Fetal Neuropathology in Zika Virus-Infected Pregnant Female Rhesus Monkeys.

The development of interventions to prevent congenital Zika syndrome (CZS) has been limited by the lack of an established nonhuman primate model. Here we show that infection of female rhesus monkeys early in pregnancy with Zika virus (ZIKV) recapitulates many features of CZS in humans. We infected 9 pregnant monkeys with ZIKV, 6 early in pregnancy (weeks 6-7 of gestation) and 3 later in pregnancy (weeks 12-14 of gestation), and compared findings with uninfected controls. 100% (6 of 6) of monkeys infected early in pregnancy exhibited prolonged maternal viremia and fetal neuropathology, including fetal loss, smaller brain size, and histopathologic brain lesions, including microcalcifications, hemorrhage, necrosis, vasculitis, gliosis, and apoptosis of neuroprogenitor cells. High-resolution MRI demonstrated concordant lesions indicative of deep gray matter injury. We also observed spinal, ocular, and neuromuscular pathology. Our data show that vascular compromise and neuroprogenitor cell dysfunction are hallmarks of CZS pathogenesis, suggesting novel strategies to prevent and to treat this disease.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

SARS-CoV and SARS-CoV-2 display limited neuronal infection and lack the ability to transmit within synaptically connected axons in stem cell-derived human neurons.

Sarbecoviruses such as SARS and SARS-CoV-2 have been responsible for two major outbreaks in humans, the latter resulting in a global pandemic. While sarbecoviruses primarily cause an acute respiratory infection, they have been shown to infect the nervous system. However, mechanisms of sarbecovirus neuroinvasion and neuropathogenesis remain unclear. In this study, we examined the infectivity and trans-synaptic transmission potential of the sarbecoviruses SARS and SARS-CoV-2 in human stem cell-derived neural model systems. We demonstrated limited ability of sarbecoviruses to infect and replicate in human stem cell-derived neurons. Furthermore, we demonstrated an inability of sarbecoviruses to transmit between synaptically connected human stem cell-derived neurons. Finally, we determined an absence of SARS-CoV-2 infection in olfactory neurons in experimentally infected ferrets. Collectively, this study indicates that sarbecoviruses exhibit low potential to infect human stem cell-derived neurons, lack an ability to infect ferret olfactory neurons, and lack an inbuilt molecular mechanism to utilise retrograde axonal trafficking and trans-synaptic transmission to spread within the human nervous system.

An orthogonal proteomic survey uncovers novel Zika virus host factors.

Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns1. However, the molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK-ERK and ATM-ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.

Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration.

In order to understand whether early epigenetic mechanisms instruct the long-term behavior of neural stem cells (NSCs) and their progeny, we examined Uhrf1 (ubiquitin-like PHD ring finger-1; also known as Np95), as it is highly expressed in NSCs of the developing brain and rapidly down-regulated upon differentiation. Conditional deletion of Uhrf1 in the developing cerebral cortex resulted in rather normal proliferation and neurogenesis but severe postnatal neurodegeneration. During development, deletion of Uhrf1 lead to global DNA hypomethylation with a strong activation of the intracisternal A particle (IAP) family of endogenous retroviral elements, accompanied by an increase in 5-hydroxymethylcytosine. Down-regulation of Tet enzymes rescued the IAP activation in Uhrf1 conditional knockout (cKO) cells, suggesting an antagonistic interplay between Uhrf1 and Tet on IAP regulation. As IAP up-regulation persists into postnatal stages in the Uhrf1 cKO mice, our data show the lack of means to repress IAPs in differentiating neurons that normally never express Uhrf1 The high load of viral proteins and other transcriptional deregulation ultimately led to postnatal neurodegeneration. Taken together, these data show that early developmental NSC factors can have long-term effects in neuronal differentiation and survival. Moreover, they highlight how specific the consequences of widespread changes in DNA methylation are for certain classes of retroviral elements.

Nitric Oxide Attenuates Human Cytomegalovirus Infection yet Disrupts Neural Cell Differentiation and Tissue Organization.

Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus that is asymptomatic in healthy individuals but can cause serious disease in immunocompromised patients. HCMV is also the leading cause of virus-mediated birth defects. Many of these defects manifest within the central nervous system and include microcephaly, sensorineural hearing loss, and cognitive developmental delays. Nitric oxide is a critical effector molecule produced as a component of the innate immune response during infection. Congenitally infected fetal brains show regions of brain damage, including necrotic foci with infiltrating macrophages and microglia, cell types that produce nitric oxide during infection. Using a 3-dimensional cortical organoid model, we demonstrate that nitric oxide inhibits HCMV spread and simultaneously disrupts neural rosette structures, resulting in tissue disorganization. Nitric oxide also attenuates HCMV replication in 2-dimensional cultures of neural progenitor cells (NPCs), a prominent cell type in cortical organoids that differentiate into neurons and glial cells. The multipotency factor SOX2 was decreased during nitric oxide exposure, suggesting that early neural differentiation is affected. Nitric oxide also reduced maximal mitochondrial respiration in both uninfected and infected NPCs. We determined that this reduction likely influences neural differentiation, as neurons (Tuj1+ GFAP- Nestin-) and glial populations (Tuj1- GFAP+ Nestin-) were reduced following differentiation. Our studies indicate a prominent, immunopathogenic role of nitric oxide in promoting developmental defects within the brain despite its antiviral activity during congenital HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of virus-mediated congenital birth defects. Congenitally infected infants can have a variety of symptoms manifesting within the central nervous system. The use of 3-dimensional (3-D) cortical organoids to model infection of the fetal brain has advanced the current understanding of development and allowed broader investigation of the mechanisms behind disease. However, the impact of the innate immune molecule nitric oxide during HCMV infection has not been explored in neural cells or cortical 3-D models. Here, we investigated the effect of nitric oxide on cortical development during HCMV infection. We demonstrate that nitric oxide plays an antiviral role during infection yet results in disorganized cortical tissue. Nitric oxide contributes to differentiation defects of neuron and glial cells from neural progenitor cells despite inhibiting viral replication. Our results indicate that immunopathogenic consequences of nitric oxide during congenital infection promote developmental defects that undermine its antiviral activity.

Zika Virus Induces Mitotic Catastrophe in Human Neural Progenitors by Triggering Unscheduled Mitotic Entry in the Presence of DNA Damage While Functionally Depleting Nuclear PNKP.

Vertical transmission of Zika virus (ZIKV) leads with high frequency to congenital ZIKV syndrome (CZS), whose worst outcome is microcephaly. However, the mechanisms of congenital ZIKV neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC or cortical neurons. NPC replicate fast, requiring efficient DNA damage responses to ensure genome stability. Like congenital ZIKV infection, mutations in the polynucleotide 5'-kinase 3'-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, result in recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there were any links between ZIKV and PNKP. Here, we show that two PNKP phosphatase inhibitors or PNKP knockout inhibited ZIKV replication. PNKP relocalized from the nucleus to the cytoplasm in infected cells, colocalizing with the marker of ZIKV replication factories (RF) NS1 and resulting in functional nuclear PNKP depletion. Although infected NPC accumulated DNA damage, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV also induced activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, supporting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors participating in ZIKV replication in NPC, and pathogenesis to neural progenitor cells. IMPORTANCE The 2015-2017 Zika virus (ZIKV) outbreak in Brazil and subsequent international epidemic revealed the strong association between ZIKV infection and congenital malformations, mostly neurodevelopmental defects up to microcephaly. The scale and global expansion of the epidemic, the new ZIKV outbreaks (Kerala state, India, 2021), and the potential burden of future ones pose a serious ongoing risk. However, the cellular and molecular mechanisms resulting in microcephaly remain incompletely understood. Here, we show that ZIKV infection of neuronal progenitor cells results in cytoplasmic sequestration of an essential DNA repair protein itself associated with microcephaly, with the consequent accumulation of DNA damage, together with an unscheduled activation of cytoplasmic CDK1/Cyclin A complexes in the presence of DNA damage. These alterations result in mitotic catastrophe of neuronal progenitors, which would lead to a depletion of cortical neurons during development.

