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Infection with herpes simplex virus 1 (HSV-1) occurs in over half the global population, causing recurrent orofacial and/or genital lesions. Individual strains of HSV-1 demonstrate differences in neurovirulence in vivo, suggesting that viral genetic differences may impact phenotype. Here differentiated SH-SY5Y human neuronal cells were infected with one of three HSV-1 strains known to differ in neurovirulence in vivo. Host and viral RNA were sequenced simultaneously, revealing strain-specific differences in both viral and host transcription in infected neurons. Neuronal morphology and immunofluorescence data highlight the pathological changes in neuronal cytoarchitecture induced by HSV-1 infection, which may reflect host transcriptional changes in pathways associated with adherens junctions, integrin signaling, and others. Comparison of viral protein levels in neurons and epithelial cells demonstrated that a number of differences were neuron-specific, suggesting that strain-to-strain variations in host and virus transcription are cell type-dependent. Together, these data demonstrate the importance of studying virus strain- and cell-type-specific factors that may contribute to neurovirulence in vivo, and highlight the specificity of HSV-1-host interactions., Competing Interests: The authors have declared that no competing interests exist.

Purines are metabolic building blocks essential for all living organisms on earth. De novo purine biosynthesis occurs in the brain and appears to play important roles in neural development. Phosphoribosyl formylglycinamidine synthase (FGAMS, also known as PFAS or FGARAT), a core enzyme involved in the de novo synthesis of purines, may play alternative roles in viral pathogenesis. To date, no thorough investigation of the endogenous expression and localization of de novo purine biosynthetic enzymes has been conducted in human neurons or in virally infected cells. In this study, we characterized expression of FGAMS using multiple neuronal models. In differentiated human SH-SY5Y neuroblastoma cells, primary rat hippocampal neurons, and in whole-mouse brain sections, FGAMS immunoreactivity was distributed within the neuronal cytoplasm. FGAMS immunolabeling in vitro demonstrated extensive distribution throughout neuronal processes. To investigate potential changes in FGAMS expression and localization following viral infection, we infected cells with the human pathogen herpes simplex virus 1. In infected fibroblasts, FGAMS immunolabeling shifted from a diffuse cytoplasmic location to a mainly perinuclear localization by 12 h post-infection. In contrast, in infected neurons, FGAMS localization showed no discernable changes in the localization of FGAMS immunoreactivity. There were no changes in total FGAMS protein levels in either cell type. Together, these data provide insight into potential purine biosynthetic mechanisms utilized within neurons during homeostasis as well as viral infection. Cover Image for this Issue: doi: 10.1111/jnc.14169., (© 2018 International Society for Neurochemistry.)

Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats, including incomplete differentiation, nonhuman origins, or the use of dividing cells that have neuropotential but lack neuronal morphology. In this study, we used a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titers similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However, terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 h postinfection (hpi), suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cyclic GMP-AMP synthase (cGAS) in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in nonepithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry and will be advantageous for the study of neurotropic viruses in vitro IMPORTANCE Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and, in rare cases, encephalitis. Presently, there is no cure available to treat those infected or prevent future transmission. Due to the ability of HSV to cause a persistent, lifelong infection in the peripheral nervous system, the virus remains within the host for life. To better understand the basis of virus-neuron interactions that allow HSV to persist within the host peripheral nervous system, improved neuronal models are required. Here we describe a cost-effective and scalable human neuronal model system that can be used to study many neurotropic viruses, such as HSV, Zika virus, dengue virus, and rabies virus., (Copyright © 2017 American Society for Microbiology.)

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.