Evidence for vagal sensory neural involvement in influenza pathogenesis and disease.

Influenza A virus (IAV) is a common respiratory pathogen and a global cause of significant and often severe morbidity. Although inflammatory immune responses to IAV infections are well described, little is known about how neuroimmune processes contribute to IAV pathogenesis. In the present study, we employed surgical, genetic, and pharmacological approaches to manipulate pulmonary vagal sensory neuron innervation and activity in the lungs to explore potential crosstalk between pulmonary sensory neurons and immune processes. Intranasal inoculation of mice with H1N1 strains of IAV resulted in stereotypical antiviral lung inflammation and tissue pathology, changes in breathing, loss of body weight and other clinical signs of severe IAV disease. Unilateral cervical vagotomy and genetic ablation of pulmonary vagal sensory neurons had a moderate effect on the pulmonary inflammation induced by IAV infection, but significantly worsened clinical disease presentation. Inhibition of pulmonary vagal sensory neuron activity via inhalation of the charged sodium channel blocker, QX-314, resulted in a moderate decrease in lung pathology, but again this was accompanied by a paradoxical worsening of clinical signs. Notably, vagal sensory ganglia neuroinflammation was induced by IAV infection and this was significantly potentiated by QX-314 administration. This vagal ganglia hyperinflammation was characterized by alterations in IAV-induced host defense gene expression, increased neuropeptide gene and protein expression, and an increase in the number of inflammatory cells present within the ganglia. These data suggest that pulmonary vagal sensory neurons play a role in the regulation of the inflammatory process during IAV infection and suggest that vagal neuroinflammation may be an important contributor to IAV pathogenesis and clinical presentation. Targeting these pathways could offer therapeutic opportunities to treat IAV-induced morbidity and mortality.

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice.

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.

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

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

Absence of signal peptide peptidase in peripheral sensory neurons affects latency-reactivation in HSV-1 ocularly infected mice.

We previously reported that HSV-1 infectivity in vitro and in vivo requires HSV glycoprotein K (gK) binding to the ER signal peptide peptidase (SPP). Anterograde-retrograde transport via peripheral nerves between the site of infection (i.e., eye) and the site of latency (neurons) is a critical process to establish latency and subsequent viral reactivation. Given the essential role of neurons in HSV-1 latency-reactivation, we generated mice lacking SPP specifically in peripheral sensory neurons by crossing Advillin-Cre mice with SPPfl/fl mice. Expression of SPP mRNA and protein were significantly lower in neurons of Avil-SPP-/- mice than in control mice despite similar levels of HSV-1 replication in the eyes of Avil-SPP-/- mice and control mice. Viral transcript levels in isolated neurons of infected mice on days 2 and 5 post infection were lower than in control mice. Significantly less LAT, gB, and PD-1 expression was seen during latency in isolated neurons and total trigeminal ganglia (TG) of Avil-SPP-/- mice than in control mice. Finally, reduced latency and reduced T cell exhaustion in infected Avil-SPP-/- mice correlated with slower and no reactivation. Overall, our results suggest that blocking SPP expression in peripheral sensory neurons does not affect primary virus replication or eye disease but does reduce latency-reactivation. Thus, blocking of gK binding to SPP may be a useful tool to reduce latency-reactivation.

Direct neuronal infection of SARS-CoV-2 reveals cellular and molecular pathology of chemosensory impairment of COVID-19 patients.

Patients with recent pandemic coronavirus disease 19 (COVID-19) complain of neurological abnormalities in sensory functions such as smell and taste in the early stages of infection. Determining the cellular and molecular mechanism of sensory impairment is critical to understand the pathogenesis of clinical manifestations, as well as in setting therapeutic targets for sequelae and recurrence. The absence of studies utilizing proper models of human peripheral nerve hampers an understanding of COVID-19 pathogenesis. Here, we report that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly infects human peripheral sensory neurons, leading to molecular pathogenesis for chemosensory impairments. An in vitro system utilizing human embryonic stem cell (hESC)-derived peripheral neurons was used to model the cellular and molecular pathologies responsible for symptoms that most COVID-19 patients experience early in infection or may develop as sequelae. Peripheral neurons differentiated from hESCs expressed viral entry factor ACE2, and were directly infected with SARS-CoV-2 via ACE2. Human peripheral neurons infected with SARS-CoV-2 exhibited impaired molecular features of chemosensory function associated with abnormalities in sensory neurons of the olfactory or gustatory organs. Our results provide new insights into the pathogenesis of chemosensory dysfunction in patients with COVID-19.

