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1.
PLoS One ; 15(1): e0227676, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935257

RESUMO

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

2.
Cell Rep ; 29(8): 2202-2216.e5, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31747595

RESUMO

Generation of protective immunity to infections and vaccinations declines with age. Studies in healthy individuals have implicated reduced miR-181a expression in T cells as contributing to this defect. To understand the impact of miR-181a expression on antiviral responses, we examined LCMV infection in mice with miR-181ab1-deficient T cells. We found that miR-181a deficiency delays viral clearance, thereby biasing the immune response in favor of CD4 over CD8 T cells. Antigen-specific CD4 T cells in mice with miR-181a-deficient T cells expand more and have a broader TCR repertoire with preferential expansion of high-affinity T cells than in wild-type mice. Importantly, generation of antigen-specific miR-181a-deficient CD8 effector T cells is particularly impaired, resulting in lower frequencies of CD8 T cells in the liver even at time points when the infection has been cleared. Consistent with the mouse model, CD4 memory T cells in individuals infected with West Nile virus at older ages tend to be more frequent and of higher affinity.

3.
J Infect Dis ; 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31616920

RESUMO

Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract has raised concerned for potential damaging effects on function. Animal studies have demonstrated that ZIKV virus can infect and damage the testis and epididymis, and these results has been correlated to lower sperm counts in ZIKV-infected humans. The prostate plays a vital role in the male reproductive tract, with acute and chronic prostatitis linked to male infertility. Here, we evaluated the effects of ZIKV virus on the prostate in mice and non-human primates. In mice, ZIKV infected the prostate and triggered inflammation that persisted even after virus clearance. Evidence of chronic prostatitis associated with ZIKV infection remained for several months. Similar histological findings were observed in the prostate of ZIKV-infected rhesus macaques. These studies establish that ZIKV replicates in the prostate and can cause acute and chronic inflammatory and proliferative changes in mouse and non-human primate models.

4.
Nat Med ; 24(8): 1104-1107, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29967348

RESUMO

Zika virus (ZIKV) infection is associated with congenital defects and pregnancy loss. Here, we found that 26% of nonhuman primates infected with Asian/American ZIKV in early gestation experienced fetal demise later in pregnancy despite showing few clinical signs of infection. Pregnancy loss due to asymptomatic ZIKV infection may therefore be a common but under-recognized adverse outcome related to maternal ZIKV infection.


Assuntos
Aborto Espontâneo/virologia , Natimorto/veterinária , Infecção por Zika virus/veterinária , Zika virus/fisiologia , Animais , Feminino , Estimativa de Kaplan-Meier , Masculino , Gravidez , Primatas
5.
Antiviral Res ; 155: 12-19, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29709563

RESUMO

Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions and cause significant morbidity and mortality worldwide. Although a partially effective vaccine is in use in several countries in which DENV are endemic, no antiviral therapeutics are approved for combating DENV-associated disease. Herein, we report the characterization of novel small molecule inhibitors of DENV replication, VGTI-A3 and VGTI-A3-03, as well as structure-activity relationship analysis of the molecules using a panel of chemical analogs. VGTI-A3 and VGTI-A3-03 are highly virus-specific, with greatest activity against DENV serotype 2. Further analysis revealed that treatment of infected cells with VGTI-A3-03 does not inhibit viral RNA replication or secretion of viral particles. Rather, the infectivity of secreted particles from A3-03 treated cells is significantly diminished compared to particles secreted from control cells. Elicitation of VGTI-A3-03-resistant mutants demonstrated a clear binding pocket in the capsid molecule at the dimerization interface. Additionally, we show that VGTI-A3-03 is incorporated into virus particles released from infected cells. In summary, these data provide detailed analysis of a potentially useful class of anti-DENV inhibitors and further identify a region of the viral capsid protein as a druggable target for other therapeutic approaches.


Assuntos
Antivirais/química , Proteínas do Capsídeo/metabolismo , Vírus da Dengue/efeitos dos fármacos , Vírion/efeitos dos fármacos , Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Dengue/virologia , Células HEK293 , Humanos , Mutagênese , Ligação Proteica , RNA Viral , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
6.
Nat Commun ; 9(1): 263, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343712

RESUMO

Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.