Heparin Protects Human Neural Progenitor Cells from Zika Virus-Induced Cell Death While Preserving Their Differentiation into Mature Neuroglial Cells.

Zika virus (ZIKV) is an arbovirus member of the Flaviviridae family that causes severe congenital brain anomalies in infected fetuses. The key target cells of ZIKV infection, human neural progenitor cells (hNPCs), are highly permissive to infection that causes the inhibition of cell proliferation and induces cell death. We have previously shown that pharmaceutical-grade heparin inhibits virus-induced cell death with negligible effects on in vitro virus replication in ZIKV-infected hNPCs at the "high" multiplicity of infection (MOI) of 1. Here, we show that heparin inhibits formation of ZIKV-induced intracellular vacuoles, a signature of paraptosis, and inhibits necrosis and apoptosis of hNPCs grown as neurospheres (NS). To test whether heparin preserved the differentiation of ZIKV-infected hNPCs into neuroglial cells, hNPCs were infected at the MOI of 0.001. In this experimental condition, heparin inhibited ZIKV replication by ca. 2 log10, mostly interfering with virion attachment, while maintaining its protective effect against ZIKV-induced cytopathicity. Heparin preserved differentiation into neuroglial cells of hNPCs that were obtained from either human-induced pluripotent stem cells (hiPSC) or by fetal tissue. Quite surprisingly, multiple additions of heparin to hNPCs enabled prolonged virus replication while preventing virus-induced cytopathicity. Collectively, these results highlight the potential neuroprotective effect of heparin that could serve as a lead compound to develop novel agents for preventing the damage of ZIKV infection on the developing brain. IMPORTANCE ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication. Here, we demonstrate that heparin also exerts antiviral activity against ZIKV replication using a much lower infectious inoculum. Moreover, heparin interferes with different modalities of virus-induced cell death. Finally, heparin-induced prevention of virus-induced NPC death allows their differentiation into neuroglial cells despite the intracellular accumulation of virions. These results highlight the potential use of heparin, or pharmacological agents derived from it, in pregnant women to prevent the devastating effects of ZIKV infection on the developing brain of their fetuses.

Neural progenitor cell pyroptosis contributes to Zika virus-induced brain atrophy and represents a therapeutic target.

Mounting evidence has associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly, which raises global alarm. Nonetheless, mechanisms by which ZIKV disrupts neurogenesis and causes microcephaly are far from being understood. In this study, we discovered direct effects of ZIKV on neural progenitor cell development by inducing caspase-1- and gasdermin D (GSDMD)-mediated pyroptotic cell death, linking ZIKV infection with the development of microcephaly. Importantly, caspase-1 depletion or its inhibitor VX-765 treatment reduced ZIKV-induced inflammatory responses and pyroptosis, and substantially attenuated neuropathology and brain atrophy in vivo. Collectively, our data identify caspase-1- and GSDMD-mediated pyroptosis in neural progenitor cells as a previously unrecognized mechanism for ZIKV-related pathological effects during neural development, and also provide treatment options for ZIKV-associated diseases.

Herpes simplex virus type 1 infection leads to neurodevelopmental disorder-associated neuropathological changes.

Neonatal herpes simplex virus type 1 (HSV-1) infections contribute to various neurodevelopmental disabilities and the subsequent long-term neurological sequelae into the adulthood. However, further understanding of fetal brain development and the potential neuropathological effects of the HSV-1 infection are hampered by the limitations of existing neurodevelopmental models due to the dramatic differences between humans and other mammalians. Here we generated in vitro neurodevelopmental disorder models including human induced pluripotent stem cell (hiPSC)-based monolayer neuronal differentiation, three-dimensional (3D) neuroepithelial bud, and 3D cerebral organoid to study fetal brain development and the potential neuropathological effects induced by the HSV-1 infections. Our results revealed that the HSV-1-infected neural stem cells (NSCs) exhibited impaired neural differentiation. HSV-1 infection led to dysregulated neurogenesis in the fetal neurodevelopment. The HSV-1-infected brain organoids modelled the pathological features of the neurodevelopmental disorders in the human fetal brain, including the impaired neuronal differentiation, and the dysregulated cortical layer and brain regionalization. Furthermore, the 3D cerebral organoid model showed that HSV-1 infection promoted the abnormal microglial activation, accompanied by the induction of inflammatory factors, such as TNF-α, IL-6, IL-10, and IL-4. Overall, our in vitro neurodevelopmental disorder models reconstituted the neuropathological features associated with HSV-1 infection in human fetal brain development, providing the causal relationships that link HSV biology with the neurodevelopmental disorder pathogen hypothesis.

