 31.7, 20, 16.7, and 12 percent, respectively and 13.1 of fluctuation in infection can be justified by different geographical locations investigated in this study (P < 0.001). Considering the significant seroprevalence of BoHV-1, present study confirmed the role of sheep in the epidemiology of this virus and control of BoHV-1 in sheep should be considered by animal health authorities in areas where BoHV-1 is prevalent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine herpesvirus type 1 (BoHV-1) is a significant cofactor for bovine respiratory disease complex (BRDC), the most important inflammatory disease in cattle. BoHV-1 infection in cell cultures induces overproduction of pathogenic reactive oxygen species (ROS) and the depletion of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a master transcriptional factor regulating a panel of antioxidant and cellular defense genes in response to oxidative stress. In this study, we reported that the virus productive infection in MDBK cells at the later stage significantly decreased the expression levels of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) proteins, the canonical downstream targets regulated by Nrf2, inhibited Nrf2 acetylation, reduced the accumulation of Nrf2 proteins in the nucleus, and relocalized nuclear Nrf2 proteins to form dot-like staining patterns in confocal microscope assay. The differential expression of Kelch-like ECH associated protein 1 (KEAP1) and DJ-1 proteins as well as the decreased association between KEAP1 and DJ-1 promoted Nrf2 degradation through the ubiquitin proteasome pathway. These data indicated that the BoHV-1 infection may significantly suppress the Nrf2 signaling pathway. Moreover, we found that there was an association between Nrf2 and LaminA/C, H3K9ac, and H3K18ac, and the binding ratios were altered following the virus infection. Taken together, for the first time, we provided evidence showing that BoHV-1 infection inhibited the Nrf2 signaling pathway by complicated mechanisms including promoting Nrf2 degradation, relocalization of nuclear Nrf2, and inhibition of Nrf2 acetylation., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2019 Xiaotian Fu et al.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate the diagnostic performance of chute-side diagnostic methods for detecting physiological and pathological changes as indicators of early bovine respiratory disease (BRD) in calves experimentally inoculated with infectious bovine rhinotracheitis virus (IBR) and Mannheimia haemolytica (Mh). A challenge study was performed over 14 d in 30 Holstein steers [average weight (±SEM) = 211 kilograms (kg) ± 2.4 kg] inoculated on day 0 with IBR and on day 6 with Mh. Diagnostic methods included clinical illness scores (CIS), lung auscultation using a computer-aided stethoscope (CAS), rectal temperature, facial thermography, pulse oximetry, and bilateral thoracic ultrasonography. Animals were randomized into 1 of 5 necropsy days (days 6, 7, 9, 11, and 13) when the percentage of lung consolidation was estimated. The effect of study day on the results of the diagnostic methods and associations between each diagnostic method's values with lung consolidation measured at necropsy were determined with mixed models. Values for all diagnostic methods differed significantly (P < 0.01) by day. During the IBR phase (days 0 to 6) calves had "normal" to "moderate" CIS, whereas during the Mh phase (days 6.5 to 13) scores were predominantly "severe" to "moribund." Similarly, CAS scores were "normal" and "mild acute" during the IBR phase and "mild acute" to "moderate acute" after the Mh challenge. Oxygen saturation did not differ significantly between days 0, 1, 2, 4, and 6; however, significantly decreased 12 h after inoculation with Mh (P < 0.05). Mean lung consolidation between animal's right and left side recorded by ultrasound was 0.13% (±0.07) before the inoculation with Mh. However, during the Mh phase, mean consolidation increased significantly over time (P < 0.05). The percentage of lung consolidation at necropsy ranged from 1.7% (±0.82) on day 6 to 55.4% (±7.49) on day 10. Clinical illness scores, rectal temperature, facial thermography, oxygen saturation, and ultrasonography were significantly associated (P < 0.05) with lung consolidation at necropsy. In addition, there was a significant trend (P = 0.07) between CAS and lung consolidation scores at necropsy. These chute-side diagnostic methods are useful for detecting disease progression on animals with early stages of