Transcriptomic analysis reveals bovine herpesvirus 1 infection regulates innate immune response resulted in restricted viral replication in neuronal cells.

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.

Bovine papular stomatitis virus as a vaccine vector for cattle.

Virus vectored vaccines are not available commercially for cattle even though compelling potential applications exist. Bovine papular stomatitis virus (BPSV), a highly prevalent parapoxvirus, causes self-limited oral lesions in cattle. Ability of virus to accommodate large amounts of foreign DNA, induce low level of antiviral immunity, and circulate and likely persist in cattle populations, make BPSV an attractive candidate viral vector. Here, recombinant BPSV were constructed expressing either Bovine herpesvirus 1 (BoHV-1) glycoprotein gD (BPSVgD), or gD and gB (BPSVgD/gB). Immunization of BPSV serologically-positive calves with BPSVgD or BPSVgD/gB induced BoHV-1 neutralization antibodies and provided protection for three of four animals following a high dose BoHV-1 challenge at day 70 pi. Results indicate BPSV suitability as a candidate virus vector for cattle vaccines.

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.

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.

A neutralizing monoclonal antibody-based blocking ELISA to detect bovine herpesvirus 1 and vaccination efficacy.

Infections caused by bovine herpesvirus 1 (BoHV-1) remain a serious global issue to the health and welfare of the bovine industry. Monitoring of neutralizing antibodies is essential not only for epidemic diagnosis, but also to assess vaccination efficacy. In this study, we generated a neutralizing monoclonal antibody, termed as 3F8, targeting glycoprotein D (gD) of BoHV-1. This monoclonal antibody could neutralize BoHV-1 with a 50% inhibitory concentration (IC50) of 37.82 ng/mL. Furthermore, 3F8 could inhibit BoHV-1 infection and cell-to-cell spread at the prebinding stage. A blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies against BoHV-1 was then developed based on 3F8 and protein gD generated using a baculovirus expression system. The sensitivity and specificity of the test were estimated to be 94.59% and 93.42%, respectively. A significant correlation (R2 = 0.9583, p < 0.01) was observed between the results obtained with the blocking ELISA and a virus neutralization test, which suggested that the blocking ELISA could detect neutralizing antibodies against BoHV-1. A serological survey was carried out in the dairy farms in Beijing district using 3F8-based blocking ELISA to monitor the annual neutralization antibody against BoHV-1 during 2012-2020. It revealed that the dairy farms in Beijing were at high risk of BoHV-1 infection during 2012-2017 but were protected since 2018 upon implementation of an immunization program. Our results demonstrated that this assay is suitable for BoHV-1 surveillance and vaccination efficacy in cattle as a replacement for the virus neutralization test. KEY POINTS: • Prevention of BoHV-1 infection requires the monitoring of neutralizing antibodies. • A blocking ELISA for the neutralizing antibody was developed based on mAb 3F8 against BoHV-1 gD. • It can replace the labor-intensive and time-consuming viral neutralizing tests.

A Pioneer Transcription Factor and Type I Nuclear Hormone Receptors Synergistically Activate the Bovine Herpesvirus 1 Infected Cell Protein 0 (ICP0) Early Promoter.