Human cytomegalovirus infection is associated with increased expression of the lissencephaly gene PAFAH1B1 encoding LIS1 in neural stem cells and congenitally infected brains.

Congenital infection of the central nervous system by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae, including mental retardation or neurodevelopmental abnormalities. The most severe complications include smooth brain or polymicrogyria, which are both indicative of abnormal migration of neural cells, although the underlying mechanisms remain to be determined. To gain better insight on the pathogenesis of such sequelae, we assessed the expression levels of a set of neurogenesis-related genes, using HCMV-infected human neural stem cells derived from embryonic stem cells (NSCs). Among the 84 genes tested, we found dramatically increased expression of the gene PAFAH1B1, encoding LIS1 (lissencephaly-1), in HCMV-infected versus uninfected NSCs. Consistent with these findings, western blotting and immunofluorescence analyses confirmed the increased levels of LIS1 in HCMV-infected NSCs at the protein level. We next assessed the migratory abilities of HCMV-infected NSCs and observed that infection strongly impaired the migration of NSCs, without detectable effect on their proliferation. Moreover, we observed increased immunostaining for LIS1 in brains of congenitally infected fetuses, but not in control samples, highlighting the clinical relevance of our findings. Of note, PAFAH1B1 mutations (resulting in either haploinsufficiency or gain of function) are primary causes of hereditary neurodevelopmental diseases. Notably, mutations resulting in PAFAH1B1 haploinsufficiency cause classic lissencephaly. Taken together, our findings suggest that PAFAH1B1 is a critical target of HCMV infection. They also shine a new light on the pathophysiological basis of the neurological outcomes of congenital HCMV infection, by suggesting that defective neural cell migration might contribute to the pathogenesis of the neurodevelopmental sequelae of infection. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Brazilian Zika virus strain causes birth defects in experimental models.

Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys. Until the twentieth century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash, arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKV(BR)) strain causes birth defects remains absent. Here we demonstrate that ZIKV(BR) infects fetuses, causing intrauterine growth restriction, including signs of microcephaly, in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. We also report that the infection of human brain organoids results in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKV(BR) crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, and impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKV(BR) outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human neurodevelopment.

Fetal Rhesus Monkey First Trimester Zika Virus Infection Impacts Cortical Development in the Second and Third Trimesters.

Zika virus is a teratogen similar to other neurotropic viruses, notably cytomegalovirus and rubella. The goal of these studies was to address the direct impact of Zika virus on fetal development by inoculating early gestation fetal rhesus monkeys using an ultrasound-guided approach (intraperitoneal vs. intraventricular). Growth and development were monitored across gestation, maternal samples collected, and fetal tissues obtained in the second trimester or near term. Although normal growth and anatomical development were observed, significant morphologic changes were noted in the cerebral cortex at 3-weeks post-Zika virus inoculation including massive alterations in the distribution, density, number, and morphology of microglial cells in proliferative regions of the fetal cerebral cortex; an altered distribution of Tbr2+ neural precursor cells; increased diameter and volume of blood vessels in the cortical proliferative zones; and a thinner cortical plate. At 3-months postinoculation, alterations in morphology, distribution, and density of microglial cells were also observed with an increase in blood vessel volume; and a thinner cortical plate. Only transient maternal viremia was observed but sustained maternal immune activation was detected. Overall, these studies suggest persistent changes in cortical structure result from early gestation Zika virus exposure with durable effects on microglial cells.

