Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons.The interactions of the human neurotrophic herpesvirus varicella-zoster virus (VZV) with neurons have proven difficult to study because the virus shows fairly strict human specificity, and small-animal models do not fully recapitulate human disease. In humans, primary VZV infection follows viral inhalation and subsequent systemic delivery to the deep dermis of the skin via hemopoietic cells. In the course of the resulting disease (chickenpox), VZV infects sensory and sympathetic ganglion neurons, where it establishes a long period of latency. The infection of neurons may take place in the ganglia by circulating VZV-infected lymphocytes, or by virus infecting cutaneous nerve endings being retrogradely transported in the axon to the neuronal somata, as is the case with herpes simplex virus (HSV). VZV reactivation often leads to herpes zoster (shingles), a disease that is frequently associated with severe, debilitating, and often long-lasting intractable pain (postherpetic neuralgia) that is more often than not refractory to therapy.Few model systems of neuronal VZV infection have been developed. Two in vitro models are VZV infection of dissociated human neurons and intact human fetal dorsal root ganglia (DRG) (8, 9, 10). These studies have shed some light on VZV-neuronal interactions, demonstrating, for example, that VZV exerts antiapoptotic activities in neurons in the short term (maximum, 5 days) and that, unlike infected fibroblasts, infectious VZV is released from neurons.A human fetal DRG-SCID mouse model (22, 29; reviewed in reference 30) has al...

Varicella zoster virus (VZV) latency in sensory and autonomic neurons has remained enigmatic and difficult to study, and experimental reactivation has not yet been achieved. We have previously shown that human embryonic stem cell (hESC)-derived neurons are permissive to a productive and spreading VZV infection. We now demonstrate that hESC-derived neurons can also host a persistent non-productive infection lasting for weeks which can subsequently be reactivated by multiple experimental stimuli. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. VZV DNA and low levels of viral transcription were detectable by qPCR for up to seven weeks. Quiescently-infected human neuronal cultures were induced to undergo renewed viral gene and protein expression by growth factor removal or by inhibition of PI3-Kinase activity. Strikingly, incubation of cultures induced to reactivate at a lower temperature (34°C) resulted in enhanced VZV reactivation, resulting in spreading, productive infections. Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. These experiments establish a powerful new system for modeling the VZV latent state, and reveal a potential role for temperature in VZV reactivation and disease.

g Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens. V aricella-zoster virus (VZV), upon encountering a naïve host, causes a primary infection commonly known as chickenpox (varicella) (1, 4, 5). The disease is generally considered mild and self-resolving even in the absence of treatment (2), although occasionally it has severe and lethal consequences (9, 23). The virus reaches sensory nerve ganglia, where it remains latent for life, unless temporary or permanent immunosuppressive conditions within the host facilitate its reactivation as shingles or herpes zoster (HZ) affecting thoracic, cranial, or lumbosacral dermatomes. Many patients report excruciating and relentless pain during HZ episodes (16,34,36,44). The reactivation is sometimes associated with postherpetic neuralgia (PHN), a severe pain along the affected sensory nerves that can linger for years even after the herpetic rash resolves (4). The drug treatments available to date against VZV-elicited diseases are useful only in alleviating some of the symptoms and in shortening the disease duration but cannot clear the virus or prevent establishment of latency (27,30). PHN is difficult to manage, especially in the elderly, who frequently suffer from other age-related conditions, and the use of the standard PHN treatment, including tricyclic antidepressants, anticonvulsants, and opioids, can be hazardous (16,36).Chickenpox was a ubiquitous childhood disease before the anti-VZV vaccination was mandated in 1995 in the United States. Since then, the numbers of hospitalizati...

The vascular endothelial growth factor (VEGF) induces pathological angiogenetic ocular diseases. It is a scientific challenge to develop carriers for the controlled release of inhibitors for VEGF present in the back of the eye domain. Carbon dots (C‐dots) functionalized with the VEGF aptamer are introduced and the hybrid nanoparticles are used for ocular nanomedicine. The C‐dots are applied as effective carriers of the anti‐VEGF aptamer across the cornea, yielding therapeutic levels upon topical administration. The hybrids show no toxicity for both in vitro and in vivo murine animal model, and further enable noninvasive intraocular concentration monitoring through the C‐dots inherent fluorescence. In addition, the hybrid C‐dots effectively inhibit VEGF‐stimulated angiogenesis in choroidal blood vessels. This inhibition is comparable to two commercially available anti‐VEGF drugs, bevacizumab and aflibercept. The hybrid aptamer‐modified C‐dots provide a versatile nanomaterial to treat age‐related macular degeneration and diabetic retinopathy.

