Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

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.

Endogenous and viral microRNAs in nasal secretions of water buffaloes (Bubalus bubalis) after Bubaline alphaherpesvirus 1 (BuHV-1) challenge infection.

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.

Cell entry of Bovine herpesvirus-1 through clathrin- and caveolin-mediated endocytosis requires activation of PI3K-Akt-NF-κB and Ras-p38 MAPK pathways as well as the interaction of BoHV-1 gD with cellular receptor nectin-1.

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 NH4Cl 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.

Phospholipase C-γ1 potentially facilitates subcellular localization of activated ß-catenin, p-ß-catenin(S552), during bovine herpesvirus 1 productive infection in MDBK cells.

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 ß-catenin are activated during BoHV-1 infection for efficient replication. However, the interplay between PLC-γ1 and ß-catenin as a consequence of virus infection remains to be elucidated. Here, we reported that PLC-γ1 interacted with ß-catenin, which was enhanced following virus infection. PLC-γ1-specific inhibitor, U73122, significantly reduced the mRNA levels of ß-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 ß-catenin [p-ß-catenin(S552)] in fractions of the cytoplasmic membrane as that observed with the treatment of methyl-ß-cyclodextrin (MßCD), which can disrupt cytoplasmic membrane structure via sequestering cholesterol. Nucleus accumulation of p-ß-catenin(S552) was increased following U73122 treatment in virus-infected cells. In addition, the association of p-ß-catenin(S552) with cytoplasmic membrane induced by the virus infection was significantly disrupted by the treatment of U73122 and MßCD. These data indicated that the PLC-γ1 signaling is potentially involved in the regulation of ß-catenin signaling stimulated by BoHV-1 infection partially via affecting the subcellular localization of p-ß-catenin(S552).

Stress Triggers Expression of Bovine Herpesvirus 1 Infected Cell Protein 4 (bICP4) RNA during Early Stages of Reactivation from Latency in Pharyngeal Tonsil.

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.

Analysis of the Function of LncRNA-MSTRG.16919.1 in BHV-1-Infected Bovine Kidney Subculture Cells by Transcriptome Sequencing.

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.

1 Cellular protein TTC4 and its cofactor HSP90 are pro-viral for bovine herpesvirus 1.

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.

Progesterone Sporadically Induces Reactivation from Latency in Female Calves but Proficiently Stimulates Bovine Herpesvirus 1 Productive Infection.

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.

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

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

VP8, the Major Tegument Protein of Bovine Herpesvirus-1, Is Partially Packaged during Early Tegument Formation in a VP22-Dependent Manner.

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.

Oncolytic Bovine Herpesvirus 1 Inhibits Human Lung Adenocarcinoma A549 Cell Proliferation and Tumor Growth by Inducing DNA Damage.

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.

Apoptosis in the late replication phase of Bovine alphaherpesvirus 1 in experimentally infected calves.

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.

Quantitative risk assessment of introduction of BVDV and BoHV-1 through indirect contacts based on implemented biosecurity measures in dairy farms of Spain.

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.

Association of BoLA DRB3 gene polymorphisms with BoHV-1 infection and zootechnical traits.

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.

Epidemiology of age-dependent prevalence of Bovine Herpes Virus Type 1 (BoHV-1) in dairy herds with and without vaccination.

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.

Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport.

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 467YTSL470 and 563YTVV566, 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.

Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection.

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.

The Role of Epidermal Growth Factor Receptor Signaling Pathway during Bovine Herpesvirus 1 Productive Infection in Cell Culture.

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.

Immunoconjugates to increase photoinactivation of bovine alphaherpesvirus 1 in semen.

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.

Bta-miR-2890 up-regulates JAK-STAT pathway to inhibit BoHV-1 replication by targeting viral gene UL41.

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.