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Background Drug repurposing is a cost-effective strategy to identify drugs with novel effects. We searched for drugs exhibiting inhibitory activity to Herpes Simplex virus 1 (HSV-1). Our strategy utilized gene expression data generated from HSV-1-infected cell cultures which was paired with drug effects on gene expression. Gene expression data from HSV-1 infected and uninfected neurons were analyzed using BaseSpace Correlation Engine (Illumina®). Based on the general Signature Reversing Principle (SRP), we hypothesized that the effects of candidate antiviral drugs on gene expression would be diametrically opposite (negatively correlated) to those effects induced by HSV-1 infection. Results We initially identified compounds capable of inducing changes in gene expression opposite to those which were consequent to HSV-1 infection. The most promising negatively correlated drugs (Valproic acid, Vorinostat) did not significantly inhibit HSV-1 infection further in African green monkey kidney epithelial cells (Vero cells). Next, we tested Sulforaphane and Menadione which showed effects similar to those caused by viral infections (positively correlated). Intriguingly, Sulforaphane caused a modest but significant inhibition of HSV-1 infection in Vero cells (IC50 = 180.4 µM, p = 0.008), but exhibited toxicity when further explored in human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells. Conclusions These results reveal the limits of the commonly used SRP strategy when applied to the identification of novel antiviral drugs and highlight the necessity to refine the SRP strategy to increase its utility.

The pathogen hypothesis of Alzheimer’s disease (AD) proposes that brain HSV-1 infection is an initial source of amyloid beta (Aβ) peptide-containing amyloid plaque development. Aβ accumulation was reported in HSV-1-infected 2D neuronal cultures and neural stem cell cultures as well as in HSV-1-infected 3D neuronal culture models., Alzheimer's disease is a progressive neurodegenerative disease characterized neuropathologically by the presence of extracellular amyloid plaques composed of fibrillar amyloid beta (Aβ) peptides and intracellular neurofibrillary tangles. Postmortem and in vivo studies implicate herpes simplex virus 1 (HSV-1) infection in the brain as a precipitating factor in disease/pathology initiation. HSV-1 infection of two-dimensional (2D) neuronal cultures causes intracellular accumulation of Aβ42 peptide, but these 2D models do not recapitulate the three-dimensional (3D) architecture of brain tissue. We employed human induced pluripotent stem cells (hiPSCs) to compare patterns of Aβ42 accumulation in HSV-1-infected 2D (neuronal monolayers) and 3D neuronal cultures (brain organoids). Akin to prior studies, HSV-1-infected 2D cultures showed Aβ42 immunoreactivity in cells expressing the HSV-1 antigen ICP4 (ICP4+). Conversely, accumulation of Aβ42 in ICP4+ cells in infected organoids was rarely observed. These results highlight the importance of considering 3D cultures to model host-pathogen interaction. IMPORTANCE The pathogen hypothesis of Alzheimer’s disease (AD) proposes that brain HSV-1 infection is an initial source of amyloid beta (Aβ) peptide-containing amyloid plaque development. Aβ accumulation was reported in HSV-1-infected 2D neuronal cultures and neural stem cell cultures as well as in HSV-1-infected 3D neuronal culture models. The current study extends these findings by showing different patterns of Aβ42 accumulation following HSV-1 infection of 2D compared to 3D neuronal cultures (brain organoids). Specifically, 2D neuronal cultures showed Aβ42 immunoreactivity mainly in HSV-1-infected cells and only rarely in uninfected cells or infected cells exposed to antivirals. Conversely, 3D brain organoids showed accumulation of Aβ42 mainly in noninfected cells surrounding HSV-1-infected cells. We suggest that because brain organoids better recapitulate architectural features of a developing brain than 2D cultures, they are a more suitable model to investigate the involvement of HSV-1 in the onset of AD pathology.

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.

Herpes simplex virus, type 1 (HSV-1) infection is highly prevalent and globally affects approximately 67% of people under age 50. It causes substantial morbidity, including recurrent ocular disease, cold sores, and rare cases of devastating encephalitis. Its pathogenicity stems primarily from the ability of the virus to cause recurrent lytic lesions, a consequence of its ability to persist for life as a latent infection in neurons. Latency is an elusive, poorly understood, and to date, untreatable process that involves a dynamic interplay between the virus and host cells. While considerable knowledge of the HSV-1 latent state has been gleaned from small animal modeling, we are now at the cusp of needing to examine HSV-1 in more human relevant models, as HSV-1 is a human-specific virus, and the underlying mechanisms of latency have presumably evolved over millennia through human host–virus interactions. Neuron-virus studies have been substantially hampered by the absence of satisfactory human models. Human induced pluripotent stem cell (hiPSC) and embryonic stem cell (hESC) technologies are profoundly changing this picture, as they can provide human neuron models to examine the interaction of pathogens with their hosts. They are also aiding in the development of novel therapeutic approaches. Herein, we describe the use of hiPSCs to derive human models of HSV-1 acute and latent infections and to generate human neuronal platforms for antiviral drug screening.

