RESUMO
Viral infection is frequently assayed by ongoing expression of viral genes. These assays fail to identify cells that have been exposed to the virus but limit or inhibit viral replication. To address this limitation, we used a dual-labeling vesicular stomatitis virus (DL-VSV), which has a deletion of the viral glycoprotein gene, to allow evaluation of primary infection outcomes. This virus encodes Cre, which can stably mark any cell with even a minimal level of viral gene expression. Additionally, the virus encodes GFP, which distinguishes cells with higher levels of viral gene expression, typically due to genome replication. Stereotactic injections of DL-VSV into the murine brain showed that different cell types had very different responses to the virus. Almost all neurons hosted high levels of viral gene expression, while glial cells varied in their responses. Astrocytes (Sox9+) were predominantly productively infected, while oligodendrocytes (Sox10+) were largely abortively infected. Microglial cells (Iba1+) were primarily uninfected. Furthermore, we monitored the early innate immune response to viral infection and identified unique patterns of interferon (IFN) induction. Shortly after infection, microglia were the main producers of IFNb, whereas later, oligodendrocytes were the main producers. IFNb+ cells were primarily abortively infected regardless of cell type. Last, we investigated whether IFN signaling had any impact on the outcome of primary infection and did not observe significant changes, suggesting that intrinsic factors are likely responsible for determining the outcome of primary infection.
Assuntos
Astrócitos , Animais , Camundongos , Astrócitos/virologia , Astrócitos/metabolismo , Replicação Viral , Microglia/virologia , Microglia/metabolismo , Microglia/imunologia , Neurônios/virologia , Neurônios/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Vesiculovirus/fisiologia , Vesiculovirus/imunologia , Vesiculovirus/genética , Oligodendroglia/virologia , Oligodendroglia/metabolismo , Estomatite Vesicular/virologia , Estomatite Vesicular/imunologia , Imunidade Inata , Camundongos Endogâmicos C57BL , Encéfalo/virologia , Encéfalo/metabolismo , Encéfalo/imunologia , Neuroglia/virologia , Neuroglia/metabolismoRESUMO
Zika virus (ZikV) infection during pregnancy can cause congenital Zika syndrome (CZS) and neurodevelopmental delay in infants, of which the pathogenesis remains poorly understood. We utilize an established female pigtail macaque maternal-to-fetal ZikV infection/exposure model to study fetal brain pathophysiology of CZS manifesting from ZikV exposure in utero. We find prenatal ZikV exposure leads to profound disruption of fetal myelin, with extensive downregulation in gene expression for key components of oligodendrocyte maturation and myelin production. Immunohistochemical analyses reveal marked decreases in myelin basic protein intensity and myelinated fiber density in ZikV-exposed animals. At the ultrastructural level, the myelin sheath in ZikV-exposed animals shows multi-focal decompaction, occurring concomitant with dysregulation of oligodendrocyte gene expression and maturation. These findings define fetal neuropathological profiles of ZikV-linked brain injury underlying CZS resulting from ZikV exposure in utero. Because myelin is critical for cortical development, ZikV-related perturbations in oligodendrocyte function may have long-term consequences on childhood neurodevelopment, even in the absence of overt microcephaly.
Assuntos
Modelos Animais de Doenças , Bainha de Mielina , Oligodendroglia , Infecção por Zika virus , Zika virus , Animais , Infecção por Zika virus/virologia , Infecção por Zika virus/patologia , Oligodendroglia/virologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Feminino , Bainha de Mielina/metabolismo , Gravidez , Zika virus/patogenicidade , Complicações Infecciosas na Gravidez/virologia , Complicações Infecciosas na Gravidez/patologia , Macaca nemestrina , Encéfalo/virologia , Encéfalo/patologia , Encéfalo/metabolismo , Humanos , Proteína Básica da Mielina/metabolismo , Proteína Básica da Mielina/genéticaRESUMO
HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affect approximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy (ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlates with the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicated in HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo, ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injection of Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment. These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin maintenance in the pathogenesis of HAND.