BRD., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Infection of cattle by bovine herpesvirus 1 (BoHV-1) can culminate in upper respiratory tract disorders, conjunctivitis, or genital disorders. Infection also consistently leads to transient immune-suppression. BoHV-1 is the number one infectious agent in cattle that is associated with abortions in cattle. BoHV-1, as other α-herpesvirinae subfamily members, establishes latency in sensory neurons. Stressful stimuli, mimicked by the synthetic corticosteroid dexamethasone, consistently induce reactivation from latency in latently infected calves and rabbits. Increased corticosteroid levels due to stress have a two-pronged effect on reactivation from latency by: (1) directly stimulating viral gene expression and replication, and (2) impairing antiviral immune responses, thus enhancing virus spread and transmission. BoHV-1 encodes several proteins, bICP0, bICP27, gG, UL49.5, and VP8, which interfere with key antiviral innate immune responses in the absence of other viral genes. Furthermore, the ability of BoHV-1 to infect lymphocytes and induce apoptosis, in particular CD4+ T cells, has negative impacts on immune responses during acute infection. BoHV-1 induced immune-suppression can initiate the poly-microbial disorder known as bovine respiratory disease complex, which costs the US cattle industry more than one billion dollars annually. Furthermore, interfering with antiviral responses may promote viral spread to ovaries and the developing fetus, thus enhancing reproductive issues associated with BoHV-1 infection of cows or pregnant cows. The focus of this review is to describe the known mechanisms, direct and indirect, by which BoHV-1 interferes with antiviral immune responses during the course of infection.

Bovine herpesvirus 1 (BoHV-1) disseminates easily, is difficult to control, and is widely spread in cattle herds worldwide. BoHV-1 causes a broad range of losses to the cattle industry, mainly concerning reproduction. Previous studies involving experimental infection of BoHV-1 in an in vitro embryo production system have reported impairment of embryonic development by BoHV-1. In this study, we evaluated the interference of BoHV-1 in the in vitro maturation system of cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) cultured with a cumulus cell suspension. Blood samples and ovaries were collected from slaughterhouse cows unvaccinated against BoHV-1. Using virus neutralization assays, the seropositive animals were classified according to their antibody titers. The oocytes were recovered by follicular aspiration and divided into two groups, COCs and DOs, which were evaluated for their nuclear maturation capacity using immunofluorescence assays by laser scanning confocal microscopy. Two experiments were carried out: (I) in vitro maturation of COCs and DOs after artificial infection of seronegative animals and (II) in vitro maturation of COCs and DOs of seropositive animals. In experiment I, a difference (P < 0.01) was observed between the maturation rates of the control group COCs (78.2%) and the infected COCs (43.6%). In experiment II, there was a difference (P < 0.01) in the maturation rate between animals with antibody titers ≥16 (56.9%) and the control group (79.4%). Immunofluorescence assays identified BoHV-1 in the COCs and DOs. Therefore, it was concluded that BoHV-1 affects the in vitro maturation process in both in vitro and natural infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes disease in cattle populations worldwide. Sphingomyelin (SM) is the most abundant sphingolipid in the mammalian cell membrane, where it preferentially associates with cholesterol to form lipid raft domains. SM is a substrate for the lysosome-resident enzyme acid sphingomyelinase, which plays a role in cell membrane repair following injury. Treatment of cells with noncytotoxic concentrations of Staphylococcus aureus -derived sphingomyelinase successfully reduced cell surface-exposed sphingomyelin but did not significantly inhibit BoHV-1 entry and infection, as measured by the beta-galactosidase reporter assay. Interestingly, entry of the porcine alphaherpesvirus pseudorabies virus (PRV) was inhibited by sphingomyelin-depletion of cells. Treatment of BoHV-1 particles with sphingomyelinase inhibited viral entry activity, suggesting that viral SM plays a role in BoHV-1 entry, while cellular SM does not. Treatment of cells with noncytotoxic concentrations of the functional inhibitors of host acid sphingomyelinase, imipramine and amitriptyline, which induce degradation of the cellular enzyme, did not significantly inhibit BoHV-1 entry. In contrast, inhibition of cellular acid sphingomyelinase inhibited PRV entry. Entry of the human alphaherpesvirus herpes simplex virus 1 (HSV-1) was independent of both host SM and acid sphingomyelinase, in a manner similar to BoHV-1. Together, the results suggest that among the alphaherpesviruses, there is variability in entry requirements for cellular sphingomyelin and acid sphingomyelinase activity. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. Available vaccines limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of BoHV-1 are significant. In order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. We recently identified a low pH endocytosis pathway for BoHV-1. Here, we examine the role of cellular factors responsible for membrane integrity and repair in alphaherpesviral entry. This study allows comparisons of the BoHV-1 entry pathway with those of other alphaherpesviruses (pseudorabies virus [PRV] and herpes simplex virus 1 [HSV-1]). Lastly, this is the first report of sphingomyelin and lysosomal sphingomyelinase playing a role in the entry of a herpesvirus. The results may lead to the development of more effective prevention and treatment regimens., (Copyright © 2019 American Society for Microbiology.)

The U L 47 gene product, VP8, is a major tegument protein of BoHV-1. While VP8 is not essential for virus replication in cell culture, a U L 47-deleted virus exhibits a smaller tegument structure and is avirulent in cattle. To obtain pure VP8 protein for structural analysis, we expressed a N-terminally truncated version of VP8 in Eschericia coli. However, the recombinant VP8 was consistently co-purified with a tightly associated bacterial protein; this protein was identified by mass spectrometry as GroEL, which has considerable homology with mammalian heat shock protein-60 (HSP60), thus suggesting a new role for VP8 in virus-host interaction. A physical interaction of HSP60 and VP8 in both VP8-transfected and BoHV-1-infected cells was demonstrated by immunoprecipitation. Analysis of different truncated VP8 constructs revealed that amino acids 259-482 and 632-741 are involved in binding to HSP60. Full-length VP8 and VP8 219-741 (containing both interacting domains, 259-482 and 632-741) co-localized with HSP60 and mitochondria. VP8 was localized in the mitochondria from 2 to 14 h post infection in BoHV-1-infected cells. The mitochondrial membrane potential was reduced in both VP8-transfected and BoHV-1-infected cells and was further diminished by overexpression of HSP60 in the presence of VP8. In addition, VP8 expression decreased the ATP concentration during transfection, as well as BoHV-1 infection. Thus, VP8 may play a role in the deregulation of mitochondrial function through interaction with HSP60. This is consistent with the fact that BoHV-1 infection is known to promote mitochondrial dysfunction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus (BVDV) and bovine herpes virus-1 (BHV-1) remain as the major pathogens with heavy economic consequences in Iran. The prevalence of antibodies against BVDV and BHV-1, the rate of BVDV persistently infected (PI) animals, and associated risk factors were evaluated in a cross-sectional study carried out in Zanjan Province, Northwest Iran, in December 2011. A total number of 562 cattle in 10 herds and five cities were randomly selected, and their serum samples were tested to detect antibodies to these viruses and also BVDV antigen-positive (PI) animals. The data were analyzed with Pearson's correlation coefficient, chi-square, and logistic regression test. In total, nine and eight of the selected herds were seropositive to BVDV and BHV-1, respectively. The overall seroprevalence of these infections were estimated at 28.6 and 10.7% for BVDV and BHV-1, respectively, and 0.53% of the samples were detected as persistently infected. Statistical analysis revealed that sex, age, and farming system are risk factors for both infections (P < 0.05), while breed was determined as a strong risk factor only for BVDV (P < 0.001). In addition, the present study certainly identifies that infection with BVDV is associated with infection to BHV-1 (OR = 4.52, 95% CI: 2.60-7.80; P ˂ 0.001). The results add our knowledge about the prevalence and associated risk factors of BVDV and BHV-1 in Iran and imply that the prophylactic and surveillance strategies need to be implemented to reduce the risk of spread of these viruses.