Nociceptive sensory neurons promote CD8 T cell responses to HSV-1 infection.

Host protection against cutaneous herpes simplex virus 1 (HSV-1) infection relies on the induction of a robust adaptive immune response. Here, we show that Nav1.8+ sensory neurons, which are involved in pain perception, control the magnitude of CD8 T cell priming and expansion in HSV-1-infected mice. The ablation of Nav1.8-expressing sensory neurons is associated with extensive skin lesions characterized by enhanced inflammatory cytokine and chemokine production. Mechanistically, Nav1.8+ sensory neurons are required for the downregulation of neutrophil infiltration in the skin after viral clearance to limit the severity of tissue damage and restore skin homeostasis, as well as for eliciting robust CD8 T cell priming in skin-draining lymph nodes by controlling dendritic cell responses. Collectively, our data reveal an important role for the sensory nervous system in regulating both innate and adaptive immune responses to viral infection, thereby opening up possibilities for new therapeutic strategies.

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

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

Innate Receptors Expression by Lung Nociceptors: Impact on COVID-19 and Aging.

The lungs are constantly exposed to non-sterile air which carries harmful threats, such as particles and pathogens. Nonetheless, this organ is equipped with fast and efficient mechanisms to eliminate these threats from the airways as well as prevent pathogen invasion. The respiratory tract is densely innervated by sensory neurons, also known as nociceptors, which are responsible for the detection of external stimuli and initiation of physiological and immunological responses. Furthermore, expression of functional innate receptors by nociceptors have been reported; however, the influence of these receptors to the lung function and local immune response is poorly described. The COVID-19 pandemic has shown the importance of coordinated and competent pulmonary immunity for the prevention of pathogen spread as well as prevention of excessive tissue injury. New findings suggest that lung nociceptors can be a target of SARS-CoV-2 infection; what remains unclear is whether innate receptor trigger sensory neuron activation during SARS-CoV-2 infection and what is the relevance for the outcomes. Moreover, elderly individuals often present with respiratory, neurological and immunological dysfunction. Whether aging in the context of sensory nerve function and innate receptors contributes to the disorders of these systems is currently unknown. Here we discuss the expression of innate receptors by nociceptors, particularly in the lungs, and the possible impact of their activation on pulmonary immunity. We then demonstrate recent evidence that suggests lung sensory neurons as reservoirs for SARS-CoV-2 and possible viral recognition via innate receptors. Lastly, we explore the mechanisms by which lung nociceptors might contribute to disturbance in respiratory and immunological responses during the aging process.

Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency.

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression.