Assuntos
Vesículas Extracelulares , HIV-1 , Oligodendroglia , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Animais , Camundongos , Células Cultivadas , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Vesículas Extracelulares/metabolismo , HIV-1/metabolismo , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Oligodendroglia/virologiaRESUMO
We herein report, by using confocal immunofluorescence, the colocalization of the SARS-CoV-2 nucleocapsid within neurons, astrocytes, oligodendrocytes and microglia in three deceased COVID-19 cases, of between 78 and 85 years of age at death. The viral nucleocapsid was detected together with its ACE2 cell entry receptor, as well as the NLRP3 inflammasome in cerebral cortical tissues. It is noteworthy that NLRP3 was colocalized with CD68 + macrophages in the brain and lung of the deceased, suggesting the critical role of this type of inflammasome in SARS-CoV-2 lesions of the nervous system/lungs and supporting its potential role as a therapeutic target.
Assuntos
Encéfalo/virologia , COVID-19/virologia , Inflamassomos/imunologia , Microglia/virologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , SARS-CoV-2/patogenicidade , Idoso , Idoso de 80 Anos ou mais , Astrócitos/virologia , Autopsia , Encéfalo/imunologia , Encéfalo/patologia , COVID-19/imunologia , COVID-19/patologia , Feminino , Humanos , Masculino , Microglia/imunologia , Neurônios/virologia , Nucleocapsídeo , Oligodendroglia/virologiaRESUMO
HIV-1 can incite activation of chemokine receptors, inflammatory mediators, and glutamate receptor-mediated excitotoxicity. The mechanisms associated with such immune activation can disrupt neuronal and glial functions. HIV-associated neurocognitive disorder (HAND) is being observed since the beginning of the AIDS epidemic due to a change in the functional integrity of cells from the central nervous system (CNS). Even with the presence of antiretroviral therapy, there is a decline in the functioning of the brain especially movement skills, noticeable swings in mood, and routine performance activities. Under the umbrella of HAND, various symptomatic and asymptomatic conditions are categorized and are on a rise despite the use of newer antiretroviral agents. Due to the use of long-lasting antiretroviral agents, this deadly disease is becoming a manageable chronic condition with the occurrence of asymptomatic neurocognitive impairment (ANI), symptomatic mild neurocognitive disorder, or HIV-associated dementia. In-depth research in the pathogenesis of HIV has focused on various mechanisms involved in neuronal dysfunction and associated toxicities ultimately showcasing the involvement of various pathways. Increasing evidence-based studies have emphasized a need to focus and explore the specific pathways in inflammation-associated neurodegenerative disorders. In the current review, we have highlighted the association of various HIV proteins and neuronal cells with their involvement in various pathways responsible for the development of neurotoxicity.
Assuntos
Complexo AIDS Demência/imunologia , Complexo AIDS Demência/virologia , Sistema Nervoso Central/virologia , HIV-1/metabolismo , Proteínas Virais/metabolismo , Complexo AIDS Demência/fisiopatologia , Antirretrovirais/uso terapêutico , Astrócitos/virologia , Sistema Nervoso Central/fisiopatologia , Genoma , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/complicações , Infecções por HIV/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Inflamação , Cinurenina/metabolismo , Macrófagos/virologia , Microglia/virologia , Neurônios/virologia , Oligodendroglia/virologia , Receptores de N-Metil-D-Aspartato/metabolismo , Carga Viral , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Viroporinas/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoAssuntos
Astrócitos , Vírus JC/isolamento & purificação , Leucoencefalopatia Multifocal Progressiva , Oligodendroglia , Idoso , Astrócitos/patologia , Astrócitos/virologia , Citodiagnóstico , Técnicas Citológicas , DNA Viral/isolamento & purificação , Feminino , Humanos , Hospedeiro Imunocomprometido , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Leucoencefalopatia Multifocal Progressiva/patologia , Oligodendroglia/patologia , Oligodendroglia/virologiaRESUMO
During persistent human beta-herpesvirus (HHV) infection, clinical manifestations may not appear. However, the lifelong influence of HHV is often associated with pathological changes in the central nervous system. Herein, we evaluated possible associations between immunoexpression of HHV-6, -7, and cellular immune response across different brain regions. The study aimed to explore HHV-6, -7 infection within the cortical lobes in cases of unspecified encephalopathy (UEP) and nonpathological conditions. We confirmed the presence of viral DNA by nPCR and viral antigens by immunohistochemistry. Overall, we have shown a significant increase (p < 0.001) of HHV antigen expression, especially HHV-7 in the temporal gray matter. Although HHV-infected neurons were found notably in the case of HHV-7, our observations suggest that higher (p < 0.001) cell tropism is associated with glial and endothelial cells in both UEP group and controls. HHV-6, predominantly detected in oligodendrocytes (p < 0.001), and HHV-7, predominantly detected in both astrocytes and oligodendrocytes (p < 0.001), exhibit varying effects on neural homeostasis. This indicates a high number (p < 0.001) of activated microglia observed in the temporal lobe in the UEP group. The question remains of whether human HHV contributes to neurological diseases or are markers for some aspect of the disease process.
