Mitochondrial DNA and Electron Transport Chain Protein Levels Are Altered in Peripheral Nerve Tissues from Donors with HIV Sensory Neuropathy: A Pilot Study.

Distal sensory polyneuropathy (DSP) and distal neuropathic pain (DNP) remain significant challenges for older people with HIV (PWH), necessitating enhanced clinical attention. HIV and certain antiretroviral therapies (ARTs) can compromise mitochondrial function and impact mitochondrial DNA (mtDNA) replication, which is linked to DSP in ART-treated PWH. This study investigated mtDNA, mitochondrial fission and fusion proteins, and mitochondrial electron transport chain protein changes in the dorsal root ganglions (DRGs) and sural nerves (SuNs) of 11 autopsied PWH. In antemortem standardized assessments, six had no or one sign of DSP, while five exhibited two or more DSP signs. Digital droplet polymerase chain reaction was used to measure mtDNA quantity and the common deletions in isolated DNA. We found lower mtDNA copy numbers in DSP+ donors. SuNs exhibited a higher proportion of mtDNA common deletion than DRGs in both groups. Mitochondrial electron transport chain (ETC) proteins were altered in the DRGs of DSP+ compared to DSP- donors, particularly Complex I. These findings suggest that reduced mtDNA quantity and increased common deletion abundance may contribute to DSP in PWH, indicating diminished mitochondrial activity in the sensory neurons. Accumulated ETC proteins in the DRG imply impaired mitochondrial transport to the sensory neuron's distal portion. Identifying molecules to safeguard mitochondrial integrity could aid in treating or preventing HIV-associated peripheral neuropathy.

Therapeutic prime/pull vaccination of HSV-2-infected guinea pigs with the ribonucleotide reductase 2 (RR2) protein and CXCL11 chemokine boosts antiviral local tissue-resident and effector memory CD4+ and CD8+ T cells and protects against recurrent genital herpes.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4+ and CD8+ T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes.

Analgesia effect of lentivirus-siSCN9A infected neurons in vincristine induced neuropathic pain rats.

At present, the mechanism of siSCN9A in Vincristine (VCR)-induced neuropathic pain (NP) is still unclear. This study aimed to explore the analgesic mechanism of lentivirus-siSCN9A (LV-siSCN9A) infected neurons against NP. 40 male Sprague-Dawley (SD) rats were divided into a control group (injected with normal saline), a model group (VCR-induced NP model), a LV-SC group (NP model mice were injected with LV-SC-infected dorsal root ganglia (DRG) neuron cells under the microscope), and a LV-siSCN9A group (NP model mice were injected with LV-siSCN9A-infected DRG neuron cells under the microscope, with 10 rats in each group. The changes of mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats in different groups were detected by behavior testing, the Nav1.7 changes in each group were detected by immunofluorescence double standard and Western-blot method. It was found that compared with the control group, the MWT and TWL of the rats in model group were significantly decreased (P < 0.05), and the expression levels of Nav1.7 messenger ribonucleic acid (mRNA) and proteins were significantly increased (P < 0.05). Compared with LV-SC group, the MWT and TWL of rats in LV-siSCN9A group were significantly increased (P < 0.05), the expression levels of Nav1.7 mRNA and proteins were significantly decreased (P < 0.05), and the CGRP expression of spinal dorsal horn was significantly decreased. It was concluded that the LV-siSCN9A infected neurons could play an analgesic role by down-regulating Nav1.7 expression induced by VCR in NP model.

Herpes Simplex Virus 1 MicroRNA miR-H8 Is Dispensable for Latency and Reactivation In Vivo.