Genome-wide CRISPR screen for Zika virus resistance in human neural cells.

Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention.

Amyloid precursor protein is a restriction factor that protects against Zika virus infection in mammalian brains.

Zika virus (ZIKV) is a neurotropic flavivirus that causes several diseases including birth defects such as microcephaly. Intrinsic immunity is known to be a frontline defense against viruses through host anti-viral restriction factors. Limited knowledge is available on intrinsic immunity against ZIKV in brains. Amyloid precursor protein (APP) is predominantly expressed in brains and implicated in the pathogenesis of Alzheimer's diseases. We have found that ZIKV interacts with APP, and viral infection increases APP expression via enhancing protein stability. Moreover, we identified the viral peptide, HGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGL, which is capable of en-hancing APP expression. We observed that aging brain tissues with APP had protective effects on ZIKV infection by reducing the availability of the viruses. Also, knockdown of APP expression or blocking ZIKV-APP interactions enhanced ZIKV replication in human neural progenitor/stem cells. Finally, intracranial infection of ZIKV in APP-null neonatal mice resulted in higher mortality and viral yields. Taken together, these findings suggest that APP is a restriction factor that protects against ZIKV by serving as a decoy receptor, and plays a protective role in ZIKV-mediated brain injuries.

Extracellular vesicles regulate gap junction-mediated intercellular communication and HIV-1 infection of human neural progenitor cells.

Human immunodeficiency virus-1 (HIV-1) has been shown to cross the blood-brain barrier and cause HIV-associated neurocognitive disorders (HAND) through a process that may involve direct or indirect interactions with the central nervous system (CNS) cells and alterations of amyloid ß (Aß) homeostasis. The present study focused on the mechanisms of HIV-1 infecting human neural progenitor cells (hNPCs) and affecting NPC intercellular communications with human brain endothelial cells (HBMEC). Despite the lack of the CD4 receptor, hNPCs were effectively infected by HIV-1 via a mechanism involving the chemokine receptors, CXCR4 and CCR5. HIV-1 infection increased expression of connexin-43 (Cx43), phosphorylated Cx43 (pCx43), and pannexin 2 (Panx2) protein levels in hNPCs, suggesting alterations in gap-junction (GJ) and pannexin channel communication. Indeed, a functional GJ assay indicated an increase in communication between HIV-infected hNPCs and non-infected HBMEC. We next analyzed the impact of HBMEC-derived extracellular vesicles (EVs) and EVs carrying Aß (EV-Aß) on the expression of Cx43, pCx43, and Panx2 in HIV-1 infected and non-infected hNPCs. Exposure to EV-Aß resulted in significant reduction of Cx43 and pCx43 protein expression in non-infected hNPCs when compared to EV controls. Interestingly, EV-Aß treatment significantly increased levels of Cx43, pCx43, and Panx2 in HIV-1-infected hNPCs when compared to non-infected controls. These results were confirmed in a GJ functional assay and an ATP release assay, which is an indicator of connexin hemichannel and/or pannexin channel functions. Overall, the current study demonstrates the importance of hNPCs in HIV-1 infection and indicates that intercellular communications between infected hNPCs and HBMEC can be effectively modulated by EVs carrying Aß as their cargo.

Patterns of Herpes Simplex Virus 1 Infection in Neural Progenitor Cells.