Assuntos
Encefalopatias/imunologia , Herpesvirus Humano 6 , Herpesvirus Humano 7 , Imunidade Celular , Neuroglia/virologia , Infecções por Roseolovirus/imunologia , Adulto , Idoso , Antígenos Virais/análise , Astrócitos/virologia , Encéfalo/imunologia , Encéfalo/virologia , Encefalopatias/virologia , Células Endoteliais/virologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Oligodendroglia/virologiaRESUMO
Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating disease of the CNS, resulting from the lytic infection of oligodendrocytes by the human neurotropic polyomavirus JC (JCPyV), typically associated with severe immunocompromised states and, in recent years, with the use of immunotherapies. Apoptosis is a homeostatic mechanism to dispose of senescent or damaged cells, including virally infected cells, triggered in the vast majority of viral infections of the brain. Previously, we showed upregulation of the normally dormant anti-apoptotic protein Survivin in cases of PML, which-in vitro-resulted in protection from apoptosis in JCPyV-infected primary cultures of astrocytes and oligodendrocytes. In the present study, we first demonstrate the absence of apoptotic DNA fragmentation and the lack of caspase activity in 16 cases of PML. We also identified the viral protein large T-Antigen as being responsible for the activation of the Survivin promoter. Chromatin Immunoprecipitation assay shows a direct binding between T-Antigen and the Survivin promoter DNA. Finally, we have identified the specific region of T-Antigen, spanning from amino acids 266 and 688, which binds to Survivin and translocates it to the nucleus, providing evidence of a mechanism that results in the efficient replication of JCPyV and a potential target for novel therapies.
Assuntos
Antígenos Virais de Tumores/genética , Apoptose , Vírus JC/genética , Regiões Promotoras Genéticas , Survivina/genética , Adulto , Idoso , Animais , Antígenos Virais de Tumores/imunologia , Astrócitos/virologia , Caspases/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Criança , Fragmentação do DNA , Feminino , Humanos , Vírus JC/imunologia , Vírus JC/patogenicidade , Leucoencefalopatia Multifocal Progressiva , Masculino , Camundongos , Pessoa de Meia-Idade , Oligodendroglia/virologia , Inclusão em Parafina , Survivina/imunologiaRESUMO
The demyelinating disease progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus, JCPyV, under conditions of prolonged immunosuppression. Initial infection is asymptomatic, and the virus establishes lifelong persistence in the host. Following the loss of immune surveillance, the virus can traffic to the central nervous system and infect oligodendrocytes to cause demyelination and PML. The mechanisms involved in glial cell infection are not completely understood. In a screen for N-glycosylated proteins that influence JCPyV pathology, we identified Adipocyte Plasma Membrane Associated Protein (APMAP) as a host cell modulator of JCPyV infection. The removal of APMAP by small interfering siRNA as well as by CRISPR-Cas9 gene editing resulted in a significant decrease in JCPyV infection. Exogenous expression of APMAP in APMAP knockout cell lines rescued susceptibility to infection. These data suggest that virus infection of glial cells is dependent on APMAP.