Assuntos
Placenta/metabolismo , Circulação Placentária , Complicações Infecciosas na Gravidez/imunologia , Infecção por Zika virus/imunologia , Imunidade Adaptativa , Animais , Encéfalo/embriologia , Encéfalo/patologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Desenvolvimento Fetal , Feto/patologia , Imunidade Inata , Macaca mulatta , Imagem por Ressonância Magnética , Oxigênio/metabolismo , Permeabilidade , Placenta/imunologia , Placenta/patologia , Placenta/virologia , Gravidez , Complicações Infecciosas na Gravidez/metabolismo , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/fisiopatologia , Carga Viral , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia , Infecção por Zika virus/fisiopatologia
7.
mBio ; 8(3)2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28465426

RESUMO

The ongoing concurrent outbreaks of Zika, Chikungunya, and dengue viruses in Latin America and the Caribbean highlight the need for development of broad-spectrum antiviral treatments. The type I interferon (IFN) system has evolved in vertebrates to generate tissue responses that actively block replication of multiple known and potentially zoonotic viruses. As such, its control and activation through pharmacological agents may represent a novel therapeutic strategy for simultaneously impairing growth of multiple virus types and rendering host populations resistant to virus spread. In light of this strategy's potential, we undertook a screen to identify novel interferon-activating small molecules. Here, we describe 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione, which we termed AV-C. Treatment of human cells with AV-C activates innate and interferon-associated responses that strongly inhibit replication of Zika, Chikungunya, and dengue viruses. By utilizing genome editing, we investigated the host proteins essential to AV-C-induced cellular states. This showed that the compound requires a TRIF-dependent signaling cascade that culminates in IFN regulatory factor 3 (IRF3)-dependent expression and secretion of type I interferon to elicit antiviral responses. The other canonical IRF3-terminal adaptor proteins STING and IPS-1/MAVS were dispensable for AV-C-induced phenotypes. However, our work revealed an important inhibitory role for IPS-1/MAVS, but not TRIF, in flavivirus replication, implying that TRIF-directed viral evasion may not occur. Additionally, we show that in response to AV-C, primary human peripheral blood mononuclear cells secrete proinflammatory cytokines that are linked with establishment of adaptive immunity to viral pathogens. Ultimately, synthetic innate immune activators such as AV-C may serve multiple therapeutic purposes, including direct antimicrobial responses and facilitation of pathogen-directed adaptive immunity.IMPORTANCE The type I interferon system is part of the innate immune response that has evolved in vertebrates as a first line of broad-spectrum immunological defense against an unknowable diversity of microbial, especially viral, pathogens. Here, we characterize a novel small molecule that artificially activates this response and in so doing generates a cellular state antagonistic to growth of currently emerging viruses: Zika virus, Chikungunya virus, and dengue virus. We also show that this molecule is capable of eliciting cellular responses that are predictive of establishment of adaptive immunity. As such, this agent may represent a powerful and multipronged therapeutic tool to combat emerging and other viral diseases.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/agonistas , Antivirais/farmacologia , Benzopiranos/farmacologia , Vírus Chikungunya/fisiologia , Vírus da Dengue/fisiologia , Tiadiazóis/farmacologia , Replicação Viral , Zika virus/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/química , Antivirais/isolamento & purificação , Benzopiranos/química , Benzopiranos/isolamento & purificação , Linhagem Celular , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/efeitos dos fármacos , Citocinas/biossíntese , Replicação do DNA/efeitos dos fármacos , Dengue/tratamento farmacológico , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/metabolismo , Descoberta de Drogas , Edição de Genes , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Imunidade Inata/efeitos dos fármacos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/efeitos dos fármacos , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Tiadiazóis/química , Tiadiazóis/isolamento & purificação , Zika virus/efeitos dos fármacos
9.
PLoS Pathog ; 13(3): e1006219, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28278237

RESUMO

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.