bPluripotent human stem cells are a powerful tool for the generation of differentiated cells that can be used for the study of human disease. We recently demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by the highly host-restricted human alphaherpesvirus varicella-zoster virus (VZV), permitting the interaction of VZV with neurons to be readily evaluated in culture. In the present study, we examine whether pluripotent hESC and neural progenitors at intermediate stages of differentiation are permissive for VZV infection. We demonstrate here that VZV infection is blocked in naïve hESC. A block to VZV replication is also seen when a bacterial artificial chromosome (BAC) containing the VZV genome is transfected into hESC. In contrast, related alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PrV) productively infect naïve hESC in a cell-free manner, and PrV replicates from a BAC transfected into hESC. Neurons differentiate from hESC via neural progenitor intermediates, as is the case in the embryo. The first in vitro stage at which permissiveness of hESC-derived neural precursors to VZV replication is observed is upon formation of "neurospheres," immediately after detachment from the inductive stromal feeder layer. These findings suggest that hESC may be useful in deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication. V aricella-zoster virus (VZV) replication is highly host restricted, growing efficiently only in human cells. In varicella, VZV typically infects and replicates in cutaneous fibroblasts and epidermal cells as well as several types of immune cells. VZV infections of central nervous system (CNS) vasculature are also not uncommonly observed, the virus infecting smooth muscle actinexpressing cells in vessel walls (16). VZV infects effectively primarily in a cell-associated manner in vitro, and it is thought that cell-to-cell spread occurs in most tissues. Cell-free virus is made in vivo by keratinocytes and is present in cutaneous vesicles (8), and released VZV appears to be an important component of T cell-toskin transmission in vivo (reviewed in reference 1).VZV infection of neurons is essential for establishment of latency and the ability to reactivate to cause herpes zoster. Initial neuronal infection by VZV is via cutaneous axons and retrograde transport to peripheral somatic and autonomic ganglia and/or by infected circulating lymphocytes that infiltrate the ganglia (28). VZV replicates in both neurons and ganglionic support cells of somatic and cranial peripheral sensory ganglia both upon initial infection and upon reactivation. Importantly, VZV causes a plethora of CNS diseases (due at least in part to infection of the vasculature) (16) and ocular diseases (reviewed in reference 9). The growth of VZV in neurons and the interactions that govern latency and reactivation have proven to be difficult to study outside the human host because of the species restriction of infection and the limited...

Many herpesviruses express small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), that may play roles in regulating lytic and latent infections. None have yet been reported in varicella-zoster virus (VZV; also known as human herpesvirus 3 [HHV-3]). Here we analyzed next-generation sequencing (NGS) data for small RNAs in VZV-infected fibroblasts and human embryonic stem cell-derived (hESC) neurons. Two independent bioinformatics analyses identified more than 20 VZV-encoded 20- to 24-nucleotide RNAs, some of which are predicted to have stem-loop precursors potentially representing miRNAs. These sequences are perfectly conserved between viruses from three clades of VZV. One NGS-identified sequence common to both bioinformatics analyses mapped to the repeat regions of the VZV genome, upstream of the predicted promoter of the immediate early gene open reading frame 63 (ORF63). This miRNA candidate was detected in each of 3 independent biological repetitions of NGS of RNA from fibroblasts and neurons productively infected with VZV using TaqMan quantitative PCR (qPCR). Importantly, transfected synthetic RNA oligonucleotides antagonistic to the miRNA candidate significantly enhanced VZV plaque growth rates. The presence of 6 additional small noncoding RNAs was also verified by TaqMan qPCR in productively infected fibroblasts and ARPE19 cells. Our results show VZV, like other human herpesviruses, encodes several sncRNAs and miRNAs, and some may regulate infection of host cells. Varicella-zoster virus is an important human pathogen, with herpes zoster being a major health issue in the aging and immunocompromised populations. Small noncoding RNAs (sncRNAs) are recognized as important actors in modulating gene expression, and this study demonstrates the first reported VZV-encoded sncRNAs. Many are clustered to a small genomic region, as seen in other human herpesviruses. At least one VZV sncRNA was expressed in productive infection of neurons and fibroblasts that is likely to reduce viral replication. Since sncRNAs have been suggested to be potential targets for antiviral therapies, identification of these molecules in VZV may provide a new direction for development of treatments for painful herpes zoster.

Aim: Longitudinal tracking of transplanted cells in clinical and experimental setups is crucial for evaluating the efficiency of retinal cell replacement therapies. Materials & methods: Gold nanoparticle-labeled photoreceptor precursors were transplanted in the vitreous and subretinal space of rats and were longitudinally tracked for over a month using optical coherence tomography, computed tomography and fluorescence fundus imaging. Results: This multimodal imaging approach enabled high-resolution long-term tracking and estimation of cell survival in the retina and vitreous, while displaying no toxic effects on the cells or the retina. Conclusion: These observations highlight the applicability of using gold nanoparticle for retinal cell tracking in existing experimental settings and its translational potential for providing more efficient retinal cell therapy in humans.

No abstract