Assuntos
Vírus JC/fisiologia , Neuroglia/metabolismo , Infecções por Polyomavirus/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Vírus JC/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Neuroglia/virologia , Oligodendroglia/metabolismo , Oligodendroglia/virologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologiaRESUMO
Endogenous retroviruses have demonstrated exaptation during long-term evolution with hosts, e.g., resulting in acquisition of antiviral effect on related extant viral infections. While empirical studies have found that an endogenous bornavirus-like element derived from viral nucleoprotein (itEBLN) in the ground squirrel genome shows antiviral effect on virus replication and de novo infection, the antiviral mechanism, dynamics, and quantitative effect of itEBLN remain unknown. In this study, we experimentally and theoretically investigated the dynamics of how an extant bornavirus, Borna disease virus 1 (BoDV-1), spreads and replicates in uninfected, BoDV-1-infected, and itEBLN-expressing cultured cells. Quantifying antiviral effect based on time course data sets, we found that the antiviral effects of itEBLN are estimated to be 75% and 34% on intercellular virus spread and intracellular virus replication, respectively. This discrepancy between intercellular virus spread and intracellular viral replication suggests that viral processes other than the replication of viral ribonucleoprotein complex (RNP) contributed to the suppression of virus spread in itEBLN-expressing cells. Because itEBLN binds to the BoDV-1 RNP, the suppression of viral RNP trafficking can be an attractive candidate explaining this discrepancy.IMPORTANCE Accumulating evidence suggests that some endogenous viral elements (EVEs), including endogenous retroviruses and endogenous nonretroviral virus elements, have acquired functions in the host as a result of long-term coevolution. Recently, an endogenous bornavirus-like element (itEBLN) found in the ground squirrel genome has been shown to have antiviral activity against exogenous bornavirus infection. In this study, we first quantified bornavirus spread in cultured cells and then calculated the antiviral activity of itEBLN on bornavirus infection. The calculated antiviral activity of itEBLN suggests its suppression of multiple processes in the viral life cycle. To our knowledge, this is the first study quantifying the antiviral activity of EVEs and speculating on a model of how some EVEs have acquired antiviral activity during host-virus arms races.
Assuntos
Vírus da Doença de Borna/genética , Genoma , Interações Hospedeiro-Patógeno/genética , Modelos Genéticos , Proteínas do Nucleocapsídeo/genética , Oligodendroglia/virologia , Adaptação Biológica , Animais , Coevolução Biológica , Doença de Borna/genética , Doença de Borna/virologia , Vírus da Doença de Borna/metabolismo , Linhagem Celular , Humanos , Proteínas do Nucleocapsídeo/metabolismo , Oligodendroglia/metabolismo , Sciuridae/genética , Sciuridae/virologia , Replicação ViralRESUMO
Highly active antiretroviral treatment has led to unprecedented efficacy and tolerability in people living with HIV. This effect was also observed in the central nervous system with the nowadays uncommon observation of dementias; yet in more recent works milder forms are still reported in 20-30% of optimally treated individuals. The idea of a subclinical neuronal toxicity induced by antiretrovirals has been proposed and was somehow supported by the late-emerging effects associated with efavirenz use. In this manuscript we are reviewing all the potential mechanisms by which antiretroviral drugs have been associated with in vitro, ex vivo, or in vivo toxicity to cells pertaining to the central nervous system (neurons, astrocytes, oligodendrocytes, and endothelial cells). These include direct or indirect effects and pathological pathways such as amyloid deposition, damage to small cerebral vessels, and impairment in neurotransmission. The aim of this review is therefore to provide a detailed description of the available literature in order to guide further clinical research for improving patients' neurocognition and quality of life.