Schizophrenia (SZ) is a common, potentially debilitating disorder. Over 70% of risk for SZ at the population level is attributable to genetic factors. Large-scale genome-wide association studies (GWAS) have revealed risk attributable to over 170 genomic loci, with varied effect sizes. Statistically, the most significant risk locus maps to the major histocompatibility complex (MHC) region on chromosome 6 (odds ratio ~1.28). One of the associated MHC loci involved the complement C4 gene. There is population-wide variation in C4 gene copy number, and SZ risk rises with the number of C4 genes that an individual’s genome encodes. C4 is a component of an immunologic defense mechanism that predates antibody-based adaptive immunity. In mammals, complement proteins also participate in neurodevelopment. Based on rodent model studies, it has been proposed that the C4-related risk for SZ could be explained by aberrant synaptic pruning—a developmental process also implicated in SZ risk. We review the synaptic pruning hypothesis and propose additional mechanisms to explain the C4-SZ link, including disruption in classical immunologic processes and changes in human endogenous retroviral sequences associated with C4. The evidence supporting these hypotheses is discussed, and empirical investigations are proposed.

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., 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.

The synthesis of a lead anti-viral cyclopropyl carboxy acyl hydrazone 4F17 (5) and three sequential arrays of structural analogues along with the initial assessment and optimization of the antiviral pharmacophore against the herpes simplex virus type 1 (HSV-1) are reported.

Acyclovir (ACV) is an effective antiviral agent for treating lytic Herpes Simplex virus, type 1 (HSV-1) infections, and it has dramatically reduced the mortality rate of herpes simplex encephalitis. However, HSV-1 resistance to ACV and its derivatives is being increasingly documented, particularly among immunocompromised individuals. The burgeoning drug resistance compels the search for a new generation of more efficacious anti-herpetic drugs. We have previously shown that trans-dihydrolycoricidine (R430), a lycorane-type alkaloid derivative, effectively inhibits HSV-1 infections in cultured cells. We now report that R430 also inhibits ACV-resistant HSV-1 strains, accompanied by global inhibition of viral gene transcription and enrichment of H3K27me3 methylation on viral gene promoters. Furthermore, we demonstrate that R430 prevents HSV-1 reactivation from latency in an ex vivo rodent model. Finally, among a panel of DNA viruses and RNA viruses, R430 inhibited Zika virus with high therapeutic index. Its therapeutic index is comparable to standard antiviral drugs, though it has greater toxicity in non-neuronal cells than in neuronal cells. Synthesis of additional derivatives could enable more efficacious antivirals and the identification of active pharmacophores.

Zika virus (ZIKV) infection during pregnancy is associated with microcephaly, a congenital malformation resulting from neuroinflammation and direct effects of virus replication on the developing central nervous system (CNS). However, the exact changes in the affected CNS remain unknown. Here, we show by transcriptome analysis (at 48 h post-infection) and multiplex immune profiling that human induced-neuroprogenitor stem cells (hiNPCs) respond to ZIKV infection with a strong induction of type-I interferons (IFNs) and several type-I IFNs stimulated genes (ISGs), notably cytokines and the pro-apoptotic chemokines CXCL9 and CXCL10. By comparing the inflammatory profile induced by a ZIKV Brazilian strain with an ancestral strain isolated from Cambodia in 2010, we observed that the response magnitude differs among them. Compared to ZIKV/Cambodia, the experimental infection of hiNPCs with ZIKV/Brazil resulted in a diminished induction of ISGs and lower induction of several cytokines (IFN-α, IL-1α/β, IL-6, IL-8, and IL-15), consequently favoring virus replication. From ZIKV-confirmed infant microcephaly cases, we detected a similar profile characterized by the presence of IFN-α, CXCL10, and CXCL9 in cerebrospinal fluid (CSF) samples collected after birth, evidencing a sustained CNS inflammation. Altogether, our data suggest that the CNS may be directly affected due to an unbalanced and chronic local inflammatory response, elicited by ZIKV infection, which contributes to damage to the fetal brain.