The regulatory functions of 10 individual viral microRNAs (miRNAs) that are abundantly expressed from the herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) region remain largely unknown. Here, we focus on HSV-1 miRNA miR-H8, which is within the LAT 3p exon, antisense to the first intron of ICP0, and has previously been shown to target a host glycosylphosphatidylinositol (GPI)-anchoring pathway. However, the functions of this miRNA have not been assessed in the context of the viral genome during infection. Therefore, we constructed a recombinant virus lacking miR-H8 (17dmiR-H8) and compared it to the parental wild-type and rescue viruses to characterize phenotypic differences. In rabbit skin cells, 17dmiR-H8 exhibited only subtle reductions in viral yields. In contrast, we found significant decreases in both viral yields (8-fold) and DNA replication (9.9-fold) in murine neuroblastoma cells, while 17dmiR-H8 exhibited a 3.6-fold increase in DNA replication in differentiated human neuronal cells (Lund human mesencephalic [LUHMES] cells). These cell culture phenotypes suggested potential host- and/or neuron-specific roles for miR-H8 in acute viral replication. To assess whether miR-H8 plays a role in HSV latency or reactivation, we used a human in vitro reactivation model as well as mouse and rabbit reactivation models. In the LUHMES cell-induced reactivation model, there was no difference in viral yields at 48 h postreactivation. In the murine dorsal root ganglion explant and rabbit ocular adrenergic reactivation models, the deletion of miR-H8 had no detectable effect on genome loads during latency or reactivation. These results indicate that miR-H8 is dispensable for the establishment of HSV-1 latency and reactivation.IMPORTANCE Herpesviruses have a remarkable ability to sustain lifelong infections by evading host immune responses, establishing a latent reservoir, and maintaining the ability to reactivate the lytic cascade to transmit the virus to the next host. The HSV-1 latency-associated transcript region is known to regulate many aspects of HSV-1 latency and reactivation, although the mechanisms for these functions remain unknown. To this end, we characterize an HSV-1 recombinant containing a deletion of a LAT-encoded miRNA, miR-H8, and demonstrate that it plays no detectable role in the establishment of latency or reactivation in differentiated human neurons (LUHMES cells) and mouse and rabbit models. Therefore, this study allows us to exclude miR-H8 from phenotypes previously attributed to the LAT region. Elucidating the genetic elements of HSV-1 responsible for establishment, maintenance, and reactivation from latency may lead to novel strategies for combating persistent herpesvirus infections.

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.

ACE2 and SCARF expression in human dorsal root ganglion nociceptors: implications for SARS-CoV-2 virus neurological effects.

SARS-CoV-2 has created a global crisis. COVID-19, the disease caused by the virus, is characterized by pneumonia, respiratory distress, and hypercoagulation and can be fatal. An early sign of infection is loss of smell, taste, and chemesthesis-loss of chemical sensation. Other neurological effects of the disease have been described, but not explained. It is now apparent that many of these neurological effects (for instance joint pain and headache) can persist for at least months after infection, suggesting a sensory neuronal involvement in persistent disease. We show that human dorsal root ganglion (DRG) neurons express the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 at the RNA and protein level. We also demonstrate that SARS-CoV-2 and coronavirus-associated factors and receptors are broadly expressed in human DRG at the lumbar and thoracic level as assessed by bulk RNA sequencing. ACE2 mRNA is expressed by a subset of nociceptors that express MRGPRD mRNA, suggesting that SARS-CoV-2 may gain access to the nervous system through entry into neurons that form free nerve endings at the outermost layers of skin and luminal organs. Therefore, DRG sensory neurons are a potential target for SARS-CoV-2 invasion of the peripheral nervous system, and viral infection of human nociceptors may cause some of the persistent neurological effects seen in COVID-19.

Pulsed radiofrequency of dorsal root ganglion of upper thoracic segment for herpes zoster neuralgia: Case report.

RATIONALE: Pulsed radiofrequency (PRF) therapy of dorsal root ganglion is effective in treating acute stage shingles neuralgia of chest and back. Herein, a case of herpetic neuralgia with difficult puncture of dorsal root ganglion of upper thoracic segment is report. PATIENT CONCERNS: A 62-year-old male patient was admitted to the hospital for 2 days for herpes zoster with paroxysmal needle-like pain in the left chest and back. The skin lesion area of herpes zoster and the superficial location of neuralgia was left T2-4, and visual analog scale (VAS) score was 6 points. DIAGNOSIS: Two days ago, the patient had paroxysmal needle-like pain in the left chest and back, without herpes, and was admitted to the hospital for emergency treatment. Chest pain and myocardial infarction were considered; however, troponin, myocardial enzyme spectrum, and blood amylase were in the normal range. On the evening of the same day, the patient presented green bean-sized blisters distributed in clusters along the left T2-4 nerve as a banded pattern. Thus, the patient was diagnosed as shingles. INTERVENTION: Oral gabapentin capsules, varaciclovir tablets, mecobalamine tablets, and amitriptyline hydrochloride tablets were administered, and topical aciclovir cream was applied. The VAS score after the above treatment was 5 points. The patient underwent computed tomography-guided PRF surgery on the dorsal root ganglion. OUTCOME: Postoperative pain was relieved. One month post-surgery, no oral analgesic drugs were administered. The VAS score was 1 point, and the pain completely disappeared at 3 months post-surgery. CONCLUSIONS: Herpes zoster is most common in the chest and back. The PRF of dorsal root ganglion cannot access the target by conventional puncture, and can be completed by thoracic sympathetic nerve radiofrequency puncture path.