Herpes simplex virus 1 (HSV-1) and HSV-2 can efficiently establish lifelong, transcriptionally silent latency states in sensory neurons to escape host detection. While host factors have previously been associated with long-range insulators in the viral genome, it is still unknown whether host transcription factors can repress viral genes more proximately to promote latency in dorsal root ganglion (DRG) neurons. Here, we assessed whether RUNX (runt-related transcription factor) transcription factors, which are critical in the development of sensory neurons, could be binding HSV-1 genome directly to suppress viral gene expression and lytic infection. Using previously published transcriptome sequencing data, we confirmed that mouse DRG neurons highly express Runx1 mRNA. Through computational analysis of HSV-1 and HSV-2 genomes, we observed that putative RUNX consensus binding sites (CBSs) were more enriched and more closely located to viral gene transcription start sites than would be expected by chance. We further found that RUNX CBSs were significantly more enriched among genomes of herpesviruses compared to those of nonherpesviruses. Utilizing an in vitro model of HSV-1 infection, we found that overexpressed RUNX1 could bind putative binding sites in the HSV-1 genome, repress numerous viral genes spanning all three kinetic classes, and suppress productive infection. In contrast, knockdown of RUNX1 in neuroblastoma cells induced viral gene expression and increased HSV-1 infection in vitro In sum, these data support a novel role for RUNX1 in directly binding herpesvirus genome, silencing the transcription of numerous viral genes, and ultimately limiting overall infection.IMPORTANCE Infecting 90% of the global population, HSV-1 and HSV-2 represent some of the most prevalent viruses in the world. Much of their success can be attributed to their ability to establish lifelong latent infections in the dorsal root ganglia (DRG). It is still largely unknown, however, how host transcription factors are involved in establishing this latency. Here, we report that RUNX1, expressed highly in DRG, binds HSV-1 genome, represses transcription of numerous viral genes, and suppresses productive in vitro infection. Our computational work further suggests this strategy may be used by other herpesviruses to reinforce latency in a cell-specific manner.

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.

Peripheral Neuronopathy Associated With Ebola Virus Infection in Rhesus Macaques: A Possible Cause of Neurological Signs and Symptoms in Human Ebola Patients.

Neurological signs and symptoms are the most common complications of Ebola virus disease. However, the mechanisms underlying the neurologic manifestations in Ebola patients are not known. In this study, peripheral ganglia were collected from 12 rhesus macaques that succumbed to Ebola virus (EBOV) disease from 5 to 8 days post exposure. Ganglionitis, characterized by neuronal degeneration, necrosis, and mononuclear leukocyte infiltrates, was observed in the dorsal root, autonomic, and enteric ganglia. By immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy, we confirmed that CD68+ macrophages are the target cells for EBOV in affected ganglia. Further, we demonstrated that EBOV can induce satellite cell and neuronal apoptosis and microglial activation in infected ganglia. Our results demonstrate that EBOV can infect peripheral ganglia and results in ganglionopathy in rhesus macaques, which may contribute to the neurological signs and symptoms observed in acute and convalescent Ebola virus disease in human patients.

Viral Transduction of DRG Neurons.

The manipulation of gene expression is an essential tool to study the function of genes or signaling pathways. Uniform and robust gene manipulation is crucial for successful assays. However, neuronal cells are generally difficult-to-transfect cells with conventional DNA/RNA transfection reagents. Therefore, virus-mediated gene delivery is a primary choice for the studies of gene functions in neurons. In this chapter, we will describe the methods for lentivirus-mediated gene expression or knockdown in DRG neurons.

Botulinum Neurotoxin Light Chains Expressed by Defective Herpes Simplex Virus Type-1 Vectors Cleave SNARE Proteins and Inhibit CGRP Release in Rat Sensory Neurons.

A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.

The bovine herpesvirus 1 regulatory proteins, bICP4 and bICP22, are expressed during the escape from latency.

Following acute infection of mucosal surfaces by bovine herpesvirus 1 (BoHV-1), sensory neurons are a primary site for lifelong latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Two viral regulatory proteins (VP16 and bICP0) are expressed within 1 h after calves latently infected with BoHV-1 are treated with dexamethasone. Since the immediate early transcription unit 1 (IEtu1) promoter regulates both BoHV-1 infected cell protein 0 (bICP0) and bICP4 expressions, we hypothesized that the bICP4 protein is also expressed during early stages of reactivation from latency. In this study, we tested whether bICP4 and bICP22, the only other BoHV-1 protein known to be encoded by an immediate early gene, were expressed during reactivation from latency by generating peptide-specific antiserum to each protein. bICP4 and bICP22 protein expression were detected in trigeminal ganglionic (TG) neurons during early phases of dexamethasone-induced reactivation from latency, operationally defined as the escape from latency. Conversely, bICP4 and bICP22 were not readily detected in TG neurons of latently infected calves. In summary, it seems clear that all proteins encoded by known BoHV-1 IE genes (bICP4, bICP22, and bICP0) were expressed during early stages of dexamethasone-induced reactivation from latency.

