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1.
Nat Med ; 25(12): 1873-1884, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31806906

RESUMO

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.


Assuntos
Encefalite por Herpes Simples/genética , Herpesvirus Humano 1/genética , Neurônios/imunologia , RNA Nucleolar Pequeno/genética , Adulto , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Pré-Escolar , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/patologia , Encefalite por Herpes Simples/virologia , Feminino , Predisposição Genética para Doença , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Humanos , Imunidade/genética , Lactente , Masculino , Metagenoma/genética , Metagenoma/imunologia , Pessoa de Meia-Idade , Neurônios/virologia , RNA Nucleolar Pequeno/imunologia
2.
BMC Infect Dis ; 19(1): 912, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664929

RESUMO

BACKGROUND: West Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia. METHODS: To better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs). RESULTS: Significant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures. CONCLUSION: NSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.


Assuntos
Surtos de Doenças , Imunidade Inata/genética , Inflamação/genética , Febre do Nilo Ocidental/epidemiologia , Vírus do Nilo Ocidental/genética , Austrália/epidemiologia , Linhagem Celular Tumoral , Quimiocina CCL2/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Interleucina-8/genética , Neurônios/virologia , Fenótipo , Virulência , Vírus do Nilo Ocidental/patogenicidade
3.
Nat Commun ; 10(1): 4439, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570731

RESUMO

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos , Integração Viral/genética , Animais , Bacteriófago lambda/genética , Encéfalo , Linhagem Celular , Mapeamento Cromossômico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cóclea , Endonucleases , Marcação de Genes/métodos , Terapia Genética/métodos , Genoma , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculos , Neurônios/virologia , Reparo Gênico Alvo-Dirigido/métodos , Resultado do Tratamento
4.
Nat Commun ; 10(1): 4430, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562326

RESUMO

Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention.


Assuntos
Moléculas de Adesão Celular/metabolismo , Monócitos/metabolismo , Monócitos/virologia , Neurônios/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Zika virus/patogenicidade , Animais , Adesão Celular/fisiologia , Sobrevivência Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Cerebelo/patologia , Cerebelo/virologia , Modelos Animais de Doenças , Células-Tronco Embrionárias , Endotélio/virologia , Feminino , Humanos , Monócitos/patologia , Neurônios/patologia , Neurônios/virologia , Organoides/metabolismo , Organoides/patologia , Peixe-Zebra , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia
5.
Nat Commun ; 10(1): 3890, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488835

RESUMO

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.


Assuntos
Encéfalo/virologia , Sinapses/virologia , Replicação Viral , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Humanos , Inflamação , Aprendizagem , Masculino , Memória , Transtornos da Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Neurônios/virologia , Terminações Pré-Sinápticas/metabolismo , Receptores Tipo I de Interleucina-1/genética , Sinapses/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
6.
BMC Vet Res ; 15(1): 317, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484573

RESUMO

BACKGROUND: Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally. RESULTS: Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels. CONCLUSION: These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.


Assuntos
Galinhas , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/virologia , Tropismo , Animais , Astrócitos/virologia , Células Cultivadas , Imunofluorescência , Microglia/virologia , Neurônios/virologia , Oligodendroglia/virologia , Especificidade da Espécie , Virulência , Replicação Viral
7.
Emerg Microbes Infect ; 8(1): 1076-1085, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339457

RESUMO

Enterovirus A71 (EV-A71) is one of the main causative agents of hand-foot-and-mouth disease and is occasionally associated with severe neurological complications. EV-A71 pathophysiology is poorly understood due to the lack of small animal models that robustly support viral replication in relevant organs/tissues. Here, we show that adult severe combined immune-deficient (SCID) mice can serve as an EV-A71 infection model to study neurotropic determinants and viral tropism. Mice inoculated intraperitoneally with an EV-A71 clinical isolate had an initial infection of the lung compartment, followed by neuroinvasion and infection of (motor)neurons, resulting in slowly progressing paralysis of the limbs. We identified a substitution (V135I) in the capsid protein VP2 as a key requirement for neurotropism. This substitution was also present in a mouse-adapted variant, obtained by passaging the clinical isolate in the brain of one-day-old mice, and induced exclusive neuropathology and rapid paralysis, confirming its role in neurotropism. Finally, we showed that this residue enhances the capacity of EV-A71 to use mouse PSGL1 for viral entry. Our data reveal that EV-A71 initially disseminates to the lung and identify viral and host determinants that define the neurotropic character of EV-A71, pointing to a hitherto understudied role of PSGL1 in EV-A71 tropism and neuropathology.


Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Neurônios/virologia , Animais , Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/genética , Infecções por Enterovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos SCID , Mutação de Sentido Incorreto , Neurônios/metabolismo , Tropismo Viral , Virulência , Internalização do Vírus
8.
Arch Virol ; 164(10): 2469-2477, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31297587

RESUMO

Rabies is a lethal viral disease that can affect a wide range of mammals. Currently, Rabies virus (RABV) in some European and American countries is maintained primarily in wild species. The regulation of viral replication is one of the critical mechanisms involved in RABV pathogenesis. However, the relationship between replication and the pathogenesis of RABV isolated from wild animals remains poorly understood. In the present study, we evaluated the pathogenicity of the street viruses Nyctinomops laticaudatus bat-associated RABV (NYBRV) and Cerdocyon thous canid-associated RABV (CECRV). Infection of mice with NYBRV led to 33% mortality with rapid disease evolution and marked histopathological changes in the CNS. In contrast, infection with CECRV led to 67% mortality and caused mild neuropathological lesions. The proportion of RABV antigen was significantly higher in the cytoplasm of neuronal cells of the cerebral cortex and in the meninges of mice infected with CECRV and NYBRV, respectively. Moreover, the replication rate of NYBRV was significantly higher (p < 0.001) than that of CECRV in neuroblastoma cells. However, CECRV replicated to a significantly higher titer in epithelial cells. Our results indicate that NYBRV infection results in rapid disease progression accompanied by frequent and intense histopathological alterations in the CNS in mice, and in a high replication rate in neuroblastoma cells. Although, CECRV is more pathogenic in mice, it caused milder histopathological changes in the CNS and replicated more efficiently in epithelial cells. Our data point to a correlation between clinical aspects of disease and the replication of RABV in different cell lines.


Assuntos
Canidae/virologia , Quirópteros/virologia , Vírus da Raiva/isolamento & purificação , Vírus da Raiva/patogenicidade , Raiva/patologia , Raiva/virologia , Animais , Linhagem Celular , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Histocitoquímica , Camundongos , Neurônios/virologia , Análise de Sobrevida , Virulência , Replicação Viral
9.
Virus Genes ; 55(5): 630-642, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31292858

RESUMO

Japanese encephalitis virus (JEV) exerts a profound burden of viral encephalitis. We have investigated the differentially expressed transcripts in the neuronal transcriptome during JEV infection by RNA sequencing (RNA-Seq) of virus-infected SH-SY5Y human neuroblastoma cells. Gene ontology analysis revealed significant enrichment from two main pathways: endoplasmic reticulum (ER)-nucleus signaling (P value: 5.75E-18; false discovery rate [FDR] 3.11E-15) and the ER unfolded protein response (P value: 7.58E-18; FDR 3.11E-15). qPCR validation showed significant upregulation and differential expression (P < 0.01) of ER stress-signaling transcripts (SESN2, TRIB3, DDIT3, DDIT4, XBP1, and ATF4) at 24 h post-infection for both low (LN) and high (HN) neurovirulence JEV strains. Immunoblot analysis following JEV infection of SH-SY5Y cells showed an increase in levels of SESN2 protein following JEV infection. Similarly, Zika virus (MR766) infection of SH-SY5Y showed a titer-dependent increase in ER stress-signaling transcripts; however, this was absent or diminished for DDIT4 and ATF4, respectively, suggestive of differences in the induction of stress-response transcripts between flaviviruses. Interestingly, SLC7A11 and SLC3A2 mRNA were also both deregulated in JEV-infected SH-SY5Y cells and encode the two constituent subunits of the plasma membrane xCT amino acid antiporter that relieves oxidative stress by export of glutamate and import of cystine. Infection of SH-SY5Y and HEK293T cells by the JEV HN strain Sw/Mie/40/2004 lead to significant upregulation of the SLC7A11 mRNA to levels comparable to DDIT3. Our findings suggest upregulation of antioxidants including SESN2 and, also, the xCT antiporter occurs to counteract the oxidative stress elicited by JEV infection.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/crescimento & desenvolvimento , Neurônios/patologia , Neurônios/virologia , Proteínas Nucleares/biossíntese , Regulação para Cima , Sistema y+ de Transporte de Aminoácidos/biossíntese , Linhagem Celular , Biologia Computacional , Perfilação da Expressão Gênica , Humanos
10.
Nat Neurosci ; 22(8): 1345-1356, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285614