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.

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

Deletion of Herpes Simplex Virus 1 MicroRNAs miR-H1 and miR-H6 Impairs Reactivation.

During all stages of infection, herpes simplex virus 1 (HSV-1) expresses viral microRNAs (miRNAs). There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. We constructed a recombinant virus lacking the sequences for miR-H1-5p and miR-H6-3p (17dmiR-H1/H6). The seed sequences for these miRNAs are antisense to each other and are transcribed from divergent noncoding RNAs in the latency-associated transcript (LAT) promoter region. Comparing phenotypes exhibited by the recombinant virus lacking these miRNAs to the wild type (17syn+), we found that during acute infection in cell culture, 17dmiR-H1/H6 exhibited a modest increase in viral yields. Analysis of pathogenesis in the mouse following footpad infection revealed a slight increase in virulence for 17dmiR-H1/H6 but no significant difference in the establishment or maintenance of latency. Strikingly, explant of latently infected dorsal root ganglia revealed a decreased and delayed reactivation phenotype. Further, 17dmiR-H1/H6 was severely impaired in epinephrine-induced reactivation in the rabbit ocular model. Finally, we demonstrated that deletion of miR-H1/H6 increased the accumulation of the LAT as well as several of the LAT region miRNAs. These results suggest that miR-H1/H6 plays an important role in facilitating efficient reactivation from latency.IMPORTANCE While HSV antivirals reduce the severity and duration of clinical disease in some individuals, there is no vaccine or cure. Therefore, understanding the mechanisms regulating latency and reactivation as a potential to elucidate targets for better therapeutics is important. There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. The present study focuses on two of the miRNAs (miR-H1/H6) that are encoded within the latency-associated transcript (LAT) region, a portion of the genome that has been associated with efficient reactivation. Here, we demonstrate that the deletion of the seed sequences of these miRNAs results in a severe reduction in reactivation of HSV-1 in the mouse and rabbit models. These results suggest a linkage between these miRNAs and reactivation.

Novel role of SARM1 mediated axonal degeneration in the pathogenesis of rabies.

Neurotropic viral infections continue to pose a serious threat to human and animal wellbeing. Host responses combatting the invading virus in these infections often cause irreversible damage to the nervous system, resulting in poor prognosis. Rabies is the most lethal neurotropic virus, which specifically infects neurons and spreads through the host nervous system by retrograde axonal transport. The key pathogenic mechanisms associated with rabies infection and axonal transmission in neurons remains unclear. Here we studied the pathogenesis of different field isolates of lyssavirus including rabies using ex-vivo model systems generated with mouse primary neurons derived from the peripheral and central nervous systems. In this study, we show that neurons activate selective and compartmentalized degeneration of their axons and dendrites in response to infection with different field strains of lyssavirus. We further show that this axonal degeneration is mediated by the loss of NAD and calpain-mediated digestion of key structural proteins such as MAP2 and neurofilament. We then analysed the role of SARM1 gene in rabies infection, which has been shown to mediate axonal self-destruction during injury. We show that SARM1 is required for the accelerated execution of rabies induced axonal degeneration and the deletion of SARM1 gene significantly delayed axonal degeneration in rabies infected neurons. Using a microfluidic-based ex-vivo neuronal model, we show that SARM1-mediated axonal degeneration impedes the spread of rabies virus among interconnected neurons. However, this neuronal defense mechanism also results in the pathological loss of axons and dendrites. This study therefore identifies a potential host-directed mechanism behind neurological dysfunction in rabies infection. This study also implicates a novel role of SARM1 mediated axonal degeneration in neurotropic viral infection.

Transmission Immunoelectron Microscopy of Herpes Simplex Virus-1-Infected Dorsal Root Ganglia Neurons Sectioned in Growth Plane.

