MOMC-1. Employing the Zika Virus to kill paediatric nervous system tumour cells

Abstract Malignant paediatric nervous system tumours, such as Medulloblastoma, Neuroblastoma and ATRT commonly harbour tumour cells with stem-like features which are highly tumorigenic and resistant to conventional cancer therapies. These tumours can exhibit high lethality and may result in severe sequelae, including cognitive and motor deficits that significantly affect patients’ quality of life. Oncolytic virotherapy is a novel therapy class that exploits viruses that preferentially infect and destroy tumour cells. These viruses present a unique advantage in targeting highly heterogeneous cancers, such as nervous system tumours, as they possess a secondary mechanism of action through which they induce a tumour-specific immune response. Clinical studies employing oncolytic virotherapy have in general reported low toxicity and minimal adverse effects, deeming oncolytic virotherapy as a potentially attractive and safer intervention against paediatric tumours. The Zika virus (ZIKV) is capable of infecting and destroying neural stem-like cancer cells from human embryonal Central Nervous System (CNS) tumours in vitro and in vivo. Infection of CNS tumour cells with ZIKV effectively inhibits tumour metastasis in mice and, in some cases, induces complete tumour remission. Neuroblastoma arises from immature nerve cells and multiple Neuroblastoma cell lines are susceptible to ZIKV infection and oncolysis. These initial findings have demonstrated the potential for a ZIKV-based virotherapy against paediatric nervous system tumours and warrants examination into the molecular mechanisms through which ZIKV executes its oncolytic ability. My research goal is to elucidate the mechanisms which are of paramount importance for ZIKV-induced oncolysis of brain tumour and Neuroblastoma cells. Utilising global expression omics profiling of ZIKV infection and mapping of viral protein-host protein interactions will identify these mechanisms both at the cellular pathway and molecular levels. These collectively will inform our understanding of how we can employ a future ZIKV-based virotherapy against paediatric nervous system tumours.

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Zika Virus Infection Leads to Demyelination and Axonal Injury in Mature CNS Cultures

Viruses ◽

10.3390/v13010091 ◽

2021 ◽

Vol 13 (1) ◽

pp. 91

Author(s):

Verena Schultz ◽

Stephanie L. Cumberworth ◽

Quan Gu ◽

Natasha Johnson ◽

Claire L. Donald ◽

...

Keyword(s):

Nervous System ◽

Neural Development ◽

Zika Virus ◽

Developmental Stages ◽

Cell Types ◽

Neural Cells ◽

Zikv Infection ◽

Axonal Pathology ◽

Time Frames

Understanding how Zika virus (Flaviviridae; ZIKV) affects neural cells is paramount in comprehending pathologies associated with infection. Whilst the effects of ZIKV in neural development are well documented, impact on the adult nervous system remains obscure. Here, we investigated the effects of ZIKV infection in established mature myelinated central nervous system (CNS) cultures. Infection incurred damage to myelinated fibers, with ZIKV-positive cells appearing when myelin damage was first detected as well as axonal pathology, suggesting the latter was a consequence of oligodendroglia infection. Transcriptome analysis revealed host factors that were upregulated during ZIKV infection. One such factor, CCL5, was validated in vitro as inhibiting myelination. Transferred UV-inactivated media from infected cultures did not damage myelin and axons, suggesting that viral replication is necessary to induce the observed effects. These data show that ZIKV infection affects CNS cells even after myelination—which is critical for saltatory conduction and neuronal function—has taken place. Understanding the targets of this virus across developmental stages including the mature CNS, and the subsequent effects of infection of cell types, is necessary to understand effective time frames for therapeutic intervention.

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CImP: Cellular Imprinting Proteomics applied to ocular disorders elicited by Congenital Zika virus Syndrome

10.1101/648600 ◽

2019 ◽

Cited By ~ 1

Author(s):

Livia Rosa-Fernandes ◽

Raquel Hora Barbosa ◽

Maria Luiza B. dos Santos ◽

Claudia B. Angeli ◽

Thiago P. Silva ◽

...