Following bovine herpesvirus 1 (BoHV-1) acute infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia are an important site for latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Expression of two key viral transcriptional regulatory proteins, BoHV-1 infected cell protein 0 (bICP0) and bICP4, are regulated by sequences within the immediate early promoter (IEtu1). A separate early promoter also drives bICP0 expression, presumably to ensure sufficient levels of this important transcriptional regulatory protein. Productive infection and bICP0 early promoter activity are cooperatively transactivated by Krüppel-like factor 4 (KLF4) and a type I nuclear hormone receptor (NHR), androgen receptor, glucocorticoid receptor, or progesterone receptor. The bICP0 early promoter contains three separate transcriptional enhancers that mediate cooperative transactivation. In contrast to the IEtu1 promoter, the bICP0 early promoter lacks consensus type I NHR binding sites. Consequently, we hypothesized that KLF4 and Sp1 binding sites are essential for type I NHR and KLF4 to transactivate the bICP0 promoter. Mutating KLF4 and Sp1 binding sites in each enhancer domain significantly reduced transactivation by KLF4 and a type I NHR. Chromatin immunoprecipitation (ChIP) studies demonstrated that occupancy of bICP0 early promoter sequences by KLF4 and type I NHR is significantly reduced when KLF4 and/or Sp1 binding sites are mutated. These studies suggest that cooperative transactivation of the bICP0 E promoter by type I NHRs and a stress-induced pioneer transcription factor (KLF4) promote viral replication and spread in neurons or nonneural cells in reproductive tissue. IMPORTANCE Understanding how stressful stimuli and changes in the cellular milieu mediate viral replication and gene expression in the natural host is important for developing therapeutic strategies that impair virus transmission and disease. For example, bovine herpesvirus 1 (BoHV-1) reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone, which mimics the effects of stress. Furthermore, BoHV-1 infection increases the incidence of abortion in pregnant cows, suggesting that sex hormones stimulate viral growth in certain tissues. Previous studies revealed that type I nuclear hormone receptors (NHRs) (androgen, glucocorticoid, or progesterone) and a pioneer transcription factor, Krüppel-like factor 4 (KLF4), cooperatively transactivate the BoHV-1 infected cell protein 0 (bICP0) early promoter. Transactivation was mediated by Sp1 and/or KLF4 consensus binding sites within the three transcriptional enhancers. These studies underscore the complexity by which BoHV-1 exploits type I NHR fluctuations to enhance viral gene expression, replication, and transmission in the natural host.

Molecular evidence for concurrent infection of goats by orf virus and bovine herpesvirus 1.

Orf is a disease of small ruminant animals, including goats and sheep, that is caused by a parapoxvirus. Although the mortality rate is low, economic losses may occur due to the clinical signs. Bovine herpesvirus 1 (BoHV-1) infection is known to cause respiratory and reproductive disorders mainly in cattle; however, it has been found to circulate among goats and sheep as well. In contrast to orf virus (ORFV), BoHV-1 does not induce clinical disease in goats. In this study, we aimed to detect the presence of ORFV by molecular methods and to uncover eventual simultaneous herpesvirus infections masked by orf disease signs. To this end, 82 goats, housed near to a cattle herd, were tested. By polymerase chain reaction (PCR), three goats (3.7%) were found to harbour both viruses, while an additional goat was positive for ORFV only. The PCR products were sequenced and phylogenetic analyses were performed. This study revealed that ORFV and BoHV-1 may be present simultaneously in an animal causing a concurrent infection. These data should be taken into consideration when looking for secondary pathogens in diseased goats, and the prevention methods should be developed accordingly.

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

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

Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency.

Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.

BVDV, BHV-1 and BLV antibodies in dromedary camels of Turkey kept without and with ruminants.

Camels are the only animals bred to sustain the tradition of wrestling in Turkey and are reared within a limited set of geographic areas. Farmers of such animals may also be engaged in ruminant breeding. The current research was aimed at documenting bovine viral diarrhoea virus (BVDV), bovine herpesvirus-1 (BHV-1), and bovine leukaemia virus (BLV) infections in sera collected from dromedary camels in four different geographical regions of Turkey during the years 2019-2021. All samples were tested for BVDV, BHV-1 and BLV antibodies as well as BVDV antigen by ELISA. Antibodies against BVDV were found in 16.8% of the camel sera tested. However, none of the camels sampled were positive in terms of BHV-1 and BLV antibodies as well as BVDV antigen. The prevalence was observed higher in the herds in which ruminants were raised in addition to camels (OR = 4.583, 95% CI, 1.298-16.182), (p = 0.018), while the prevalence was observed lower in the herds in which only camels were raised. This study showed that BVDV infection was more prevalent than BHV-1 and BLV infections in Turkish dromedary camels. Herewith, the camels, being a susceptible species to numerous viral ruminant diseases, may also serve as an important source of BVDV infection for other ruminant animals in the same flock.