Peste des petits ruminants is an emerging, often fatal viral disease of domestic and wild small ruminants caused by peste des petits ruminants virus. The haemagglutinin and the fusion protein are viral envelope glycoproteins and essential for the infection process and both induce a protective immune response in infected or vaccinated animals. Attempts to generate pseudotyped bovine herpesvirus 1 recombinants firstly by integration of expression cassettes for PPRV-H and PPRV-F into the herpesviral genome or secondly to generate pseudotyped BHV-1 by genetically fusing relevant parts of both PPRV glycoproteins to the amino-terminal subunit of glycoprotein B, approaches that had been successful for heterologous viral membrane glycoproteins in the past, failed repeatedly. We therefore analyzed at which intracellular stage generation of viable BHV-1 hybrid-gB recombinants might be inhibited. Results obtained from transient protein expression experiments revealed that, dependent on the fusion protein, transport of the hybrid glycoproteins beyond the endoplasmic reticulum is impeded. Thus, expression of heterologous glycoproteins using BHV-1 interferes more than expected from published experience with BHV-1 gB transport and consequently with virus replication., (Copyright © 2018 Elsevier B.V. All rights reserved.)

During bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures, partial of intranuclear viral DNA is present in nucleosomes, and viral protein VP22 associates with histones and decreases histone H4 acetylation, indicating the involvement of histone H4 acetylation in virus replication. In this study, we demonstrated that BoHV-1 infection at the late stage (at 24 h after infection) dramatically decreased histone H3 acetylation [at residues K9 (H3K9ac) and K18 (H3K18ac)], which was supported by the pronounced depletion of histone acetyltransferases (HATs) including CBP/P300 (CREB binding protein and p300), GCN5L2 (general control of amino acid synthesis yeast homolog like 2) and PCAF (P300/CBP-associated factor). The depletion of GCN5L2 promoted by virus infection was partially mediated by ubiquitin-proteasome pathway. Interestingly, the viral replication was enhanced by HAT (histone acetyltransferase) activator CTPB [ N -(4-Chloro-3-trifluoromethylphenyl)-2-ethoxy-6-pentadecylbenzamide], and vice versa, inhibited by HAT inhibitor Anacardic acid (AA), suggesting that BoHV-1 may take advantage of histone acetylation for efficient replication. Taken together, we proposed that the HAT-dependent histone H3 acetylation plays an important role in BoHV-1 replication in MDBK (Madin-Darby bovine kidney) cells.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low-pH-mediated endocytosis pathway. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (50% uptake by ∼5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times postinfection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low-pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Lastly, treatment of cells with the proteasome inhibitor MG132 inhibited BoHV-1 entry in a concentration-dependent manner. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane and necessary for successful infection of the target cell. IMPORTANCE BoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. While there are vaccines on the market, they only limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of this virus are significant, and in order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. This research establishes the initial entry pathway of BoHV-1, which provides a foundation for future development of effective treatments and preventative vaccines. Additionally, it allows comparisons to the entry pathways of other alphaherpesviruses, such as HSV-1., (Copyright © 2018 American Society for Microbiology.)