A recent report suggested Complement 4 (C4A) gene copy numbers (GCN) as risk factors for schizophrenia. Rodent model showed association of C4 with synaptic pruning suggesting its pathophysiological significance (Sekar, A. et al. (2016)). We, therefore, predicted that C4A GCN would be positively correlated with neuropil contraction in the human brain among schizophrenia patients showing more prominent correlations in ventral regions among young adults and dorsal regions among adolescents since neuromaturation progresses dorsoventrally. Whole-brain, multi-voxel, in vivo phosphorus magnetic resonance spectroscopy (31P MRS) assessed neuropil changes by estimating levels of membrane phospholipid (MPL) precursors and catabolites. Increased MPL catabolites and/or decreased MPL precursors indexed neuropil contraction. Digital droplet PCR-based assay was used to estimate C4A and C4B GCN. We evaluated two independent cohorts (young adult-onset early-course schizophrenia (YASZ = 15) and adolescent-onset schizophrenia (AOSZ = 12) patients), and controls matched for each group, n = 22 and 15, respectively. Separate forward stepwise linear regression models with Akaike information Criterion were built for MPL catabolites and precursors. YASZ cohort: Consistent with the rodent model (Sekar, A. et al. 2016)), C4A GCN positively correlated with neuropil contraction (increased pruning/decreased formation) in the inferior frontal cortex and inferior parietal lobule. AOSZ cohort: C4A GCN positively correlated with neuropil contraction in the dorsolateral prefrontal cortex and thalamus. Exploratory analysis of C4B GCN showed positive correlation with neuropil contraction in the cerebellum and superior temporal gyrus among YASZ while AOSZ showed neuropil contraction in the prefrontal and subcortical structures. Thus, C4A and C4B GCN are associated with neuropil contraction in regions often associated with schizophrenia, and may be neuromaturationally dependent.

Background A variety of neurological disorders including neurodegenerative diseases and infection by neurotropic viruses can cause structural and functional changes in the central nervous system (CNS), resulting in long-term neurological sequelae. An improved understanding of the pathogenesis of these disorders is important for developing efficacious interventions. Human induced pluripotent stem cells (hiPSCs) offer an extraordinary window for modeling pathogen-CNS interactions, and other cellular interactions, in three-dimensional (3D) neuronal cultures that can recapitulate several aspects of in vivo brain tissue. Methods Herein, we describe a prototype of scaffold-free hiPSC-based adherent 3D (A-3D) human neuronal cultures in 96-well plates. To test their suitability for drug screening, A-3D neuronal cultures were infected with herpes simplex virus type 1 (HSV-1) with or without acyclovir. Results The half maximal inhibitory concentration (IC50) of acyclovir was 3.14 μM and 3.12 μM determined using flow cytometry and the CX7 High Content Screening platform, respectively. Conclusions Our A-3D neuronal cultures provide an unprecedented opportunity for high-content drug screening programs to treat human CNS infections. Electronic supplementary material The online version of this article (10.1186/s13287-018-0881-6) contains supplementary material, which is available to authorized users.

Background Herpes simplex virus, type 1 (HSV-1) commonly produces lytic mucosal lesions. It invariably initiates latent infection in sensory ganglia enabling persistent, lifelong infection. Acute HSV-1 encephalitis is rare and definitive evidence of latent infection in the brain is lacking. However, exposure untraceable to encephalitis has been repeatedly associated with impaired working memory and executive functions, particularly among schizophrenia patients. Methods Patterns of HSV-1 infection and gene expression changes were examined in human induced pluripotent stem cell (iPSC)-derived neurons. Separately, differences in blood oxygenation level-dependent (BOLD) responses to working memory challenges using letter n-back tests were investigated using functional magnetic resonance imaging (fMRI) among schizophrenia cases/controls. Results HSV-1 induced lytic changes in iPSC-derived glutamatergic neurons and neuroprogenitor cells. In neurons, HSV-1 also entered a quiescent state following coincubation with antiviral drugs, with distinctive changes in gene expression related to functions such as glutamatergic signaling. In the fMRI studies, main effects of schizophrenia (P = .001) and HSV-1 exposure (1-back, P = 1.76 × 10(-4); 2-back, P = 1.39 × 10(-5)) on BOLD responses were observed. We also noted increased BOLD responses in the frontoparietal, thalamus, and midbrain regions among HSV-1 exposed schizophrenia cases and controls, compared with unexposed persons. Conclusions The lytic/quiescent cycles in iPSC-derived neurons indicate that persistent neuronal infection can occur, altering cellular function. The fMRI studies affirm the associations between nonencephalitic HSV-1 infection and functional brain changes linked with working memory impairment. The fMRI and iPSC studies together provide putative mechanisms for the cognitive impairments linked to HSV-1 exposure.

The Amaryllidaceae alkaloid trans-dihydrolycoricidine 7 and three analogues 8-10 were produced via asymmetric chemical synthesis. Alkaloid 7 proved superior to acyclovir, the current standard for herpes simplex virus, type 1 (HSV-1) infection. Compound 7 potently inhibited lytic HSV-1 infection, significantly reduced HSV-1 reactivation, and more potently inhibited varicella zoster virus (VZV) lytic infection. A configurationally defined (3R)-secondary alcohol at C3 proved crucial for efficacious inhibition of lytic HSV-1 infection.