Transmission immunoelectron microscopy allows for the ultrastructural detection and localization of herpes simplex virus-1 (HSV-1) particles and viral proteins within the infected cell and their relation to the cell cytoskeleton, cellular proteins, vesicles, membranes, and organelles. For the successful application of immunoelectron microscopy, preservation of cell ultrastructure and of epitope antigenicity is essential during sample preparation. This chapter describes the use of chemical fixation followed by rapid cooling of HSV-1 infected sensory neurons in the presence of sucrose as a cryoprotectant to achieve optimal preservation of cell morphology and the use of freeze substitution and resin polymerization at low temperatures for preservation of protein antigenicity. In order to examine HSV-1 infection in the specialized compartments of the neurons (cell body, axons, and growth cones), neurons cultured on plastic coverslips are flat embedded prior to resin polymerization. Overall, this method allows for the ultrathin sectioning and immunogold labeling of the neurons and their axons in growth plane.

The Use of Microfluidic Neuronal Devices to Study the Anterograde Axonal Transport of Herpes Simplex Virus-1.

Understanding how herpes simplex virus-1 (HSV-1) interacts with different parts of the neuron is fundamental in understanding the mechanisms behind HSV-1 transport during primary and recurrent infections. In this chapter, we describe a unique neuronal culture system that is capable of compartmentalizing neuronal cell bodies from their axons to study the transport of HSV-1 along axons. The ability to separate neuronal cell bodies and axons provides a unique model to investigate the mechanisms used by HSV-1 for viral transport, assembly, and exit from different parts of the neuron.

Modulation of Voltage-Gated Sodium Channel Activity in Human Dorsal Root Ganglion Neurons by Herpesvirus Quiescent Infection.

The molecular mechanisms of pain associated with alphaherpesvirus latency are not clear. We hypothesize that the voltage-gated sodium channels (VGSC) on the dorsal root ganglion (DRG) neurons controlling electrical impulses may have abnormal activity during latent viral infection and reactivation. We used herpes simplex virus 1 (HSV-1) to infect the human DRG-derived neuronal cell line HD10.6 in order to study the establishment and maintenance of viral latency, viral reactivation, and changes in the functional expression of VGSCs. Differentiated cells exhibited robust tetrodotoxin (TTX)-sensitive sodium currents, and acute infection significantly reduced the functional expression of VGSCs within 24 h and completely abolished VGSC activity within 3 days. A quiescent state of infection mimicking latency can be achieved in the presence of acyclovir (ACV) for 7 days followed by 5 days of ACV washout, and then the viruses can remain dormant for another 3 weeks. It was noted that during the establishment of HSV-1 latency, the loss of VGSC activity caused by HSV-1 infection could not be blocked by ACV treatment. However, neurons with continued ACV treatment for another 4 days showed a gradual recovery of VGSC functional expression. Furthermore, the latently infected neurons exhibited higher VGSC activity than controls. The overall regulation of VGSCs by HSV-1 during quiescent infection was proved by increased transcription and possible translation of Nav1.7. Together, these observations demonstrated a very complex pattern of electrophysiological changes during HSV infection of DRG neurons, which may have implications for understanding of the mechanisms of virus-mediated pain linked to latency and reactivation.IMPORTANCE The reactivation of herpesviruses, most commonly varicella-zoster virus (VZV) and pseudorabies virus (PRV), may cause cranial nerve disorder and unbearable pain. Clinical studies have also reported that HSV-1 causes postherpetic neuralgia and chronic occipital neuralgia in humans. The current work meticulously studies the functional expression profile changes of VGSCs during the processes of HSV-1 latency establishment and reactivation using human dorsal root ganglion-derived neuronal HD10.6 cells as an in vitro model. Our results indicated that VGSC activity was eliminated upon infection but steadily recovered during latency establishment and that latent neurons exhibited even higher VGSC activity. This finding advances our knowledge of how ganglion neurons generate uncharacteristic electrical impulses due to abnormal VGSC functional expression influenced by the latent virus.

[Cutaneous Herpesvirus Infection].

Among human herpes viruses, those known to cause neurological symptoms are the herpes simplex virus (HSV) types 1 and 2 and the varicella-zoster virus (VZV), which remain latent in the dorsal root ganglion. HSV causes herpes labialis, genital herpes, etc. VZV causes varicella during the primary infection, and shingles when reactivated. In this review, we present to the typical cases of each disease; in addition, we present several types of diseases that are closely related to neurological diseases.

Mechanisms of Immune Control of Mucosal HSV Infection: A Guide to Rational Vaccine Design.