Bovine herpesvirus 1 (BoHV-1) is one of several microbes that contributes to the development of the bovine respiratory disease (BRD) and can also induce abortions in cattle. As other alpha- herpesvirinae subfamily members, BoHV-1 efficiently replicates in many cell types and subsequently establishes a life-long latent infection in sensory neurons. BoHV-1 encodes more than 70 proteins that are expressed in a well-defined manner during productive infection. However, in silico open reading frame (ORF) prediction of the BoHV-1 genome suggests that the virus may encode more than one hundred proteins. In this study we used mass spectrometry followed by proteogenomic mapping to reveal the existence of 92 peptides that map to previously un-annotated regions of the viral genome. Twenty-one of the newly termed "intergenic peptides" were predicted to have a viable ORF around them. Twelve of these produced an mRNA transcript as demonstrated by strand-specific RT-PCR. We further characterized the 5' and 3' termini of one mRNA transcript, ORF-A, and detected a 55 kDa protein produced during active infection using a custom-synthesized antibody. We conclude that the coding potential of BoHV-1 is underestimated.

Following productive infection, bovine herpesvirus 1 (BoHV-1) establishes latency in sensory neurons. As in other alphaherpesviruses, expression of BoHV-1 immediate early (IE) genes is regulated by an enhancer complex containing the viral IE activator VP16, the cellular transcription factor Oct-1, and transcriptional coactivator HCF-1, which is assembled on an IE enhancer core element (TAATGARAT). Expression of the IE transcription unit that encodes the viral IE activators bICP0 and bICP4 may also be induced by the activated glucocorticoid receptor (GR) via two glucocorticoid response elements (GREs) located upstream of the enhancer core. Strikingly, lytic infection and reactivation from latency are consistently enhanced by glucocorticoid treatment in vivo As the coactivator HCF-1 is essential for IE gene expression of alphaherpesviruses and recruited by multiple transcription factors, we tested whether HCF-1 is required for glucocorticoid-induced IE gene expression. Depletion of HCF-1 reduced GR-mediated activation of the IE promoter in mouse neuroblastoma cells (Neuro-2A). More importantly, HCF-1-mediated GR activation of the promoter was dependent on the presence of GRE sites but independent of the TAATGARAT enhancer core element. HCF-1 was also recruited to the GRE region of a promoter lacking the enhancer core, consistent with a direct role of the coactivator in mediating GR-induced transcription. Similarly, during productive lytic infection, HCF-1 and GR occupied the IE region containing the GREs. These studies indicate HCF-1 is critical for GR activation of the viral IE genes and suggests that glucocorticoid induction of viral reactivation proceeds via an HCF-1-GR mechanism in the absence of the viral IE activator VP16. IMPORTANCE BoHV-1 transcription is rapidly activated during stress-induced reactivation from latency. The immediate early transcription unit 1 (IEtu1) promoter is regulated by the GR via two GREs. The IEtu1 promoter regulates expression of two viral transcriptional regulatory proteins, infected cell proteins 0 and 4 (bICP0 and bICP4), and thus must be stimulated during reactivation. This study demonstrates that activation of the IEtu1 promoter by the synthetic corticosteroid dexamethasone requires HCF-1. Interestingly, the GRE sites, but not the IE enhancer core element (TAATGARAT), were required for HCF-1-mediated GR promoter activation. The GR and HCF-1 were recruited to the IEtu1 promoter in transfected and infected cells. Collectively, these studies indicate that HCF-1 is critical for GR activation of the viral IE genes and suggest that an HCF-1-GR complex can stimulate the IEtu1 promoter in the absence of the viral IE activator VP16., (Copyright © 2018 American Society for Microbiology.)