Herpes simplex virus 1 (HSV-1) can induce damage in brain regions that include the hippocampus and associated limbic structures. These neurogenic niches are important because they are associated with memory formation and are highly enriched with neural progenitor cells (NPCs). The susceptibility and fate of HSV-1-infected NPCs are largely unexplored. We differentiated human induced pluripotent stem cells (hiPSCs) into NPCs to generate two-dimensional (2D) and three-dimensional (3D) culture models to examine the interaction of HSV-1 with NPCs. Here, we show that (i) NPCs can be efficiently infected by HSV-1, but infection does not result in cell death of most NPCs, even at high multiplicities of infection (MOIs); (ii) limited HSV-1 replication and gene expression can be detected in the infected NPCs; (iii) a viral silencing mechanism is established in NPCs exposed to the antivirals (E)-5-(2-bromovinyl)-2'-deoxyuridine (5BVdU) and alpha interferon (IFN-α) and when the antivirals are removed, spontaneous reactivation can occur at low frequency; (iv) HSV-1 impairs the ability of NPCs to migrate in a dose-dependent fashion in the presence of 5BVdU plus IFN-α; and (v) 3D cultures of NPCs are less susceptible to HSV-1 infection than 2D cultures. These results suggest that NPC pools could be sites of HSV-1 reactivation in the central nervous system (CNS). Finally, our results highlight the potential value of hiPSC-derived 3D cultures to model HSV-1-NPC interaction.IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model the interaction of HSV-1 with NPCs, which reside in the neurogenic niches of the CNS and play a fundamental role in adult neurogenesis. Herein, we provide evidence that in NPCs infected at an MOI as low as 0.001, HSV-1 can establish a latent state, suggesting that (i) a variant of classical HSV-1 latency can be established during earlier stages of neuronal differentiation and (ii) neurogenic niches in the brain may constitute additional sites of viral reactivation. Lytic HSV-1 infections impaired NPC migration, which represents a critical step in neurogenesis. A difference in susceptibility to HSV-1 infection between two-dimensional (2D) and three-dimensional (3D) NPC cultures was observed, highlighting the potential value of 3D cultures for modeling host-pathogen interactions. Together, our results are relevant in light of observations relating HSV-1 infection to postencephalitic cognitive dysfunction.

Human induced pluripotent stem cell-derived glial cells and neural progenitors display divergent responses to Zika and dengue infections.

Maternal Zika virus (ZIKV) infection during pregnancy is recognized as the cause of an epidemic of microcephaly and other neurological anomalies in human fetuses. It remains unclear how ZIKV accesses the highly vulnerable population of neural progenitors of the fetal central nervous system (CNS), and which cell types of the CNS may be viral reservoirs. In contrast, the related dengue virus (DENV) does not elicit teratogenicity. To model viral interaction with cells of the fetal CNS in vitro, we investigated the tropism of ZIKV and DENV for different induced pluripotent stem cell-derived human cells, with a particular focus on microglia-like cells. We show that ZIKV infected isogenic neural progenitors, astrocytes, and microglia-like cells (pMGLs), but was only cytotoxic to neural progenitors. Infected glial cells propagated ZIKV and maintained ZIKV load over time, leading to viral spread to susceptible cells. DENV triggered stronger immune responses and could be cleared by neural and glial cells more efficiently. pMGLs, when cocultured with neural spheroids, invaded the tissue and, when infected with ZIKV, initiated neural infection. Since microglia derive from primitive macrophages originating in proximity to the maternal vasculature, they may act as a viral reservoir for ZIKV and establish infection of the fetal brain. Infection of immature neural stem cells by invading microglia may occur in the early stages of pregnancy, before angiogenesis in the brain rudiments. Our data are also consistent with ZIKV and DENV affecting the integrity of the blood-brain barrier, thus allowing infection of the brain later in life.

Zika Virus: A New Therapeutic Candidate for Glioblastoma Treatment.

Glioblastoma (GBM) is the most aggressive among the neurological tumors. At present, no chemotherapy or radiotherapy regimen is associated with a positive long-term outcome. In the majority of cases, the tumor recurs within 32-36 weeks of initial treatment. The recent discovery that Zika virus (ZIKV) has an oncolytic action against GBM has brought hope for the development of new therapeutic approaches. ZIKV is an arbovirus of the Flaviviridae family, and its infection during development has been associated with central nervous system (CNS) malformations, including microcephaly, through the targeting of neural stem/progenitor cells (NSCs/NPCs). This finding has led various groups to evaluate ZIKV's effects against glioblastoma stem cells (GSCs), supposedly responsible for GBM onset, progression, and therapy resistance. While preliminary data support ZIKV tropism toward GSCs, a more accurate study of ZIKV mechanisms of action is fundamental in order to launch ZIKV-based clinical trials for GBM patients.