Assuntos
Alcinos/toxicidade , Fármacos Anti-HIV/toxicidade , Benzoxazinas/toxicidade , Sistema Nervoso Central/efeitos dos fármacos , Disfunção Cognitiva/induzido quimicamente , Ciclopropanos/toxicidade , Infecções por HIV/tratamento farmacológico , Neurônios/efeitos dos fármacos , Terapia Antirretroviral de Alta Atividade/métodos , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Astrócitos/virologia , Sulfato de Atazanavir/toxicidade , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Disfunção Cognitiva/patologia , Disfunção Cognitiva/prevenção & controle , Disfunção Cognitiva/virologia , Didesoxinucleosídeos/toxicidade , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/virologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Neurônios/patologia , Neurônios/virologia , Nevirapina/toxicidade , Nitrilas/toxicidade , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Oligodendroglia/virologia , Pirimidinas/toxicidadeRESUMO
Endocytosis is a pathway used by viruses to enter cells that can be classified based on the proteins involved, such as dynamin, clathrin or caveolin. Although the entry of herpes simplex type 1 (HSV-1) by endocytosis has been documented in different cell types, its dependence on clathrin has not been described whereas its dependence on dynamin has been shown according to the cell line used. The present work shows how clathrin-mediated endocytosis (CME) is one way that HSV-1 infects the human oligodendroglial (HOG) cell line. Partial dynamin inhibition using dynasore revealed a relationship between decrease of infection and dynamin inhibition, measured by viral titration and immunoblot. Co-localization between dynamin and HSV-1 was verified by immunofluorescence at the moment of viral entry into the cell. Inhibition by chlorpromazine revealed that viral progeny also decreased when clathrin was partially inhibited in our cell line. RT-qPCR of immediately early viral genes, specific entry assays and electron microscopy all confirmed clathrin's participation in HSV-1 entry into HOG cells. In contrast, caveolin entry assays showed no effect on the entry of this virus. Therefore, our results suggest the participation of dynamin and clathrin during endocytosis of HSV-1 in HOG cells.
Assuntos
Caveolinas/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose/fisiologia , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Oligodendroglia/virologia , Internalização do Vírus , Linhagem Celular , Clorpromazina/farmacologia , Endocitose/efeitos dos fármacos , Herpes Simples/metabolismo , Humanos , Hidrazonas/farmacologia , Microscopia Eletrônica , Microscopia de Fluorescência , Nistatina/farmacologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Myelin and lymphocyte protein (MAL) is a tetraspan integral membrane protein that resides in detergent-insoluble membrane fractions enriched in condensed membranes. MAL is expressed in oligodendrocytes, in Schwann cells, where it is essential for the stability of myelin, and at the apical membrane of epithelial cells, where it has a critical role in transport. In T lymphocytes, MAL is found at the immunological synapse and plays a crucial role in exosome secretion. However, no involvement of MAL in viral infections has been reported so far. Here, we show that herpes simplex virus 1 (HSV-1) virions travel in association with MAL-positive structures to reach the end of cellular processes, which contact uninfected oligodendrocytes. Importantly, the depletion of MAL led to a significant decrease in infection, with a drastic reduction in the number of lytic plaques in MAL-silenced cells. These results suggest a significant role for MAL in viral spread at cell contacts. The participation of MAL in the cell-to-cell spread of HSV-1 may shed light on the involvement of proteolipids in this process.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establish latent infections in neurons. HSV-1 may spread from infected to uninfected cells by two main routes: by cell-free virus or by cell-to-cell spread. In the first case, virions exit into the extracellular space and then infect another cell from the outside. In the second case, viral transmission occurs through cell-to-cell contacts via a mechanism that is still poorly understood. A third mode of spread, using extracellular vesicles, also exists. In this study, we demonstrate the important role for a myelin protein, myelin and lymphocyte protein (MAL), in the process of cell-to-cell viral spread in oligodendrocytes. We show that MAL is involved in trafficking of virions along cell processes and that MAL depletion produces a significant alteration in the viral cycle, which reduces cell-to cell spread of HSV-1.
Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Humanos , Linfócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Mielina/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/química , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/fisiologia , Neurônios/metabolismo , Neurônios/virologia , Oligodendroglia/metabolismo , Oligodendroglia/virologia , Proteolipídeos/química , Proteolipídeos/metabolismo , Linfócitos T/metabolismoRESUMO
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 ViralRESUMO
Treatment options for chronic spinal cord injury (SCI) remain limited due to unfavourable changes in the microenvironment. Gene therapy can overcome these barriers through continuous delivery of therapeutic gene products to the target tissue. In particular, adeno-associated virus (AAV) vectors are potential candidates for use in chronic SCI, considering their safety and stable gene expression in vivo. Given that different AAV serotypes display different cellular tropisms, it is extremely important to select an optimal serotype for establishing a gene transfer system during the chronic phase of SCI. Therefore, we generated multiple AAV serotypes expressing ffLuc-cp156, a fusion protein of firefly luciferase and Venus, a variant of yellow fluorescent protein with fast and efficient maturation, as a reporter, and we performed intraparenchymal injection in a chronic SCI mouse model. Among the various serotypes tested, AAVrh10 displayed the highest photon count on bioluminescence imaging. Immunohistological analysis revealed that AAVrh10 showed favourable tropism for neurons, astrocytes, and oligodendrocytes. Additionally, with AAVrh10, the area expressing Venus was larger in the injury epicentre and extended to the surrounding tissue. Furthermore, the fluorescence intensity was significantly higher with AAVrh10 than with the other vectors. These results indicate that AAVrh10 may be an appropriate serotype for gene delivery to the chronically injured spinal cord. This promising tool may be applied for research and development related to the treatment of chronic SCI.
Assuntos
Proteínas de Bactérias/genética , Dependovirus/fisiologia , Luciferases de Vaga-Lume/genética , Proteínas Luminescentes/genética , Traumatismos da Medula Espinal/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/virologia , Proteínas de Bactérias/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Luciferases de Vaga-Lume/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Neurônios/metabolismo , Neurônios/virologia , Oligodendroglia/metabolismo , Oligodendroglia/virologia , Proteínas Recombinantes de Fusão/administração & dosagem , Traumatismos da Medula Espinal/genética , Tropismo ViralRESUMO
Herpes simplex virus type 1 (HSV-1) is a ubiquitous infectious agent that can establish latency in neurons, and in some cases, viral retrograde transport results in infection of the central nervous system (CNS). Several antivirals have been identified with the ability to inhibit HSV-1 replication in human cells to a greater or lesser degree, most of which are nucleoside analogues that unfortunately exhibit teratogenic potential, embryotoxicity, carcinogenic or antiproliferative activities and resistances in immunocompromised patients, specially. In the present study, we assessed two amidic derivatives of valproic acid (VPA) - valpromide (VPD) and valnoctamide (VCD) - which are already used in clinic treatments, as feasible HSV-1 antivirals in glial cells. Both VPD and VCD have exhibited increased efficacy in bipolar disorders and as anticonvulsant drugs compared to VPA, while being less teratogenic and hepatotoxic. Cytotoxicity assays carried out in our laboratory showed that VPD and VCD were not toxic in a human oligodendroglioma cell line (HOG), at least at the concentrations established for human treatments. Infectivity assays showed a significant inhibition of HSV-1 infection in HOG cells after VPD and VCD treatment, being more pronounced in VPD-treated cells, comparable to the effects obtained with acyclovir. Furthermore, the same antiherpetic effects of VPD were observed in other oligodendrocytic cell lines and rat primary oligodendrocytes (OPCs), confirming the results obtained in HOG cells. Altogether, our results allow us to propose VPD as a potential antiherpetic drug that is able to act directly on oligodendrocytes of the CNS.
Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Oligodendroglia/virologia , Ácido Valproico/análogos & derivados , Amidas/química , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Estrutura Molecular , Oligodendroglia/efeitos dos fármacos , Ratos , Ácido Valproico/química , Ácido Valproico/farmacologia , Proteínas Virais/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Fatal neurological syndromes can occur after measles virus (MeV) infection of the brain. The mechanisms controlling MeV spread within the central nervous system (CNS) remain poorly understood. We analyzed the role of type I interferon (IFN-I) receptor (IFNAR) signaling in the control of MeV infection in a murine model of brain infection. Using organotypic brain cultures (OBC) from wild-type and IFNAR-knockout (IFNARKO) transgenic mice ubiquitously expressing the human SLAM (CD150) receptor, the heterogeneity of the permissiveness of different CNS cell types to MeV infection was characterized. In the absence of IFNAR signaling, MeV propagated significantly better in explant slices. In OBC from IFNAR-competent mice, while astrocytes and microglia were infected on the day of explant preparation, they became refractory to infection with time, in contrast to neurons and oligodendrocytes, which remained permissive to infection. This selective loss of permissiveness to MeV infection was not observed in IFNARKO mouse OBC. Accordingly, the development of astrogliosis related to the OBC procedure was exacerbated in the presence of IFNAR signaling. In the hippocampus, this astrogliosis was characterized by a change in the astrocyte phenotype and by an increase of IFN-I transcripts. A proteome analysis showed the upregulation of 84 out of 111 secreted proteins. In the absence of IFNAR, only 27 secreted proteins were upregulated, and none of these were associated with antiviral activities. Our results highlight the essential role of the IFN-I response in astrogliosis and in the permissiveness of astrocytes and microglia that could control MeV propagation throughout the CNS.IMPORTANCE Measles virus (MeV) can infect the central nervous system (CNS), with dramatic consequences. The mechanisms controlling MeV invasion of the CNS remain ill-defined since most previous data were obtained from postmortem analysis. Here, we highlight for the first time the crucial role of the type I interferon (IFN-I) response not only in the control of CNS invasion but also in the early permissiveness of glial cells to measles virus infection.
Assuntos
Astrócitos/virologia , Vírus do Sarampo/metabolismo , Sarampo/metabolismo , Microglia/virologia , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/fisiologia , Animais , Antivirais/farmacologia , Astrócitos/patologia , Encéfalo/virologia , Sistema Nervoso Central/virologia , Citocinas , Feminino , Hipocampo/patologia , Hipocampo/virologia , Humanos , Masculino , Sarampo/patologia , Sarampo/virologia , Camundongos , Camundongos Knockout , Neurônios/virologia , Oligodendroglia/virologia , Receptor de Interferon alfa e beta/genética , Transdução de Sinais/genética , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismoRESUMO
JC virus (JCV) can cause a lytic infection of oligodendrocytes and astrocytes in the central nervous system (CNS) leading to progressive multifocal leukoencephalopathy (PML). JCV can also infect meningeal and choroid plexus cells causing JCV meningitis (JCVM). Whether JCV also infects meningeal and choroid plexus cells in PML patients and other immunosuppressed individuals with no overt symptoms of meningitis remains unknown. We therefore analyzed archival formalin-fixed, paraffin-embedded brain samples from PML patients, and HIV-seropositive and seronegative control subjects by immunohistochemistry for the presence of JCV early regulatory T Ag and JCV VP1 late capsid protein. In meninges, we detected JCV T Ag in 11/48 (22.9%) and JCV VP1 protein in 8/48 (16.7%) PML patients. In choroid plexi, we detected JCV T Ag in 1/7 (14.2%) and JCV VP1 protein in 1/8 (12.5%) PML patients. Neither JCV T Ag nor VP1 protein could be detected in meninges or choroid plexus of HIV-seropositive and HIV-seronegative control subjects without PML. In addition, examination of underlying cerebellar cortex of PML patients revealed JCV-infected cells in the molecular layer, including GAD 67+ interneurons, but not in HIV-seropositive and HIV-seronegative control subjects without PML. Our findings suggest that productive JCV infection of meningeal cells and choroid plexus cells also occurs in PML patients without signs or symptoms of meningitis. The phenotypic characterization of JCV-infected neurons in the molecular layer deserves further study. This data provides new insight into JCV pathogenesis in the CNS.