Alphaherpesvirus envelope glycoprotein N (gN) and gM form a covalently linked complex. Bovine herpesvirus type 1 (BHV-1) U L 49.5 (a gN homolog) contains two predicted cysteine residues, C42 and C78. The C42 is highly conserved among the alphaherpesvirus gN homologs (e.g., herpes simplex virus 1 and pseudorabies virus). To identify which cysteine residue is required for the formation of the U L 49.5/gM complex and to characterize the functional significance of the U L 49.5/gM complex, we constructed and analyzed C42S and C78S substitution mutants in either a BHV-1 wild type (wt) or BHV-1 U L 49.5 cytoplasmic tail-null (CT-null) virus background. The results demonstrated that BHV-1 U L 49.5 residue C42 but not C78 was essential for the formation of the covalently linked functional U L 49.5/gM complex, gM maturation in the Golgi compartment, and efficient cell-to-cell spread of the virus. Interestingly, the C42S and CT-null mutations separately did not affect mutant U L 49.5 virion incorporation. However, when both of the mutations were introduced simultaneously, the U L 49.5 C42S/CT-null protein virion incorporation was severely reduced. Incidentally, the anti-VP22 antibody coimmunoprecipitated the U L 49.5 C42S/CT-null mutant protein at a noticeably reduced level compared to that of the individual U L 49.5 C42S and CT-null mutant proteins. As expected, in a dual U L 49.5 C42S/VP22Δ virus with deletion of VP22 (VP22Δ), the U L 49.5 C42S virion incorporation was also severely reduced while in a gMΔ virus, U L 49.5 virion incorporation was affected only slightly. Together, these results suggested that U L 49.5 virion incorporation is mediated redundantly, by both U L 49.5/gM functional complex and VP22, through a putative gM-independent novel U L 49.5 and VP22 interaction. IMPORTANCE Bovine herpesvirus 1 (BHV-1) envelope protein U L 49.5 is an important virulence determinant because it downregulates major histocompatibility complex class I (MHC-I). U L 49.5 also forms a covalently linked complex with gM. The results of this study demonstrate that U L 49.5 regulates gM maturation and virus cell-to-cell spread since gM maturation in the Golgi compartment depends on covalently linked U L 49.5/gM complex. The results also show that the U L 49.5 residue cysteine 42 (C42) mediates the formation of the covalently linked U L 49.5-gM interaction. Furthermore, a C42S mutant virus in which U L 49.5 cannot interact with gM has defective cell-to-cell spread. Interestingly, U L 49.5 also interacts with the tegument protein VP22 via its cytoplasmic tail (CT). The putative U L 49.5 CT-VP22 interaction is essential for a gM-independent U L 49.5 virion incorporation and is revealed when U L 49.5 and gM are not linked. Therefore, U L 49.5 virion incorporation is mediated by U L 49.5-gM complex interaction and through a gM-independent interaction between U L 49.5 and VP22., (Copyright © 2018 Pannhorst et al.)

Various types of intercellular connections that are essential for communication between cells are often utilized by pathogens. Recently, a new type of cellular connection, consisting of long, thin, actin-rich membrane extensions named tunneling nanotubes (TNTs), has been shown to play an important role in cell-to-cell spread of HIV and influenza virus. In the present report, we show that TNTs are frequently formed by cells infected by an alphaherpesvirus, bovine herpesvirus 1 (BoHV-1). Viral proteins, such as envelope glycoprotein E (gE), capsid protein VP26, and tegument protein Us3, as well as cellular organelles (mitochondria) were detected by immunofluorescence and live-cell imaging of nanotubes formed by bovine primary fibroblasts and oropharynx cells (KOP cells). Time-lapse confocal studies of live cells infected with fluorescently labeled viruses showed that viral particles were transmitted via TNTs. This transfer also occurred in the presence of neutralizing antibodies, which prevented free entry of BoHV-1. We conclude that TNT formation contributes to successful cell-to-cell spread of BoHV-1 and demonstrate for the first time the participation of membrane nanotubes in intercellular transfer of a herpesvirus in live cells. IMPORTANCE Efficient transmission of viral particles between cells is an important factor in successful infection by herpesviruses. Herpesviruses can spread by the free-entry mode or direct cell-to-cell transfer via cell junctions and long extensions of neuronal cells. In this report, we show for the first time that an alphaherpesvirus can also spread between various types of cells using tunneling nanotubes, intercellular connections that are utilized by HIV and other viruses. Live-cell monitoring revealed that viral transmission occurs between the cells of the same type as well as between epithelial cells and fibroblasts. This newly discovered route of herpesviruses spread may contribute to efficient transmission despite the presence of host immune responses, especially after reactivation from latency that developed after primary infection. Long-range communication provided by TNTs may facilitate the spread of herpesviruses between many tissues and organs of an infected organism., (Copyright © 2018 American Society for Microbiology.)

Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with β-catenin and a β-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased β-catenin-dependent transcription and cell survival. β-Catenin and HMGA1 are readily detected in a subset of latently infected TG neurons but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/β-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/β-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but these levels decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 (BMPR2) was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to herpes simplex virus 1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady-state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2, to promote latency. IMPORTANCE Lifelong BoHV-1 latency primarily occurs in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. RNA sequencing studies revealed 102 genes associated with the Wnt/β-catenin signaling pathway are differentially regulated during the latency-reactivation cycle. Two protein kinases associated with the Wnt pathway, Akt3 and BMPR2, were expressed at higher levels during latency but were repressed during reactivation. Furthermore, five genes encoding soluble Wnt antagonists and β-catenin-dependent transcription inhibitors were induced during reactivation from latency. These findings are important because Wnt, BMPR2, and Akt3 promote neurogenesis and cell survival, processes crucial for lifelong viral latency. In transfected neuroblastoma cells, a viral protein expressed during latency (ORF2) interacts with and enhances Akt3 protein kinase activity. These findings provide insight into how cellular factors associated with the Wnt signaling pathway cooperate with LR gene products to regulate the BoHV-1 latency-reactivation cycle., (Copyright © 2018 American Society for Microbiology.)

Plant-derived flavonoids contain large amount of compounds with pharmacological effects. In this study, we showed the compound Kaempferol to have robust antiviral activity against bovine herpesvirus 1 (BoHV-1) replication in vitro. Kaempferol at a concentration of 100 μmol/l completely inhibited viral replication in MDBK cells. It mainly affects the viral replication at the post-entry stages. The inhibition of Akt signaling is a potential mechanism underlying the antiviral effect of Kaempferol. In addition, at a concentration of 25 and 50 μmol/l Kaempferol could significantly reduce the expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and macrophage inflammatory protein 1 alpha (MIP-1α) in human promonocytic U937 cells-derived macrophages (dU937) in response to lipopolysaccharides (LPS) stimulation. Overall, our results indicated that Kaempferol provides a potent protection against BoHV-1 infection and LPS-induced inflammatory response.

Us3 protein is a serine/threonine kinase conserved within the Alphaherpesvirinae subfamily of herpesviruses. The Us3 homologs of herpes simplex virus, pseudorabies virus, and bovine herpesvirus type 5 have been shown to block apoptosis triggered by viral infection or exogenous inducers. To determine whether these characteristics are shared by bovine herpesvirus type 1 Us3, we constructed two viral mutants: BHV-1 Us3 deletion mutant (BHV-1ΔUs3) and a kinase-dead mutant (BHV-1KD). Flow cytometry analysis and TUNEL assay clearly demonstrated, that only BHV-1 wild type virus suppressed infection-induced apoptosis and protected cells from apoptosis triggered by exogenous factors: sorbitol or staurosporine. Us3 of BHV-1 was directly capable of blocking apoptosis without the presence of other viral proteins. The presence of Us3 correlated with phosphorylation of BAD, a pro-apoptotic Bcl-2 family member. Our results clearly indicate that BHV-1 Us3 is necessary for efficient blocking of apoptosis triggered by viral infection and exogenous factors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bovine respiratory disease (BRD) is the most common infectious disease of beef and dairy cattle and is characterized by a complex infectious etiology that includes a variety of viral and bacterial pathogens. We examined the global changes in mRNA abundance in healthy lung and lung lesions and in the lymphoid tissues bronchial lymph node, retropharyngeal lymph node, nasopharyngeal lymph node and pharyngeal tonsil collected at the peak of clinical disease from beef cattle experimentally challenged with either bovine respiratory syncytial virus, infectious bovine rhinotracheitis, bovine viral diarrhea virus, Mannheimia haemolytica or Mycoplasma bovis. We identified signatures of tissue-specific transcriptional responses indicative of tropism in the coordination of host's immune tissue responses to infection by viral or bacterial infections. Furthermore, our study shows that this tissue tropism in host transcriptional response to BRD pathogens results in the activation of different networks of response genes. The differential crosstalk among genes expressed in lymphoid tissues was predicted to be orchestrated by specific immune genes that act as 'key players' within expression networks. The results of this study serve as a basis for the development of innovative therapeutic strategies and for the selection of cattle with enhanced resistance to BRD.