Feedback inhibition of bovine herpesvirus 5 replication by dual-copy bhv5-miR-B10-3p.

Bovine herpesvirus 5 (BoHV-5) is a pathogen of cattle responsible for fatal meningoencephalitis. Like alpha herpesvirus subfamily members, BoHV-5 also encodes microRNA in lytic infections of epithelial cells. BoHV-5-miR-B10 was the most abundant miRNA detected in a high-throughput sequencing study. Here, we evaluated the kinetics of miR-B10 expression after BoHV-5 productive infection by stem-loop real-time quantitative PCR. miR-B10 candidate target sites in the virus were predicted, and BoHV-5 UL39 was confirmed as a target gene by dual-luciferase assay with the design of an miR-B10 tough decoy (TuD). The UL39 gene encoding ribonucleotide reductase (RR) large subunit plays an important role in the early stage of BoHV-5 lytic infection. As BoHV-5-miR-B10 is located in internal and terminal repeat regions, we generated a TuD gene-integrated BoHV-5 strain, which effectively down-regulated miR-B10-3p. Strikingly, the suppression of miR-B10-3p significantly improved BoHV-5 replication. Taking these findings together, our study established an efficient method to deliver and express TuD RNA for viral miRNA suppression, and demonstrated that virus-encoded miRNA suppresses viral-genome biogenesis with a feedback mode, which might serve as a brake for viral replication. Herpesviruses infect humans and a variety of animals. Almost all herpesviruses can encode miRNAs, but the functions of these miRNAs remain to be elucidated. Most herpesvirus-encoded miRNA harbours dual copies, which is difficult to be deleted by current genetic modulation. Here, we developed an efficient method to deliver and express TuD RNA to efficiently suppress viral miRNA with multiple copies. Using this method, we demonstrated for the first time that viral miRNA feedback regulates viral replication by suppressing the expression of RR.

US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells.

Bovine herpesvirus 1 (BoHV-1) infects bovine species, causing respiratory infections, genital disorders and abortions. VP8 is the most abundant tegument protein of BoHV-1 and is critical for virus replication in cattle. In this study, the cellular transport of VP8 in BoHV-1-infected cells and its ability to alter the cellular lipid metabolism were investigated. A viral kinase, US3, was found to be involved in regulating these processes. In the early stages of infection VP8 was localized in the nucleus. Subsequently, presumably after completion of its role in the nucleus, VP8 was translocated to the cytoplasm. When US3 was deleted or the essential US3 phosphorylation site of VP8 was mutated in BoHV-1, the majority of VP8 was localized in the nuclei of infected cells. This suggests that phosphorylation by US3 may be critical for cytoplasmic localization of VP8. Eventually, the cytoplasmic VP8 was accumulated in the cis-Golgi apparatus but not in the trans-Golgi network, implying that VP8 was not involved in virion transport toward and budding from the cell membrane. VP8 caused lipid droplet (LD) formation in the nuclei of transfected cells and increased cellular cholesterol levels. Lipid droplets were not found in the nuclei of BoHV-1-infected cells when VP8 was cytoplasmic in the presence of US3. However, when US3 was deleted or phosphorylation residues in VP8 were mutated, nuclear VP8 and LDs appeared in BoHV-1-infected cells. The total cholesterol level was increased in BoHV-1-infected cells but not in ΔUL47-BoHV-1-infected cells, further supporting a role for VP8 in altering the cellular lipid metabolism during infection.IMPORTANCE Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important elements directing VP8 to the desired locations in the BoHV-1-infected cell. In this study, a critical regulator that switches the nuclear and cytoplasmic localization of VP8 in BoHV-1-infected cells was identified. BoHV-1 used viral kinase US3 to regulate the cellular localization of VP8. Early during BoHV-1 infection VP8 was localized in the nucleus, where it performs various functions; once US3 was expressed, phosphorylated VP8 was cytoplasmic and ultimately accumulated in the cis-Golgi apparatus, presumably to be incorporated into virions. The Golgi localization of VP8 was only observed in virus-infected cells and not in US3-cotransfected cells, suggesting that this is mediated by other viral factors. Interestingly, VP8 was shown to cause increased cholesterol levels, which is a novel function for VP8 and a potential strategy to supply lipid for viral replication.