Keyword(s):

Zika Virus ◽

Ocular Surface ◽

Molecular Mechanisms ◽

Membrane Filter ◽

Neurological Diseases ◽

Specific Protein ◽

Protein Markers ◽

Impression Cytology ◽

Zikv Infection ◽

Molecular Alterations

AbstractIMPORTANCEOcular complications in infants with Congenital Zika Syndrome (CZS) have been reported. However, the molecular mechanisms underlying of eye dysfunctions are presently unknown.OBJECTIVEA method (termed Cellular Imprinting Proteomics, CImP) for the identification and quantification of the ocular surface proteome using a minimally invasive membrane filter device is described. Moreover, The CImP method was applied to profile the molecular alterations in the eyes of infants exposed to Zika virus (ZIKV) infection during gestation.DESIGN, SETTINGS AND PARTICIMPANTSThe CImP method was applied to a cohort divided into three conditions: 1) Ctrl (infants with no infectious diseases, n=5). 2) Zikv (infants exposed to ZIKV gestation, with no microcephaly, n=5). 3) ZikvCZS(infants exposed to ZIKV, with microcephaly, n=3). All conditions were age and sex-matched. An improved impression cytology method was used to capture the outermost ocular surface cells. The number of impression cytology membrane collected was: Ctrl (12), Zikv (14) and ZikvCZS(8). Proteins were extracted and analysed using mass spectrometry-based proteomics technology followed by statistical analysis. Parallel reaction monitoring was performed to validate the expression of specific protein markers.RESULTSUsing the CImP method, 2209 proteins were identified on the membrane-captured conjunctiva epithelial cells. Modulation of neutrophil degranulation, cell death, ocular and neurodevelopment pathways are reported in infants with CZS compared to matched controls. Moreover, the molecular pattern of ocular surface cells retrieved from infants infected during the gestation but with no CZS was different from matched controls.CONCLUSIONS AND PERSPECTIVESMolecular alterations in the ocular cell surface associated to ZIKV infection with and without CZS complications are reported for the first time. We predict that this method will be introduced successfully in the study of several neurological diseases with the aim to identify novel diagnostic and therapeutic biomarkers.

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Molecular Mechanisms of Central Nervous System Axonal Regeneration and Remyelination: A Review

International Journal of Molecular Sciences ◽

10.3390/ijms21218116 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8116

Author(s):

Akiko Uyeda ◽

Rieko Muramatsu

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Molecular Mechanisms ◽

Motor Deficits ◽

Cns Injury ◽

Cellular Mechanisms ◽

Cns Regeneration ◽

Cord Injury ◽

Extracellular Environment ◽

Injury Causes

Central nervous system (CNS) injury, including stroke, spinal cord injury, and traumatic brain injury, causes severe neurological symptoms such as sensory and motor deficits. Currently, there is no effective therapeutic method to restore neurological function because the adult CNS has limited capacity to regenerate after injury. Many efforts have been made to understand the molecular and cellular mechanisms underlying CNS regeneration and to establish novel therapeutic methods based on these mechanisms, with a variety of strategies including cell transplantation, modulation of cell intrinsic molecular mechanisms, and therapeutic targeting of the pathological nature of the extracellular environment in CNS injury. In this review, we will focus on the mechanisms that regulate CNS regeneration, highlighting the history, recent efforts, and questions left unanswered in this field.

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#71: Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Neuroprogenitor Cells and Cerebral Organoids

Journal of the Pediatric Infectious Diseases Society ◽

10.1093/jpids/piaa170.042 ◽

2021 ◽

Vol 10 (Supplement_1) ◽

pp. S14-S14

Author(s):

K E Ocwieja ◽

T K Hughes ◽

J M Antonucci ◽

A L Richards ◽

A C Stanton ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Zika Virus ◽

Molecular Mechanisms ◽

Sequencing Analysis ◽

Human Stem Cell ◽

Zikv Infection ◽

Single Cell Sequencing ◽

Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not observed prior to the recent epidemics in French Polynesia and the Americas, despite evidence that the Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively. Methods Due to advances in stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neuroprogenitor cells and cerebral organoids, which recapitulate complex three-dimensional neural architecture. We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells. Results In neuroprogenitor cells, single-cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these are largely suppressed in cells infected with ZIKV within the same culture dish. In our organoid model, single-cell sequencing allows us to identify multiple cellular populations, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. In this model of the developing brain, we identify preferred tropisms of ZIKV infection. Our data additionally reveal differences in cell-type frequencies and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. Conclusions These findings may help explain phenotypic differences attributed to the viruses, including variable propensities to cause microcephaly. Overall, our work provides insight into normal and diseased human brain development and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