RESUMO

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.


Assuntos
Dependovirus/genética , Marcação de Genes/métodos , Neuroglia/virologia , Neurônios/virologia , Animais , Técnicas de Transferência de Genes , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Retina/virologia
11.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180044

RESUMO

ErbB-3 binding protein 1 (Ebp1) is a host protein which binds ErbB-3 receptor to induce signalling events for cell growth regulation. In addition, Ebp1 also interacts with ribonucleoprotein complexes. In recent times, Ebp1 was found to play an antagonistic role in viral infections caused by Influenza and Rinderpest viruses. In our present work we have tried to understand the role of Ebp1 in Chandipura virus (CHPV) infection. We have observed an induction in Ebp1 expression upon CHPV infection similar to other viruses. However, unlike other viruses an overexpressed Ebp1 only reduces viral protein expression, but does not affect its progeny formation. Additionally, this effect is being carried out in an indirect manner, as there is no interaction between Ebp1 and viral proteins. This is despite Ebp1's presence in viral inclusion bodies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Interações Hospedeiro-Patógeno/genética , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Vesiculovirus/genética , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Corpos de Inclusão Viral/química , Neurônios/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transfecção , Células Vero , Vesiculovirus/crescimento & desenvolvimento , Vesiculovirus/metabolismo , Ensaio de Placa Viral
12.
PLoS Negl Trop Dis ; 13(6): e0007537, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31251739

RESUMO

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.


Assuntos
Fatores Imunológicos/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Zika virus/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Neurônios/imunologia , Neurônios/virologia , Ligação Proteica , Replicação Viral
13.
Viral Immunol ; 32(4): 170-178, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31063043

RESUMO

Hand, foot, and mouth disease (HFMD) is a common infection for children younger than the age of five. HFMD is mainly induced by coxsackievirus A16 and enterovirus 71 (EV71). EV71-associated HFMD often has serious neurological disease complications. The purpose of this study was to reveal the mechanisms of action of EV71 on neurons. SH-SY5Y cells transfected or untransfected with EV71 were sequenced. After data preprocessing, differentially expressed genes (DEGs) were screened using the limma package in R, and clustering analysis was then performed using the ComplexHeatmap package in R. The DAVID tool was used for EDG enrichment analysis. Protein-protein interactions (PPIs) were predicted using the STRING database and PPI networks were then constructed using Cytoscape software. After pathways involved in the key PPI network nodes were enriched, pathway deviation scores were calculated. Clustering analysis was also conducted for these pathways. There were 978 DEGs in the transfected samples. Upregulated TNF was enriched in NF-kappa B signaling pathway. Among the top 20 nodes in the PPI network, CDK1, STAT3, CCND1, TNF, and MYC had the highest degrees. A total of 28 pathways were enriched for the top 20 nodes, including Epstein-Barr virus infection (p = 3.78E-06), proteoglycans in cancer (p = 4.96E-06), and melanoma (p = 1.99E-05). In addition, clustering analysis showed that these pathways could clearly differentiate the two groups of samples. EV71 may affect neurons by mediating CDK1, STAT3, CCND1, TNF, and MYC, indicating that these genes are promising targets for preventing the neuronal complications of HFMD.


