IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a ubiquitous virus and a significant cause of morbidity and some mortality. It is the causative agent of benign cold sores, but it can also cause blindness and life-threatening encephalitis. The success of HSV-1 is largely due to its ability to establish lifelong latent infections in neurons and to occasionally reactivate. The exact mechanisms by which neurons defend against virus infection is poorly understood, but such defense is at least partially mediated by autophagy, an intracellular pathway by which pathogens and other unwanted cargoes are degraded. The study demonstrates and investigates a new autophagic structure that appears to be specific to the interaction between neurotropic herpesviruses and murine primary sensory neurons. This work may therefore have important implications for our understanding of latency and reactivation. Herpes simplex virus 1 (HSV-1) is a highly prevalent human pathogen that establishes lifelong infection in the neurons of sensory and autonomic ganglia (1, 2). Initial infection and lytic replication at mucosal sites are followed by infection of innervating axonal termini of sensory neurons. Virions then travel in a retrograde direction to the soma, where they may replicate or immediately establish latency, depending partly on the infected neuronal subtype (3). Virions resulting from periodic reactivation events travel in an anterograde direction along the axon, allowing reinfection of the oral epithelium, thereby facilitating viral shedding and host-to-host transmission (2).Innate immune responses are critical in controlling virulence of HSV-1 and many other viruses through a variety of antiviral pathways (4-12), and viruses have coevolved to counter these host responses (13)(14)(15)(16)(17)(18)(19)(20). Cells detect the presence of incoming virus through pattern recognition receptors (21, 22) that, in turn, lead to the activation of pivotal transcription factors such as IRF (5, 23) and NF-B (24). These factors subsequently induce the production of type I interferon (IFN), which drives IFN-stimulated gene (ISG) expression (25) through a JAK-STAT-dependent pathway (26,27). This further stimulates production of type I IFN and ISGs to establish an antiviral state that consists of transcriptional and translational arrest, cytokine production, and apoptosis (28,29).Defects in innate immunity are often associated with increased susceptibility to HSV infection in both mice and humans, with frequent neurological sequelae (8,12,(30)(31)(32). That said, the IFNdriven responses of neurons themselves are muted and atypical (12,33). Nondestructive clearance is especially critical for postmitotic adult neurons, a population of irreplaceable cells that is essential for both the success and survival of the host. Indeed, there is mounting evidence that neurons depend on autophagy rather than inflammatory or cell-destructive responses for the control of intracellular pathogens (34-37). Autophagy is a highly conserved response to starvation, during...

Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection.

Microvesicles shedding from cell membrane affect inflammation, apoptosis, and angiogenesis. We hypothesize that microvesicles of women with gestational vascular complications reflect pathophysiological state of the patients and affect their endothelial and trophoblast cell function. Microvesicles of healthy pregnant women, women with gestational hypertension, mild, or severe preeclampsia/toxemia, were characterized, and their effects on early-stage or term trophoblasts and endothelial cells were evaluated using apoptosis, migration, and tube formation assays. Patient subgroups differed significantly only in proteinuria levels, therefore their microvesicles were assessed as 1 group, demonstrating higher levels of inflammatory and angiogenic proteins compared with those of healthy pregnant women. In endothelial cells, microvesicles of healthy pregnant women reduced caspase 3/7 activity, increased migration, and induced tube formation. These processes were suppressed by microvesicles of women with gestational vascular complications. In early-stage trophoblasts, microvesicles of healthy pregnant women decreased apoptosis compared with untreated cells (6±5% versus 13.8±5.8%; P <0.001) and caspase 3/7 activity and induced higher migration (39.7±10.1 versus 20.3±8.3 mm 2 ; P <0.001). This effect was mediated through extracellular signal-regulated kinase pathway. Conversely, microvesicles of women with gestational vascular complications increased term trophoblast apoptosis compared with cells exposed to microvesicles of healthy pregnant women (15.1±3.3% versus 6.5±2.1%; P <0.001) and inhibited early-stage trophoblasts migration (21.4±18.5 versus 39.7±10.1 mm 2 ; P <0.001). In conclusion, microvesicle content and effects on endothelial and trophoblast cells vary according to the physiological/pathological state of a pregnant woman. Microvesicles seem to play a pivotal role in the course of pregnancy, which could potentially result in gestational vascular complications.

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