Role of Sphingomyelin in Alphaherpesvirus Entry.

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.

Mapping a highly conserved linear neutralizing epitope at the N-terminus of the gD glycoprotein of bovine herpesvirus type I using a monoclonal antibody.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae subfamily, causes significant economic losses to the cattle industry worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an essential role in the viral entry into permissive cells and possibly cooperates with other envelope glycoproteins. The herpesvirus gD induces a protective immune response against diseases in cattle or animal models. Mapping epitopes on gD will facilitate the understanding of the BoHV-1 pathogenesis and development of alternative vaccines against various diseases associated with the virus. In this study, a monoclonal antibody (MAb), designated as 3C1, was generated using naive BoHV-1 in vaccination of mice, demonstrating that 3C1 was specific to gD and represents a neutralizing activity against BoHV-1 infection in Madin-Darby bovine kidney cells. Panels of overlapping gD recombinant proteins with glutathione S-transferase tag were prepared to define the epitope recognized by 3C1. The data demonstrated that the N-terminus of gD 23APRVTVYVD31 was recognized by 3C1. Furthermore, the 26VTVYVD31 motif was the minimal amino acid sequence for the recognition. The epitope identified in this study is highly conserved among the typical strains of BoHV-1 and BoHV-5, suggesting that this epitope may be useful in the diagnosis of diseases. In addition, the defined region on gD of BoHV-1 might be essential in viral entry upon comparison with the prototype virus in herpes simplex virus (Alphaherpesvirinae). The data will elucidate the roles of gD of BoHV-1 in viral entry and pathogenesis and its potential application for the development of vaccine candidates and diagnostic techniques based on the conserved epitopes on gD or in combination with those of other herpesvirus glycoproteins.

Protective immunity following vaccination with a recombinant multiple-epitope protein of bovine herpesvirus type I in a rabbit model.

Bovine herpesvirus type 1 (BoHV-1) causes considerable economic losses to the cow industry. Vaccination remains an effective strategy to control the diseases associated with BoHV-1. However, live vaccines present safety concerns, especially in pregnant cows; thus, nonreplicating vaccines have been developed to control the disease. The envelope glycoproteins of BoHV-1 induce a protective immune response. In this work, selected epitopes on glycoproteins gD, gC, and gB were constructed in triplicate with linker peptides. Vaccination of rabbits demonstrated that P2-gD/gC/gB with AAYAAY induced higher specific antibodies than that with GGGGS linker. P2-gD/gC/gB with AAYAAY linker was fused with bovine interleukin-6 (BoIL-6) or rabbit IL-6 (RaIL-6) and bacterially expressed. Rabbits were intramuscularly immunized with 100 µg of P2-gD/gC/gB-BoIL-6, P2-gD/gC/gB-RaIL-6, P2-gD/gC/gB, P2-gD/gC/gB plus BoIL-6, P2-(gD-a)3-BoIL-6, or P2-(gD-a)3 emulsified with ISA 206 adjuvant thrice at 3-week intervals. P2-gD/gC/gB-BoIL-6 generated a higher titer of BoHV-1-specific antibodies, neutralizing antibodies, interferon (IFN)-γ, and IL-4 compared with P2-gD/gC/gB plus BoIL-6, P2-gD/gC/gB-RaIL-6, or other formulation. P2-gD/gC/gB-BoIL-6 triggered similar levels of antibodies and significantly higher titer of IFN-γ and IL-4 compared with inactivated bovine viral diarrhea (BVD)-infectious bovine rhinotracheitis (IBR) vaccine. Rabbits vaccinated with P2-gD/gC/gB-BoIL-6 dramatically reduced viral shedding and tissue lesions in lungs and trachea after viral challenge and reactivation compared with those with P2-gD/gC/gB plus BoIL-6 or P2-gD/gC/gB-RaIL-6. P2-gD/gC/gB-BoIL-6 provided protective effects against viral shedding and tissue pathogenesis similar to those of the inactivated vaccine. The data confirmed the safety and immunogenicity of multiple-epitope recombinant protein and a potential vaccine candidate to control the disease, especially for pregnant cattle.