Assuntos
Enterovirus Humano A/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Neurônios/virologia , Análise de Sequência de RNA , Linhagem Celular , Biologia Computacional , Redes Reguladoras de Genes , Humanos , Mapas de Interação de Proteínas
14.
Neuroimage ; 197: 133-142, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31022567

RESUMO

The elucidation of neural networks is essential to understanding the mechanisms of brain functions and brain disorders. Neurotropic virus-based trans-synaptic tracing tools have become an effective method for dissecting the structure and analyzing the function of neural-circuitry. However, these tracing systems rely on fluorescent signals, making it hard to visualize the panorama of the labeled networks in mammalian brain in vivo. One MRI method, Diffusion Tensor Imaging (DTI), is capable of imaging the networks of the whole brain in live animals but without information of anatomical connections through synapses. In this report, a chimeric gene coding for ferritin and enhanced green fluorescent protein (EGFP) was integrated into Vesicular stomatitis virus (VSV), a neurotropic virus that is able to spread anterogradely in synaptically connected networks. After the animal was injected with the recombinant VSV (rVSV), rVSV-Ferritin-EGFP, into the somatosensory cortex (SC) for four days, the labeled neural-network was visualized in the postmortem whole brain with a T2-weighted MRI sequence. The modified virus transmitted from SC to synaptically connected downstream regions. The results demonstrate that rVSV-Ferritin-EGFP could be used as a bimodal imaging vector for detecting synaptically connected neural-network with both ex vivo MRI and fluorescent imaging. The strategy in the current study has the potential to longitudinally monitor the global structure of a given neural-network in living animals.


Assuntos
Mapeamento Encefálico/métodos , Imagem por Ressonância Magnética , Neurônios/citologia , Córtex Somatossensorial/citologia , Vesiculovirus/fisiologia , Animais , Ferritinas/genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/virologia , Neurônios/virologia , Córtex Somatossensorial/virologia , Vesiculovirus/genética
15.
Mol Cell ; 74(3): 466-480.e4, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30930055

RESUMO

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.


Assuntos
DNA Topoisomerases Tipo II/genética , Herpesvirus Humano 1/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-akt/genética , Latência Viral/genética , Animais , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Proteína Homóloga a MRE11/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neurônios/metabolismo , Neurônios/virologia , Fosforilação , Ratos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
16.
Oxid Med Cell Longev ; 2019: 2302835, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30886672

RESUMO

Herpes simplex virus type 1 (HSV-1) has the ability to replicate in neurons and glial cells and to produce encephalitis leading to neurodegeneration. Accumulated evidence suggests that nitric oxide (NO) is a key molecule in the pathogenesis of neurotropic virus infections. NO can exert both cytoprotective as well as cytotoxic effects in the central nervous system (CNS) depending on its concentration, time course exposure, and site of action. In this study, we used an in vitro model of HSV-1-infected primary neuronal and mixed glial cultures as well as an intranasal model of HSV-1 in BALB/c mice to elucidate the role of NO and nonapoptotic Fas signalling in neuroinflammation and neurodegeneration. We found that low, nontoxic concentration of NO decreased HSV-1 replication in neuronal cultures together with production of IFN-alpha and proinflammatory chemokines. However, in HSV-1-infected glial cultures, low concentrations of NO supported virus replication and production of IFN-alpha and proinflammatory chemokines. HSV-1-infected microglia downregulated Fas expression and upregulated its ligand, FasL. Fas signalling led to production of proinflammatory cytokines and chemokines as well as induced iNOS in uninfected bystander glial cells. On the contrary, NO reduced production of IFN-alpha and CXCL10 through nonapoptotic Fas signalling in HSV-1-infected neuronal cultures. Here, we also observed colocalization of NO production with the accumulation of ß-amyloid peptide in HSV-1-infected neurons both in vitro and in vivo. Low levels of the NO donor increased accumulation of ß-amyloid in uninfected primary neuronal cultures, while the NO inhibitor decreased its accumulation in HSV-1-infected neuronal cultures. This study shows for the first time the existence of a link between NO and Fas signalling during HSV-1-induced neuroinflammation and neurodegeneration.