Herpesvirus infections cause considerable morbidity and mortality through lifelong recurrent cycles of lytic and latent infection in several tissues, including the human nervous system. Acyclovir (ACV) and its prodrug, the current antivirals of choice for herpes simplex virus (HSV) and, to some extent, varicella zoster virus (VZV) infections are nucleoside analogues that inhibit viral DNA replication. Rising viral resistance and the need for more effective second-line drugs have motivated searches for additional antiviral agents, particularly non-nucleoside based agents. We evaluated the antiviral activity of five compounds with predicted lysosomotropic activity using conventional and human induced pluripotent stem cell-derived neuronal (iPSC-neurons) cultures. Their potency and toxicity were compared with ACV and the lysosomotropic agents chloroquine and bafilomycin A1. Out of five compounds tested, micromolar concentrations of 30N12, 16F19, and 4F17 showed antiviral activity comparable to ACV (50μM) during lytic herpes simplex virus type 1 (HSV-1) infections, reduced viral DNA copy number, and reduced selected HSV-1 protein levels. These compounds also inhibited the reactivation of 'quiescent' HSV-1 infection established in iPSC-neurons, but did not inhibit viral entry into host cells. The same compounds had greater potency than ACV against lytic VZV infection; they also inhibited replication of human cytomegalovirus. The anti-herpetic effects of these non-nucleoside agents merit further evaluation in vivo.

Fluorescence in situ hybridization (FISH) with probes representing sheep satellite I and satellite II DNAs shows a different distribution of the two repetitive DNA families in the centromeric region of most chromosomes. The single signal per chromosome produced by the satellite I probe suggests close proximity of this DNA family to the primary constriction. Satellite II produces two separate signals on the sister chromatids, and large blocks of satellite II DNA constitute most of the short arm of all acrocentric chromosomes. We have isolated and sequenced a phage clone containing a junction between discrete blocks of satellite I and satellite II sequences. The junction is characterized by an abrupt juxtaposition of arrays of the two satellites. The possibility that the peculiar structural features of this junction could have a functional significance is discussed.

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.

Roberto Di Maio1, Kommu Naga Mohan2, Brianna Heath3 and Leonardo D’Aiuto4 1 Department of Neurology, University of Pittsburgh, & Ri.Med Foundation, Palermo 2 Department of Molecular and Human Genetics, Baylor College of Medicine 3 Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, 4 Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic 1Italy 2,3,4United States

We have developed a telomere-based episome by large-scale amplification in Escherichia coli cells. This episome consists of a PAC vector in which a 6 Kb sequence, containing an array of telomeric repeats spaced by a synthetic sequence, is tandemly repeated by large-scale multimerization in E. coli. After transfection in human HT1080 cells, the construct, called clone 106, was able to persist in episomal form or integrated into some endogenous chromosomes. Integrations occurred exclusively at the telomeres. Episomes were still present in HT1080 cells after more than 100 days in the absence of selection. Integrations of clone 106 into the telomeric regions were retained only under selective conditions, and when the selection was removed the construct was progressively eliminated from the chromosome. The long-term maintenance of clone 106 into human cells as an episome and its ability to integrate transiently into the telomeres of the host chromosomes suggest that this PAC-based episome is potentially a good candidate vector for gene therapy applications.

We have isolated and characterized a new alphoid probe, named p190.22. Its chromosomal location was investigated using fluorescence in situ hybridization. Under high stringency conditions p190.22 recognizes specifically the centromere of chromosome 22. A chromosome 22-specific alphoid subset has been previously reported in the literature (p22/1∶2.1). The partial sequence and the genomic organization comparison strongly suggests that they recognize distinct subsets both specific for chromosome 22. The comparative mapping of probes p190.22 and p22/1∶2.1 on chimpanzee (PTR and PPA) and gorilla (GGO) chromosomes was investigated. The two probes showed different hybridization results. p190.22, in particular, did not show any hybridization signal in these three species, suggesting a recent evolution.

The causative agent of Severe Acute Respiratory Syndrome (SARS) has been identified as a new type of coronavirus. Here we have examined the ability of adenoviral gene transfer of codon-optimized SARS-CoV strain Urbani structural antigens spike (S) protein S1 fragment, membrane protein (M), and nucleocapsid protein (N) to induce broad immunity in the non-human primate. Six rhesus macaques were immunized intramuscularly with a combination of the three Ad5-SARS-CoV vectors and boosted on day 28. The vaccinated animals all had antibody responses against S1 and T-cell responses against the antigens. Importantly, all six vaccinated animals showed strong neutralizing antibody responses to SARS-CoV infection in vitro. These results demonstrate that an adenoviral-based vaccine can induce strong SARS-CoV-specific immune responses in the monkey, and hold promise for development of a protective vaccine against the SARS etiologic agent.