Development of a quantitative risk assessment of bovine viral diarrhea virus and bovine herpesvirus-1 introduction in dairy cattle herds to improve biosecurity.

A quantitative risk assessment model was developed to estimate the annual probability of introducing bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) at the farm level through animal movements. Data from 2017 official animal movements, biosecurity questionnaires, scientific literature, and expert opinion from field veterinarians were taken into consideration for model input parameters. Purchasing or introducing cattle, rearing replacement heifers offsite, showing cattle at competitions, sharing transport vehicles with other herds, and transporting cattle in vehicles that have not been cleaned and disinfected were considered in the model. The annual probability of introducing BVDV or BoHV-1 through infected animals was very heterogeneous between farms. The median likelihoods of BVDV and BoHV-1introduction were 12 and 9%, respectively. Farms that purchased cattle from within their region (i.e., local movements) and shared transport with other farms had a higher probability for BVDV and BoHV-1 introduction. This model can be a useful tool to support decision-making on biosecurity measures that should be prioritized to reduce the probability of introduction of these 2 diseases in dairy herds.

Parainfluenza Virus 5 Infection in Neurological Disease and Encephalitis of Cattle.

The etiology of viral encephalitis in cattle often remains unresolved, posing a potential risk for animal and human health. In metagenomics studies of cattle with bovine non-suppurative encephalitis, parainfluenza virus 5 (PIV5) was identified in three brain samples. Interestingly, in two of these animals, bovine herpesvirus 6 and bovine astrovirus CH13 were additionally found. We investigated the role of PIV5 in bovine non-suppurative encephalitis and further characterized the three cases. With traditional sequencing methods, we completed the three PIV5 genomes, which were compared to one another. However, in comparison to already described PIV5 strains, unique features were revealed, like an 81 nucleotide longer open reading frame encoding the small hydrophobic (SH) protein. With in situ techniques, we demonstrated PIV5 antigen and RNA in one animal and found a broad cell tropism of PIV5 in the brain. Comparative quantitative analyses revealed a high viral load of PIV5 in the in situ positive animal and therefore, we propose that PIV5 was probably the cause of the disease. With this study, we clearly show that PIV5 is capable of naturally infecting different brain cell types in cattle in vivo and therefore it is a probable cause of encephalitis and neurological disease in cattle.

Transmembrane regions of bovine herpesvirus 1-encoded UL49.5 and glycoprotein M regulate complex maturation and ER-Golgi trafficking.

Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.

The Wnt Signaling Pathway Is Differentially Expressed during the Bovine Herpesvirus 1 Latency-Reactivation Cycle: Evidence That Two Protein Kinases Associated with Neuronal Survival, Akt3 and BMPR2, Are Expressed at Higher Levels during Latency.

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 ß-catenin and a ß-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased ß-catenin-dependent transcription and cell survival. ß-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/ß-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/ß-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/ß-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 ß-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.

Bovine Herpesvirus 1 Entry by a Low-pH Endosomal Pathway.

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.