Assuntos
Infecção por Zika virus/patologia , Infecção por Zika virus/virologia , Animais , Separação Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hibridização In Situ , Macaca mulatta , Masculino , Testes de Neutralização , Reação em Cadeia da Polimerase , Viremia/virologia , Zika virus
10.
J Virol ; 91(8)2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28148804

RESUMO

The impact of mosquito-borne flavivirus infections worldwide is significant, and many critical aspects of these viruses' biology, including virus-host interactions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent reemergence and spread of flaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlight the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression posttranscriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and antiflaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on the replication of three flaviviruses, DENV, WNV, and Japanese encephalitis virus (JEV), using a high-content immunofluorescence screen. Several families of miRNAs were identified as inhibiting multiple flaviviruses, including the miRNA miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/ß-catenin signaling, demonstrated strong antiflaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and confirmed that these miRNAs potentiate interferon regulatory factor 3 (IRF3) phosphorylation and translocation to the nucleus, the induction of IFN-responsive genes, and the release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of glycogen synthase kinase 3ß (GSK3ß)-TANK-binding kinase 1 (TBK1) binding, inducing TBK1 to phosphorylate IRF3 and initiate downstream IFN signaling. In this way, we have identified a novel cellular signaling network with a critical role in regulating the replication of multiple virus families. These findings highlight the opportunities for using miRNAs as tools to discover and characterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research.IMPORTANCE MicroRNAs are a class of small regulatory RNAs that modulate cellular processes through the posttranscriptional repression of multiple transcripts. We hypothesized that individual miRNAs may be capable of inhibiting viral replication through their effects on host proteins or pathways. To test this, we performed a high-content screen for miRNAs that inhibit the replication of three medically relevant members of the flavivirus family: West Nile virus, Japanese encephalitis virus, and dengue virus 2. The results of this screen identify multiple miRNAs that inhibit one or more of these viruses. Extensive follow-up on members of the miR-34 family of miRNAs, which are active against all three viruses as well as the closely related Zika virus, demonstrated that miR-34 functions through increasing the infected cell's ability to respond to infection through the interferon-based innate immune pathway. Our results not only add to the knowledge of how viruses interact with cellular pathways but also provide a basis for more extensive data mining by providing a comprehensive list of miRNAs capable of inhibiting flavivirus replication. Finally, the miRNAs themselves or cellular pathways identified as modulating virus infection may prove to be novel candidates for the development of therapeutic interventions.


Assuntos
Vírus da Dengue/imunologia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Interações Hospedeiro-Patógeno , Interferons/imunologia , MicroRNAs/metabolismo , Vírus do Nilo Ocidental/imunologia , Via de Sinalização Wnt , Vírus da Dengue/fisiologia , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Regulação da Expressão Gênica , MicroRNAs/genética , Replicação Viral , Vírus do Nilo Ocidental/fisiologia
11.
J Virol ; 88(10): 5533-42, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24599995

RESUMO

UNLABELLED: Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds-dihydrodibenzothiepines (DHBTs), identified through high-throughput screening-with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds. IMPORTANCE: The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.


Assuntos
Antivirais/metabolismo , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Dopamina D4/antagonistas & inibidores , Transdução de Sinais , Replicação Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Vírus Sindbis/efeitos dos fármacos , Vírus Sindbis/fisiologia , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/fisiologia
12.
J Virol ; 87(17): 9411-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23824813

RESUMO

Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.


Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/metabolismo , Flavivirus/efeitos dos fármacos , Flavivirus/metabolismo , Timidina/biossíntese , Animais , Linhagem Celular , Vírus de DNA/efeitos dos fármacos , Vírus da Dengue/fisiologia , Modelos Animais de Doenças , Flavivirus/fisiologia , Infecções por Flavivirus/tratamento farmacológico , Floxuridina/farmacologia , Células HEK293 , Células HeLa , Humanos , Leucovorina/farmacologia , Metotrexato/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Vírus de RNA/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/fisiologia
13.
J Virol ; 86(9): 5278-87, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22345437

RESUMO

MicroRNAs (miRNAs) are a class of noncoding small RNAs that regulate multiple cellular processes, as well as the replication and pathogenesis of many DNA viruses and some RNA viruses. Examination of cellular miRNA profiles in West Nile virus (WNV)-infected HEK293 and SK-N-MC cells revealed increased expression of multiple miRNA species. One of these miRNAs, Hs_154, was significantly induced not only in WNV-infected neuronal cells in culture but also in the central nervous system tissues of infected mice and, upon transfection, caused a significant reduction in viral replication. Analysis of mRNA transcripts enriched through immunoprecipitation of the RNA-induced silencing complex identified several transcripts that contain seed sequence matches to Hs_154 in their 3' untranslated regions (UTRs). Two of these targets, the CCCTC-binding factor (CTCF) and the epidermal growth factor receptor (EGFR)-coamplified and overexpressed protein (ECOP/VOPP1) proteins display reduced expression in WNV-infected cells, and the 3' UTRs of these transcripts were sufficient to cause downregulation of expression in infected cells or in cells transfected with Hs_154, findings consistent with miRNA targeting of these transcripts. CTCF and ECOP have been shown to be associated with cell survival, implicating miRNA-directed repression of these targets in WNV-induced cell death. Consistent with this hypothesis, expression of these genes in WNV-infected cells results in a reduction in the number of cells undergoing apoptosis. These observations suggest that induction of Hs_154 expression after WNV infection modulates the apoptotic response to WNV and that cellular miRNA expression can be quickly altered during WNV infection to control aspects of the host response.


