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Background: Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. Methods: Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. Results: Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37 °C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death—especially necroptosis—in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. Conclusions: We developed a novel human brain culture model in which the viability of the major brain-resident cells—including neurons, microglia, astrocytes and

Background: Trigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized. Methods: Paired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts. Results: VZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses. Conclusions: The low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.

Herpes zoster is a localized skin infection caused by reactivation of latent varicella-zoster virus. Tissue-resident T cells likely control skin infections. Zoster provides a unique opportunity to determine if focal reinfection of human skin boosts local or disseminated antigen-specific tissue-resident T cells. Here, we show virus-specific T cells are retained over one year in serial samples of rash site and contralateral unaffected skin of individuals recovered from zoster. Consistent with zoster resolution, viral DNA is largely undetectable on skin from day 90 and virus-specific B and T cells decline in blood. In skin, there is selective infiltration and long-term persistence of varicella-zoster virus-specific T cells in the rash site relative to the contralateral site. The skin T cell infiltrates express the canonical tissue-resident T cell markers CD69 and CD103. These findings show that zoster promotes spatially-restricted long-term retention of antigen-specific tissue-resident T cells in previously infected skin.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed direct long-read RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines t

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.