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An Update on Sexual Transmission of Zika Virus

Pathogens ◽

10.3390/pathogens7030066 ◽

2018 ◽

Vol 7 (3) ◽

pp. 66 ◽

Cited By ~ 18

Author(s):

Hercules Sakkas ◽

Petros Bozidis ◽

Xenofon Giannakopoulos ◽

Nikolaos Sofikitis ◽

Chrissanthy Papadopoulou

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Reproductive System ◽

Zika Virus ◽

Rna Virus ◽

Sexual Transmission ◽

Limited Disease ◽

Zikv Infection ◽

Blood Sucking

Zika virus (ZIKV) is a single-stranded RNA virus belonging to the arthropod-borne flaviviruses (arboviruses) which are mainly transmitted by blood-sucking mosquitoes of the genus Aedes. ZIKV infection has been known to be rather asymptomatic or presented as febrile self-limited disease; however, during the last decade the manifestation of ZIKV infection has been associated with a variety of neuroimmunological disorders including Guillain–Barré syndrome, microcephaly and other central nervous system abnormalities. More recently, there is accumulating evidence about sexual transmission of ZIKV, a trait that has never been observed in any other mosquito-borne flavivirus before. This article reviews the latest information regarding the latter and emerging role of ZIKV, focusing on the consequences of ZIKV infection on the male reproductive system and the epidemiology of human-to-human sexual transmission.

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Zika Virus Production Is Resistant to RNase L Antiviral Activity

Journal of Virology ◽

10.1128/jvi.00313-19 ◽

2019 ◽

Vol 93 (16) ◽

Cited By ~ 9

Author(s):

Jillian N. Whelan ◽

Yize Li ◽

Robert H. Silverman ◽

Susan R. Weiss

Keyword(s):

Antiviral Activity ◽

Zika Virus ◽

Viral Genome ◽

Molecular Mechanisms ◽

Virus Production ◽

Type I ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Zikv Infection ◽

Initial Report

SUMMARYThere is currently no knowledge of how the emerging human pathogen Zika virus (ZIKV) interacts with the antiviral endoribonuclease L (RNase L) pathway during infection. Since activation of RNase L during infection typically limits virus production dramatically, we used CRISPR-Cas9 gene editing technology to knockout (KO) targeted host genes involved in the RNase L pathway to evaluate the effects of RNase L on ZIKV infection in human A549 cells. RNase L was activated in response to ZIKV infection, which degraded ZIKV genomic RNA. Surprisingly, despite viral genome reduction, RNase L activity did not reduce ZIKV infectious titers. In contrast, both the flavivirus dengue virus and the alphavirus Sindbis virus replicated to significantly higher titers in RNase L KO cells compared to wild-type (WT) cells. Using MAVS/RNase L double KO cells, we demonstrated that the absence of increased ZIKV production in RNase L KO cells was not due to compensation by enhanced type I interferon transcripts to thus inhibit virus production. Finally, when synthetic double-stranded RNA was detected by OAS3 to induce RNase L antiviral activity prior to ZIKV infection, we observed reduced ZIKV replication factory formation, as well as a 42-fold reduction in virus yield in WT but not RNase L KO cells. This study proposes that ZIKV evades RNase L antiviral activity by generating a viral genome reservoir protected from RNase L cleavage during early infection, allowing for sufficient virus production before RNase L activation is detectable.IMPORTANCEWith the onset of the 2015 ZIKV outbreak, ZIKV pathogenesis has been of extreme global public health interest, and a better understanding of interactions with the host would provide insight into molecular mechanisms driving the severe neurological outcomes of ZIKV disease. Here is the initial report on the relationship between ZIKV and the host oligoadenylate synthetase-RNase L (OAS-RNase L) system, a potent antiviral pathway effective at restricting replication of diverse viruses. Our study elucidated a unique mechanism whereby ZIKV production is impervious to antiviral RNase L activity, through a mechanism of viral RNA protection that is not mimicked during infection with numerous other RNase L-activating viruses, thus identifying a distinct replication strategy potentially important for ZIKV pathogenesis.