Assuntos
Astrócitos/virologia , Plexo Corióideo/virologia , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/virologia , Meninges/virologia , Neurônios/virologia , Oligodendroglia/virologia , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Astrócitos/patologia , Autopsia , Biomarcadores/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Córtex Cerebelar/patologia , Córtex Cerebelar/virologia , Plexo Corióideo/patologia , Expressão Gênica , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , HIV/genética , HIV/patogenicidade , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Imuno-Histoquímica , Vírus JC/patogenicidade , Leucoencefalopatia Multifocal Progressiva/patologia , Meninges/patologia , Neurônios/patologia , Oligodendroglia/patologiaRESUMO
Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in the neurons of sensory ganglia. In some cases, the virus spreads into the central nervous system, causing encephalitis or meningitis. Cells infected with several different types of viruses may secrete microvesicles (MVs) containing viral proteins and RNAs. In some instances, extracellular microvesicles harboring infectious virus have been found. Here we describe the features of shedding microvesicles released by the human oligodendroglial HOG cell line infected with HSV-1 and their participation in the viral cycle. Using transmission electron microscopy, we detected for the first time microvesicles containing HSV-1 virions. Interestingly, the Chinese hamster ovary (CHO) cell line, which is resistant to infection by free HSV-1 virions, was susceptible to HSV-1 infection after being exposed to virus-containing microvesicles. Therefore, our results indicate for the first time that MVs released by infected cells contain virions, are endocytosed by naive cells, and lead to a productive infection. Furthermore, infection of CHO cells was not completely neutralized when virus-containing microvesicles were preincubated with neutralizing anti-HSV-1 antibodies. The lack of complete neutralization and the ability of MVs to infect nectin-1/HVEM-negative CHO-K1 cells suggest a novel way for HSV-1 to spread to and enter target cells. Taken together, our results suggest that HSV-1 could spread through microvesicles to expand its tropism and that microvesicles could shield the virus from neutralizing antibodies as a possible mechanism to escape the host immune response.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons. Extracellular vesicles are a heterogeneous group of membrane vesicles secreted by most cell types. Microvesicles, which are extracellular vesicles which derive from the shedding of the plasma membrane, isolated from the supernatant of HSV-1-infected HOG cells were analyzed to find out whether they were involved in the viral cycle. The importance of our investigation lies in the detection, for the first time, of microvesicles containing HSV-1 virions. In addition, virus-containing microvesicles were endocytosed into CHO-K1 cells and were able to actively infect these otherwise nonpermissive cells. Finally, the infection of CHO cells with these virus-containing microvesicles was not completely neutralized by anti-HSV-1 antibodies, suggesting that these extracellular vesicles might shield the virus from neutralizing antibodies as a possible mechanism of immune evasion.
Assuntos
Micropartículas Derivadas de Células/virologia , Herpes Simples/transmissão , Herpesvirus Humano 1/fisiologia , Oligodendroglia/virologia , Replicação Viral/fisiologia , Animais , Anticorpos Antivirais/imunologia , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetulus , Endocitose , Células HeLa , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Microscopia Eletrônica de Transmissão , Oligodendroglia/citologia , Células Vero , Internalização do VírusRESUMO
Progressive multifocal leukoencephalopathy (PML) is an often-fatal demyelinating disease of the central nervous system. PML results when oligodendrocytes within immunocompromised individuals are infected with the human JC virus (JCV). We have identified an oligodendrocyte precursor cell line, termed G144, that supports robust levels of JCV DNA replication, a central part of the JCV life cycle. In addition, we have determined that JC virus readily infects G144 cells. Furthermore, we have determined that JCV DNA replication in G144 cells is stimulated by myristoylated (i.e., constitutively active) Akt and reduced by the Akt-specific inhibitor MK2206. Thus, this oligodendrocyte-based model system will be useful for a number of purposes, such as studies of JCV infection, establishing key pathways needed for the regulation of JCV DNA replication, and identifying inhibitors of this process.IMPORTANCE The disease progressive multifocal leukoencephalopathy (PML) is caused by the infection of particular brain cells, termed oligodendrocytes, by the JC virus. Studies of PML, however, have been hampered by the lack of an immortalized human cell line derived from oligodendrocytes. Here, we report that the G144 oligodendrocyte cell line supports both infection by JC virus and robust levels of JCV DNA replication. Moreover, we have established that the Akt pathway regulates JCV DNA replication and that JCV DNA replication can be inhibited by MK2206, a compound that is specific for Akt. These and related findings suggest that we have established a powerful oligodendrocyte-based model system for studies of JCV-dependent PML.