Herpes Simplex Virus (HSV) is a highly prevalent sexually transmitted infection that aside from causing cold sores and genital lesions, causes complications in the immunocompromised and has facilitated a large proportion of HIV acquisition globally. Despite decades of research, there is no prophylactic HSV vaccine ready for use in humans, leaving many questioning whether a prophylactic vaccine is an achievable goal. A previous HSV vaccine trial did have partial success in decreasing acquisition of HSV2-promising evidence that vaccines can prevent acquisition. However, there is still an incomplete understanding of the immune response pathways elicited by HSV after initial mucosal infection and how best to replicate these responses with a vaccine, such that acquisition and colonization of the dorsal root ganglia could be prevented. Another factor to consider in the rational design of an HSV vaccine is adjuvant choice. Understanding the immune responses elicited by different adjuvants and whether lasting humoral and cell-mediated responses are induced is important, especially when studies of past trial vaccines found that a sufficiently protective cell-mediated response was lacking. In this review, we discuss what is known of the immune control involved in initial herpes lesions and reactivation, including the importance of CD4 and CD8 T cells, and the interplay between innate and adaptive immunity in response to primary infection, specifically focusing on the viral relay involved. Additionally, a summary of previous and current vaccine trials, including the components used, immune responses elicited and the feasibility of prophylactic vaccines looking forward, will also be discussed.

Herpes Simplex Virus 2 in Autonomic Ganglia: Evidence for Spontaneous Reactivation.

Herpes simplex virus 2 (HSV-2) can be transmitted in the presence or absence of lesions, allowing efficient spread among the general population. Recurrent HSV genital lesions are thought to arise from reactivated latent virus in sensory cell bodies of the dorsal root ganglia (DRG). However, HSV-2 has also been found latent in autonomic ganglia. Spontaneous reactivation or a low level of chronic infection could theoretically also occur in these peripheral nervous tissues, contributing to the presence of infectious virus in the periphery and to viral transmission. Use of a recently described, optimized virus with a monomeric mNeonGreen protein fused to viral capsid protein 26 (VP26) permitted detection of reactivating virus in explanted ganglia and cryosections of DRG and the sacral sympathetic ganglia (SSG) from latently infected guinea pigs. Immediate early, early, and late gene expression were quantified by droplet digital reverse transcription-PCR (ddRT-PCR), providing further evidence of viral reactivation not only in the expected DRG but also in the sympathetic SSG. These findings indicate that viral reactivation from autonomic ganglia is a feature of latent viral infection and that these reactivations likely contribute to viral pathogenesis.IMPORTANCE HSV-2 is a ubiquitous important human pathogen that causes recurrent infections for the life of its host. We hypothesized that the autonomic ganglia have important roles in viral reactivation, and this study sought to determine whether this is correct in the clinically relevant guinea pig vaginal infection model. Our findings indicate that sympathetic ganglia are sources of reactivating virus, helping explain how the virus causes lifelong recurrent disease.

Cryopreservation of Canine Primary Dorsal Root Ganglion Neurons and Its Impact upon Susceptibility to Paramyxovirus Infection.

Canine dorsal root ganglion (DRG) neurons, isolated post mortem from adult dogs, could provide a promising tool to study neuropathogenesis of neurotropic virus infections with a non-rodent host spectrum. However, access to canine DRG is limited due to lack of donor tissue and the cryopreservation of DRG neurons would greatly facilitate experiments. The present study aimed (i) to establish canine DRG neurons as an in vitro model for canine distemper virus (CDV) infection; and (ii) to determine whether DRG neurons are cryopreservable and remain infectable with CDV. Neurons were characterized morphologically and phenotypically by light microscopy, immunofluorescence, and functionally, by studying their neurite outgrowth and infectability with CDV. Cryopreserved canine DRG neurons remained in culture for at least 12 days. Furthermore, both non-cryopreserved and cryopreserved DRG neurons were susceptible to infection with two different strains of CDV, albeit only one of the two strains (CDV R252) provided sufficient absolute numbers of infected neurons. However, cryopreserved DRG neurons showed reduced cell yield, neurite outgrowth, neurite branching, and soma size and reduced susceptibility to CDV infection. In conclusion, canine primary DRG neurons represent a suitable tool for investigations upon the pathogenesis of neuronal CDV infection. Moreover, despite certain limitations, cryopreserved canine DRG neurons generally provide a useful and practicable alternative to address questions regarding virus tropism and neuropathogenesis.

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.