Assuntos
Herpesvirus Humano 1/fisiologia , Inflamação/virologia , Neurônios/patologia , Neurônios/virologia , Óxido Nítrico/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Quimiocinas , Proteína Ligante Fas/metabolismo , Herpesvirus Humano 1/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos , Receptor fas/metabolismo
17.
mBio ; 10(2)2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837340

RESUMO

Interferons (IFNs) and autophagy are critical neuronal defenses against viral infection. IFNs alter neuronal autophagy by promoting the accumulation of IFN-dependent LC3-decorated autophagic structures, termed LC3 clusters. Here, we analyzed LC3 clusters in sensory ganglia following herpes simplex virus 1 (HSV-1) infection. In the vicinity of acutely infected neurons, antigen-negative neurons contained structures resembling accumulated autophagosomes and autolysosomes that culminated in LC3 clusters. This accumulation reflects a delayed completion of autophagy. The endosomal sorting complexes required for transport (ESCRT) machinery participates in autophagosome closure and is also required for HSV-1 replication. In this study, our results showed that HSV-1 infection in vivo and in primary neurons caused a decrease in Vps4 (a key ESCRT pathway ATPase) RNA and protein with concomitant Stat1 activation and LC3 cluster induction. We also observed that IFNs were sufficient to decrease RNA and protein levels of Vps4 in primary neurons and in other cell types. The accumulation of ubiquitin was also observed at the LC3 cluster sites. Together, our results show that IFNs modulate the ESCRT machinery in neurons in response to HSV-1 infections.IMPORTANCE Neurons rely on IFNs and autophagy as major defenses against viral infections, and HSV must overcome such defenses in order to replicate. In addition to controlling host immunity, HSV must also control host membranes in order to complete its life cycle. HSV uses the host ESCRT membrane scission machinery for viral production and transport. Here we present evidence of a new IFN-dependent mechanism used by the host to prevent ESCRT subversion by HSV. This activity also impacts the dynamics of autophagy, possibly explaining the presence of recently described LC3 clusters in the HSV-infected nervous system. The induced accumulations of ubiquitin observed in these LC3 clusters resembled those observed in certain neurodegenerative diseases, suggesting possible mechanistic parallels between these conditions.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Herpes Simples/patologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Interferons/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Autofagia , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Perfilação da Expressão Gênica , Camundongos , Neurônios/patologia , Neurônios/virologia , Fator de Transcrição STAT1/metabolismo
18.
Emerg Microbes Infect ; 8(1): 307-326, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866785

RESUMO

The harmful effects of ZIKA virus (ZIKV) infection are reflected by severe neurological manifestations such as microcephaly in neonates and other complications associated with Guillain-Barré syndrome in adults. The transmission dynamics of ZIKV in or between neurons, or within the developing brains of the foetuses are not fully understood. Using primary cultures of murine cortical neurons, we show that ZIKV uses exosomes as mediators of viral transmission between neurons. Cryo-electron microscopy showed heterogeneous population of neuronal exosomes with a size range of 30-200 nm. Increased production of exosomes from neuronal cells was noted upon ZIKV infection. Neuronal exosomes contained both ZIKV viral RNA and protein(s) that were highly infectious to naïve cells. RNaseA and neutralizing antibodies treatment studies suggest the presence of viral RNA/proteins inside exosomes. Exosomes derived from time- and dose-dependent incubations showed increasing viral loads suggesting higher packaging and delivery of ZIKV RNA and proteins. Furthermore, we noted that ZIKV induced both activity and gene expression of neutral Sphingomyelinase (nSMase)-2/SMPD3, an important molecule that regulates production and release of exosomes. Silencing of SMPD3 in neurons resulted in reduced viral burden and transmission through exosomes. Treatment with SMPD3 specific inhibitor GW4869, significantly reduced ZIKV loads in both cortical neurons and in exosomes derived from these neuronal cells. Taken together, our results suggest that ZIKV modulates SMPD3 activity in cortical neurons for its infection and transmission through exosomes perhaps leading to severe neuronal death that may result in neurological manifestations such as microcephaly in the developing embryonic brains.