Infection of kidney cells (MDBK) with Bovine Herpesvirus 1 (BoHV-1) is affected by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which accelerates BoHV-1-induced apoptosis and increases virus replication. Herein, to elucidate the mechanism through TCDD modifies BoHV-1 infection, we analyzed the modulation of a members of Sirtuin proteins family in MDBK cells. We found that mitochondrial SIRT3 was upregulated during infection. This change was accompanied by cytoskeletal rearrangements and cell extensions. All these trends were drastically modified by TCDD. We hypothesize that, taken together, these results might further clarify the processes responsible for the action of TCDD on the BoHV-1 replication, resulting in enhanced virus production.

Bovine herpesvirus 1 (BoHV-1) can provoke conjunctivitis, abortions and shipping fever. BoHV-1 infection can also cause immunosuppression and increased susceptibility to secondary bacterial infections, leading to pneumonia and occasionally to death. Herein, we investigated the influence of MG-132, a proteasome inhibitor, on BoHV-1 infection in bovine kidney (MDBK) cells. Infection of MDBK cells with BoHV-1 induces apoptotic cell death that enhances virus release. Whereas, MG-132 inhibited virus-induced apoptosis and stimulated autophagy. Protein expression of viral infected cell protein 0 (bICP0), which is constitutively expressed during infection and is able to stimulate Nuclear factor kappa B (NF-κB), was completely inhibited by MG-132. These results were accompanied by a significant delay in the NF-κB activation. Interestingly, the efficient virus release provoked by BoHV-1-induced apoptosis was significantly reduced by MG-132. Overall, this study suggests that MG-132, through the activation of autophagy, may limit BoHV-1 replication during productive infection, by providing an antiviral defense mechanism.

Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bovine ICP0 (bICP0) and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency, and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same trigeminal ganglion (TG) neuron during reactivation and cooperatively stimulated productive infection and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are transactivated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL52; a component of the DNA primase/helicase complex), bICP4, IEtu2, and the unique short region were stimulated by KLF15 and the GR. Chromatin immunoprecipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and the UL52 fragment. Coimmunoprecipitation studies demonstrated that KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest that these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that causes respiratory disease and suppresses immune responses in cattle; consequently, life-threatening bacterial pneumonia can occur. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons. Reactivation from latency is initiated by the synthetic corticosteroid dexamethasone. Dexamethasone stimulates lytic cycle viral gene expression in sensory neurons of calves latently infected with BoHV-1, culminating in virus shedding and transmission. Two stress-induced cellular transcription factors, Krüppel-like transcription factor 15 (KLF15) and the glucocorticoid receptor (GR), cooperate to stimulate productive infection and viral transcription. Additional studies demonstrated that KLF15 and the GR form a stable complex and that these stress-induced transcription factors bind to viral DNA sequences, which correlates with transcriptional activation. The ability of the GR and KLF15 to synergistically stimulate viral gene expression and productive infection may be critical for the ability of BoHV-1 to reactivate from latency following stressful stimuli., (Copyright © 2017 American Society for Microbiology.)