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MicroRNAs 145 and 148a Are Upregulated During Congenital Zika Virus Infection

ASN NEURO ◽

10.1177/1759091419850983 ◽

2019 ◽

Vol 11 ◽

pp. 175909141985098 ◽

Cited By ~ 3

Author(s):

Fernanda L. Castro ◽

Victor E. V. Geddes ◽

Fábio L. L. Monteiro ◽

Raphael M. D. T. Gonçalves ◽

Loraine Campanati ◽

...

Keyword(s):

Zika Virus ◽

Target Genes ◽

Neuroblastoma Cell ◽

In Silico Analysis ◽

Neuroblastoma Cell Line ◽

Cellular Migration ◽

Postmortem Brain ◽

Zikv Infection ◽

Cellular Mirnas ◽

Congenital Zika Syndrome

Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) member of the Flaviviridae family, which has been associated with the development of the congenital Zika syndrome (CZS). RNA viruses, such as flaviviruses, have been reported to exert a profound impact on host microRNAs (miRNAs). Cellular miRNAs modulated by ZIKV may help identify cellular pathways of relevance to pathogenesis. Here, we screened 754 human cellular miRNAs modulated by ZIKV infection (Brazilian PE strain) in a neuroblastoma cell line. Seven miRNAs (miR-99a*, miR-126*, miR-190b, miR-361-3p, miR-522-3p, miR-299-5p, and miR-1267) were downregulated during ZIKV infection, while miR-145 was upregulated. Furthermore, 11 miRNAs were exclusively expressed in ZIKV-infected (miR-148a, miR-342-5p, miR-598, and miR-708-3p) or mock cells (miR-208, miR-329, miR-432-5p, miR-488, miR-518b, miR-520g, and miR-767-5p). Furthermore, in silico analysis indicated that some central nervous system, cellular migration, and adhesion function-related biological processes were overrepresented in the list of target genes of the miRNAs regulated in ZIKV-infected cells, especially for miR-145 and miR-148a. The induction of miR-145 and miR-148a was confirmed in postmortem brain samples from stillborn with severe CZS. Finally, we determined the expression regulation of microcephaly related genes through RNA interference pathway caused by ZIKV directly on neuron cells.

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Zika Virus sfRNA Plays an Essential Role in the Infection of Insects and Mammals

Proceedings ◽

10.3390/proceedings2020050112 ◽

2020 ◽

Vol 50 (1) ◽

pp. 112

Author(s):

Andrii Slonchak ◽

Leon E. Hugo ◽

Morgan Freney ◽

Alberto A Amarilla ◽

Sonja Hall-Mendelin ◽

...

Keyword(s):

Human Brain ◽

Brain Development ◽

Zika Virus ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Terminal Deoxynucleotidyl Transferase ◽

Rnai Pathway ◽

Type I ◽

Zikv Infection

Similar to other flaviviruses, Zika virus (ZIKV) produces abundant subgenomic flavivirus RNA (sfRNA) derived from the 3’ untranslated region. The molecular mechanisms that determine the functions of sfRNA are currently not completely understood. Here, we created ZIKV mutants deficient in sfRNA production and employed them to investigate the role of this RNA in virus interactions with mammalian and insect hosts. We found that in mosquitoes, sfRNA facilitates virus replication and is required for ZIKV dissemination into saliva and virus transmission. The production of sfRNA was found to have no effect on the RNAi pathway, but instead downregulated the expression of genes involved in the regulation of apoptosis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of histological sections from infected mosquitoes confirmed that sfRNA prevents the apoptotic death of infected cells, thus identifying inhibition of apoptosis as a novel mechanism of sfRNA action in mosquitoes. We also found that sfRNA facilitates ZIKV replication in mammalian cells, mice, and human brain organoids. Moreover, ZIKV mutants deficient in sfRNA production were unable to form plaques, cause the death of human brain organoids, or establish infection in the mouse foetal brain. We then found that the proviral activity of sfRNA in mammalian cells relies on its ability to suppress type I interferon signalling. We showed that this is achieved via the inhibition of phosphorylation and the nuclear translocation of STAT1. In addition, we found that the production of sfRNA in the ZIKV infection of human brain organoids is associated with the suppression of multiple genes involved in brain development, indicating that sfRNA can be involved in the disruption of brain development associated with ZIKV infection.