Assuntos
Apoptose/genética , MicroRNAs/biossíntese , Vírus do Nilo Ocidental/fisiologia , Animais , Sequência de Bases , Fator de Ligação a CCCTC , Caspases/metabolismo , Linhagem Celular , Análise por Conglomerados , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Interferons/metabolismo , Cinética , Camundongos , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Replicação Viral/genética
14.
J Virol ; 85(19): 10154-66, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21795337

RESUMO

The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5' cyclization sequence (5'CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an "in vivo-ready" version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.


Assuntos
Antivirais/administração & dosagem , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Animais , Anticorpos Facilitadores , Produtos Biológicos/administração & dosagem , Produtos Biológicos/farmacologia , Peso Corporal , Técnicas de Cultura de Células , Sequência Conservada , Dengue/patologia , Dengue/virologia , Vírus da Dengue/genética , Modelos Animais de Doenças , Humanos , Camundongos , RNA Interferente Pequeno/genética , Doenças dos Roedores/tratamento farmacológico , Doenças dos Roedores/patologia , Doenças dos Roedores/virologia , Análise de Sobrevida
15.
Assay Drug Dev Technol ; 8(5): 553-70, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20973722

RESUMO

Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration-approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC50 potencies ranging from 60 nM to 9 µM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.


Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Animais , Antivirais/química , Antivirais/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Vírus da Dengue/fisiologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Microscopia Confocal , Miniaturização , Software , Proteínas Virais/metabolismo , Ligação Viral/efeitos dos fármacos
16.
Future Microbiol ; 5(2): 303-11, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20143951

RESUMO

The recent development of RNAi-based techniques for protein knockdown in mammalian cells has allowed for unprecedented flexibility in the study of protein function. Currently, large siRNA libraries are available that allow the knockdown of all proteins known to be encoded by the human genome. These libraries have been used to identify the host proteins required for the replication of several clinically important viruses, including HIV, flaviviruses and influenza. This review summarizes the methods used in RNAi-based screening for host factors involved in virus replication, and discusses published examples of such screens.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Interações Hospedeiro-Patógeno , Proteínas/antagonistas & inibidores , Interferência de RNA , Replicação Viral , Vírus/patogenicidade , Humanos , Proteínas/genética
17.
J Virol ; 83(12): 6125-34, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19369347

RESUMO

During acute infection, West Nile virus (WNV) has been reported to infect a variety of cell types in various tissues of both experimentally and naturally infected hosts. Virus infects epithelial cells in the skin, kidney, intestine, and testes, although the importance of these findings is unclear. In the current study, we have observed that WNV infection of kidney tubules in mice coincides with the loss of expression of several members of the claudin family. Proteins of this family are often involved in epithelial barrier formation and function. WNV infection of epithelial cells in culture resulted in a decrease in the transepithelial electrical resistance, an increase in the efflux of mannitol across the monolayer, and a loss of intracellular levels of claudin-1 to -4. WNV capsid alone was sufficient for the degradation event, which was mediated through lysosomal proteases. Since epithelial cells are frequent sites of WNV infection, these observations imply a potential mechanism for virus dissemination and extraneural pathogenesis.


Assuntos
Capsídeo/metabolismo , Túbulos Renais/virologia , Proteínas de Membrana/metabolismo , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Células CACO-2 , Permeabilidade da Membrana Celular , Claudina-1 , Claudina-3 , Claudinas , Células Epiteliais/virologia , Humanos , Manitol/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Junções Íntimas/virologia , Células Vero
18.
J Virol ; 82(11): 5212-9, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18385233