Assuntos
Córtex Cerebral/virologia , Exossomos/virologia , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Zika virus/fisiologia , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Microscopia Crioeletrônica , Exossomos/genética , Exossomos/metabolismo , Regulação Enzimológica da Expressão Gênica , Camundongos , Neurônios/citologia , Neurônios/virologia , Fatores de Tempo , Carga Viral/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos
19.
J Virol ; 93(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30787148

RESUMO

Herpes simplex virus 1 (HSV-1) establishes latency in both peripheral nerve ganglia and the central nervous system (CNS). The outcomes of acute and latent infections in these different anatomic sites appear to be distinct. It is becoming clear that many of the existing culture models using animal primary neurons to investigate HSV-1 infection of the CNS are limited and not ideal, and most do not recapitulate features of CNS neurons. Human induced pluripotent stem cells (hiPSCs) and neurons derived from them are documented as tools to study aspects of neuropathogenesis, but few have focused on modeling infections of the CNS. Here, we characterize functional two-dimensional (2D) CNS-like neuron cultures and three-dimensional (3D) brain organoids made from hiPSCs to model HSV-1-human-CNS interactions. Our results show that (i) hiPSC-derived CNS neurons are permissive for HSV-1 infection; (ii) a quiescent state exhibiting key landmarks of HSV-1 latency described in animal models can be established in hiPSC-derived CNS neurons; (iii) the complex laminar structure of the organoids can be efficiently infected with HSV, with virus being transported from the periphery to the central layers of the organoid; and (iv) the organoids support reactivation of HSV-1, albeit less efficiently than 2D cultures. Collectively, our results indicate that hiPSC-derived neuronal platforms, especially 3D organoids, offer an extraordinary opportunity for modeling the interaction of HSV-1 with the complex cellular and architectural structure of the human CNS.IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model acute and latent HSV-1 infections in two-dimensional (2D) and three-dimensional (3D) CNS neuronal cultures. We successfully established acute HSV-1 infections and infections showing features of latency. HSV-1 infection of the 3D organoids was able to spread from the outer surface of the organoid and was transported to the interior lamina, providing a model to study HSV-1 trafficking through complex neuronal tissue structures. HSV-1 could be reactivated in both culture systems; though, in contrast to 2D cultures, it appeared to be more difficult to reactivate HSV-1 in 3D cultures, potentially paralleling the low efficiency of HSV-1 reactivation in the CNS of animal models. The reactivation events were accompanied by dramatic neuronal morphological changes and cell-cell fusion. Together, our results provide substantive evidence of the suitability of hiPSC-based neuronal platforms to model HSV-1-CNS interactions in a human context.


Assuntos
Sistema Nervoso Central/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Animais , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Herpes Simples/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Neurônios/patologia , Neurônios/virologia , Células Vero
20.
Acta Histochem ; 121(3): 368-375, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30771905

RESUMO

Lentiviral transduction is a powerful tool and widely used in neuroscience research to manipulate gene expression of cells. However, the injection of lentiviral vectors in the brain is not totally benign, it potentially induces focal neuroinflammation. Upon inflammation, microglial cells get activated and can induce major changes in tissue environment, which may interfere with experimental results. In the current study, two weeks after the injection of control viral construction in the dentate gyrus (DG) of rats, an activation of microglia was detected. To access the activation status, we used a fast and accurate method of phenotype detection - measurement of fractal dimension (FD). Microglial morphology is a key indicator of neuroinflammation, therefore FD of microglial cells may serve as a reliable index of inflammation status in the brain. Here we present a detailed description of image processing procedure of images of individual microglial cells. The method allows to preserve the complex structure of microglial cells and their thin processes on the output image, which is important for accurate FD assessment.


Assuntos
Encéfalo/citologia , Giro Denteado/citologia , Microglia/citologia , Neurônios/citologia , Animais , Encéfalo/virologia , Giro Denteado/virologia , Lentivirus/isolamento & purificação , Masculino , Microglia/virologia , Neurônios/virologia , Ratos Wistar
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