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A Rat Model of Prenatal Zika Virus Infection and Associated Long-Term Outcomes

Viruses ◽

10.3390/v13112298 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2298

Author(s):

Morgan L. Sherer ◽

Elise A. Lemanski ◽

Rita T. Patel ◽

Shannon R. Wheeler ◽

Mark S. Parcells ◽

...

Keyword(s):

Rat Model ◽

Zika Virus ◽

Fold Increase ◽

Interferon Response ◽

Motor Deficits ◽

Long Term Outcomes ◽

Zikv Infection ◽

Full Spectrum ◽

The Brain

Zika virus (ZIKV) is a mosquito-borne flavivirus that became widely recognized due to the epidemic in Brazil in 2015. Since then, there has been nearly a 20-fold increase in the incidence of microcephaly and birth defects seen among women giving birth in Brazil, leading the Centers for Disease Control and Prevention (CDC) to officially declare a causal link between prenatal ZIKV infection and the serious brain abnormalities seen in affected infants. Here, we used a unique rat model of prenatal ZIKV infection to study three possible long-term outcomes of congenital ZIKV infection: (1) behavior, (2) cell proliferation, survival, and differentiation in the brain, and (3) immune responses later in life. Adult offspring that were prenatally infected with ZIKV exhibited motor deficits in a sex-specific manner, and failed to mount a normal interferon response to a viral immune challenge later in life. Despite undetectable levels of ZIKV in the brain and serum in these offspring at P2, P24, or P60, these results suggest that prenatal exposure to ZIKV results in lasting consequences that could significantly impact the health of the offspring. To help individuals already exposed to ZIKV, as well as be prepared for future outbreaks, we need to understand the full spectrum of neurological and immunological consequences that could arise following prenatal ZIKV infection.

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Skeletal muscle is an early site of Zika virus replication and injury impairing myogenesis

Journal of Virology ◽

10.1128/jvi.00904-21 ◽

2021 ◽

Author(s):

Daniel Gavino-Leopoldino ◽

Camila Menezes Figueiredo ◽

Mariana Oliveira Lopes da Silva ◽

Letícia Gonçalves Barcellos ◽

Rômulo Leão Silva Neris ◽

...

Keyword(s):

Skeletal Muscle ◽

Central Nervous System ◽

Nervous System ◽

Blood Brain Barrier ◽

Zika Virus ◽

Muscle Development ◽

Brain Barrier ◽

Zikv Infection ◽

Viral Spread ◽

Blood Brain

Zika virus (ZIKV) infection became a worldwide concern due to its correlation with the development of microcephaly and other neurological disorders. ZIKV neurotropism is well characterized, but the role of peripheral viral amplification to brain infection remains unknown. Here we found that ZIKV replicates in human primary skeletal muscle myoblasts, impairing its differentiation into myotubes but not interfering with the integrity of the already formed muscle fibers. Using mouse models, we showed ZIKV tropism to muscle tissue either during embryogenesis after maternal transmission or when infection occurred after birth. Interestingly, ZIKV replication in the mouse skeletal muscle started immediately after ZIKV inoculation, preceding viral RNA detection in the brain and causing no disruption to the integrity of the blood brain barrier, and remained active for more than two weeks, while replication in spleen and liver were not sustained over time. In addition, ZIKV infection of the skeletal muscle induces necrotic lesions, inflammation and fiber atrophy. We also found a reduction in the expression of regulatory myogenic factors that are essential for muscle repair after injury. Taken together our results indicate that the skeletal muscle is an early site of viral amplification and lesion that may result in late consequences in muscle development after ZIKV infection. Importance Zika Virus (ZIKV) neurotropism and its deleterious effects on central nervous system have been well characterized. But, investigations of the initial replication sites for the establishment of infection and viral spread to neural tissues remain under explored. The complete description of the range of ZIKV induced lesions and others factors that can influence the severity of the disease are necessary to prevent ZIKV deleterious effects. ZIKV has been shown to access the central nervous system without significantly affecting blood-brain barrier permeability. Here we demonstrated that skeletal muscle is an earlier site of ZIKV replication, contributing to the increase of peripheral ZIKV load. ZIKV replication in muscle promotes necrotic lesions, inflammation and also impairs myogenesis. Overall, our findings showed that skeletal muscle is involved in pathogenesis and opens new fields in the investigation of the long-term consequence of early infection.

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