Neuronal Subtype Determines Herpes Simplex Virus 1 Latency-Associated-Transcript Promoter Activity during Latency.

Herpes simplex virus (HSV) latency in neurons remains poorly understood, and the heterogeneity of the sensory nervous system complicates mechanistic studies. In this study, we used primary culture of adult trigeminal ganglion (TG) mouse neurons in microfluidic devices and an in vivo model to examine the subtypes of sensory neurons involved in HSV latency. HSV-infected neurofilament heavy-positive (NefH+) neurons were more likely to express latency-associated transcripts (LATs) than infected neurofilament heavy-negative (NefH-) neurons. This differential expression of the LAT promoter correlated with differences in HSV-1 early infection that manifested as differences in the efficiency with which HSV particles reached the cell body following infection at the distal axon. In vivo, we further identified a specific subset of NefH+ neurons which coexpressed calcitonin gene-related peptide α (NefH+ CGRP+ neurons) as the sensory neuron subpopulation with the highest LAT promoter activity following HSV-1 infection. Finally, an early-phase reactivation assay showed HSV-1 reactivating in NefH+ CGRP+ neurons, although other sensory neuron subpopulations were also involved. Together, these results show that sensory neurons expressing neurofilaments exhibit enhanced LAT promoter activity. We hypothesize that the reduced efficiency of HSV-1 invasion at an early phase of infection may promote efficient establishment of latency in NefH+ neurons due to initiation of the antiviral state preceding arrival of the virus at the neuronal cell body. While the outcome of HSV-1 infection of neurons is determined by a broad variety of factors in vivo, neuronal subtypes are likely to play differential roles in modulating the establishment of latent infection.IMPORTANCE Two pivotal properties of HSV-1 make it a successful pathogen. First, it infects neurons, which are immune privileged. Second, it establishes latency in these neurons. Together, these properties allow HSV to persist for the lifetime of its host. Neurons are diverse and highly organized cells, with specific anatomical, physiological, and molecular characteristics. Previous work has shown that establishment of latency by HSV-1 does not occur equally in all types of neurons. Our results show that the kinetics of HSV infection and the levels of latency-related gene expression differ in certain types of neurons. The neuronal subtype infected by HSV is therefore a critical determinant of the outcome of infection and latency.

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.

Sensory neuronopathy heralding human T cell lymphotropic virus type I myelopathy.

Neurological phenotypes of human T cell lymphotropic virus type I (HTLV-1) are numerous and rarely may not manifest the classic HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We report a case of HTLV-1-related sensory neuronopathy heralding the classic HAM/TSP.

Adeno-associated virus serotype rh10 is a useful gene transfer vector for sensory nerves that innervate bone in immunodeficient mice.

Adeno-associated virus (AAV) is frequently used to manipulate gene expression in the sensory nervous system for the study of pain mechanisms. Although some serotypes of AAV are known to have nerve tropism, whether AAV can distribute to sensory nerves that innervate the bone or skeletal tissue has not been shown. This information is crucial, since bone pain, including cancer-induced bone pain, is an area of high importance in pain biology. In this study, we found that AAVrh10 transduces neurons in the spinal cord and dorsal root ganglia of immunodeficient mice with higher efficacy than AAV2, 5, 6, 8, and 9 when injected intrathecally. Additionally, AAVrh10 has tropism towards sensory neurons in skeletal tissue, such as bone marrow and periosteum, while it occasionally reaches the sensory nerve fibers in the mouse footpad. Moreover, AAVrh10 has higher tropic affinity to large myelinated and small peptidergic sensory neurons that innervate bone, compared to small non-peptidergic sensory neurons that rarely innervate bone. Taken together, these results suggest that AAVrh10 is a useful gene delivery vector to target the sensory nerves innervating bone. This finding may lead to a greater understanding of the molecular mechanisms of chronic bone pain and cancer-induced bone pain.