RESUMO

West Nile virus (WNV) has been the leading cause of viral encephalitis in the United States since 1999. The endocytic processes involved in the internalization of infectious WNV by various cell types are not well characterized, and the involvement of cholesterol-rich membrane microdomains, or lipid rafts, in the life cycle of WNV has not been investigated previously. In this study, we found that the depletion of cellular cholesterol levels by brief treatment with methyl-beta-cyclodextrin resulted in a 100-fold reduction of the titers of infectious WNV released into the culture supernatant, as well as a reduction in the number of WNV genome copies in the cholesterol-depleted cells. The addition of exogenous cholesterol to cholesterol-depleted cells reversed this effect. Cholesterol depletion postinfection did not affect WNV growth, suggesting that the effect occurs at the level of WNV entry. We also showed that while WNV entry did not require alphavbeta3 integrin and focal adhesion kinase, WNV particles failed to be internalized by cholesterol-depleted cells. Finally, we showed the colocalization of the WNV envelope protein and cholera toxin B, which is internalized in a lipid raft-dependent pathway, in microdomain clusters at the plasma membrane. These data suggest that WNV utilizes lipid rafts during initial stages of internalization and that the lipid rafts may contain a factor(s) that may enhance WNV endocytosis.


Assuntos
Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Internalização do Vírus , Vírus do Nilo Ocidental/metabolismo , Animais , Linhagem Celular , Toxina da Cólera/farmacologia , Humanos , Integrina alfaVbeta3/deficiência , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , RNA Viral/genética , RNA Viral/metabolismo , Titulometria , Vírion/metabolismo , Vírus do Nilo Ocidental/genética
19.
J Virol ; 81(20): 10849-60, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17686866

RESUMO

West Nile virus (WNV)-mediated neuronal death is a hallmark of WNV meningitis and encephalitis. However, the mechanisms of WNV-induced neuronal damage are not well understood. We investigated WNV neuropathogenesis by using human neuroblastoma cells and primary rat hippocampal neurons. We observed that WNV activates multiple unfolded protein response (UPR) pathways, leading to transcriptional and translational induction of UPR target genes. We evaluated the role of the three major UPR pathways, namely, inositol-requiring enzyme 1-dependent splicing of X box binding protein 1 (XBP1) mRNA, activation of activating transcription factor 6 (ATF6), and protein kinase R-like endoplasmic reticulum (ER) kinase-dependent eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, in WNV-infected cells. We show that XBP1 is nonessential or can be replaced by other UPR pathways in WNV replication. ATF6 was rapidly degraded by proteasomes, consistent with induction of ER stress by WNV. We further observed a transient phosphorylation of eIF2alpha and induction of the proapoptotic cyclic AMP response element-binding transcription factor homologous protein (CHOP). WNV-infected cells exhibited a number of apoptotic phenotypes, such as (i) induction of growth arrest and DNA damage-inducible gene 34, (ii) activation of caspase-3, and (iii) cleavage of poly(ADP-ribose) polymerase. The expression of WNV nonstructural proteins alone was sufficient to induce CHOP expression. Importantly, WNV grew to significantly higher viral titers in chop(-)(/)(-) mouse embryonic fibroblasts (MEFs) than in wild-type MEFs, suggesting that CHOP-dependent premature cell death represents a host defense mechanism to limit viral replication that might also be responsible for the widespread neuronal loss observed in WNV-infected neuronal tissue.


Assuntos
Apoptose , Neurônios/virologia , Fator de Transcrição CHOP/genética , Febre do Nilo Ocidental/patologia , Fator 6 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Neurônios/patologia , Proteínas Nucleares/genética , Ratos , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição , Replicação Viral , Febre do Nilo Ocidental/etiologia , Febre do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/patogenicidade , Proteína 1 de Ligação a X-Box
20.
Antimicrob Agents Chemother ; 51(7): 2642-5, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17452483

RESUMO

We have identified sultam thioureas as novel inhibitors of West Nile virus (WNV) replication. One such compound inhibited WNV, with a 50% effective concentration of 0.7 microM, and reduced reporter expression from cells that harbored a WNV-based replicon. Our results demonstrate that sultam thioureas can block a postentry, preassembly step of WNV replication.


Assuntos
Antivirais/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Vírus do Nilo Ocidental/efeitos dos fármacos , Animais , Antivirais/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Estrutura Molecular , RNA Viral/efeitos dos fármacos , Replicon/efeitos dos fármacos , Replicon/genética , Relação Estrutura-Atividade , Tioureia/química , Células Vero , Replicação Viral/efeitos dos fármacos , Vírus do Nilo Ocidental/fisiologia
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