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1.
Nat Immunol ; 22(11): 1428-1439, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34471264

RESUMO

Coordinated local mucosal and systemic immune responses following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection either protect against coronavirus disease 2019 (COVID-19) pathologies or fail, leading to severe clinical outcomes. To understand this process, we performed an integrated analysis of SARS-CoV-2 spike-specific antibodies, cytokines, viral load and bacterial communities in paired nasopharyngeal swabs and plasma samples from a cohort of clinically distinct patients with COVID-19 during acute infection. Plasma viral load was associated with systemic inflammatory cytokines that were elevated in severe COVID-19, and also with spike-specific neutralizing antibodies. By contrast, nasopharyngeal viral load correlated with SARS-CoV-2 humoral responses but inversely with interferon responses, the latter associating with protective microbial communities. Potential pathogenic microorganisms, often implicated in secondary respiratory infections, were associated with mucosal inflammation and elevated in severe COVID-19. Our results demonstrate distinct tissue compartmentalization of SARS-CoV-2 immune responses and highlight a role for the nasopharyngeal microbiome in regulating local and systemic immunity that determines COVID-19 clinical outcomes.


Assuntos
COVID-19/imunologia , Microbiota/imunologia , Nasofaringe/imunologia , SARS-CoV-2/fisiologia , Doença Aguda , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Estudos de Coortes , Feminino , Humanos , Imunidade Humoral , Imunidade nas Mucosas , Interferons/sangue , Masculino , Pessoa de Meia-Idade , Nasofaringe/microbiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Carga Viral , Adulto Jovem
2.
Nature ; 580(7802): 274-277, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269344

RESUMO

Tandem repeat elements such as the diverse class of satellite repeats occupy large parts of eukaryotic chromosomes, mostly at centromeric, pericentromeric, telomeric and subtelomeric regions1. However, some elements are located in euchromatic regions throughout the genome and have been hypothesized to regulate gene expression in cis by modulating local chromatin structure, or in trans via transcripts derived from the repeats2-4. Here we show that a satellite repeat in the mosquito Aedes aegypti promotes sequence-specific gene silencing via the expression of two PIWI-interacting RNAs (piRNAs). Whereas satellite repeats and piRNA sequences generally evolve extremely quickly5-7, this locus was conserved for approximately 200 million years, suggesting that it has a central function in mosquito biology. piRNA production commenced shortly after egg laying, and inactivation of the more abundant piRNA resulted in failure to degrade maternally deposited transcripts in the zygote and developmental arrest. Our results reveal a mechanism by which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing that is essential for embryonic development.


Assuntos
Aedes/embriologia , Aedes/genética , DNA Satélite/genética , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Feminino , Inativação Gênica
3.
PLoS Genet ; 16(5): e1008794, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463828

RESUMO

Although specific interactions between host and pathogen genotypes have been well documented in invertebrates, the identification of host genes involved in discriminating pathogen genotypes remains a challenge. In the mosquito Aedes aegypti, the main dengue virus (DENV) vector worldwide, statistical associations between host genetic markers and DENV types or strains were previously detected, but the host genes underlying this genetic specificity have not been identified. In particular, it is unknown whether DENV type- or strain-specific resistance relies on allelic variants of the same genes or on distinct gene sets. Here, we investigated the genetic architecture of DENV resistance in a population of Ae. aegypti from Bakoumba, Gabon, which displays a stronger resistance phenotype to DENV type 1 (DENV-1) than to DENV type 3 (DENV-3) infection. Following experimental exposure to either DENV-1 or DENV-3, we sequenced the exomes of large phenotypic pools of mosquitoes that are either resistant or susceptible to each DENV type. Using variation in single-nucleotide polymorphism (SNP) frequencies among the pools, we computed empirical p values based on average gene scores adjusted for the differences in SNP counts, to identify genes associated with infection in a DENV type-specific manner. Among the top 5% most significant genes, 263 genes were significantly associated with resistance to both DENV-1 and DENV-3, 287 genes were only associated with DENV-1 resistance and 290 were only associated with DENV-3 resistance. The shared significant genes were enriched in genes with ATP binding activity and sulfur compound transmembrane transporter activity, whereas the genes uniquely associated with DENV-3 resistance were enriched in genes with zinc ion binding activity. Together, these results indicate that specific resistance to different DENV types relies on largely non-overlapping sets of genes in this Ae. aegypti population and pave the way for further mechanistic studies.


Assuntos
Aedes/genética , Vírus da Dengue/classificação , Resistência à Doença , Sequenciamento do Exoma/métodos , Proteínas de Insetos/genética , Aedes/virologia , Animais , Células Cultivadas , Vírus da Dengue/patogenicidade , Feminino , Gabão , Genótipo , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Especificidade da Espécie
4.
PLoS Biol ; 17(3): e2006146, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30860988

RESUMO

Stress responses are crucial processes that require activation of genetic programs that protect from the stressor. Stress responses are also energy consuming and can thus be deleterious to the organism. The mechanisms coordinating energy consumption during stress response in multicellular organisms are not well understood. Here, we show that loss of the epigenetic regulator G9a in Drosophila causes a shift in the transcriptional and metabolic responses to oxidative stress (OS) that leads to decreased survival time upon feeding the xenobiotic paraquat. During OS exposure, G9a mutants show overactivation of stress response genes, rapid depletion of glycogen, and inability to access lipid energy stores. The OS survival deficiency of G9a mutants can be rescued by a high-sugar diet. Control flies also show improved OS survival when fed a high-sugar diet, suggesting that energy availability is generally a limiting factor for OS tolerance. Directly limiting access to glycogen stores by knocking down glycogen phosphorylase recapitulates the OS-induced survival defects of G9a mutants. We propose that G9a mutants are sensitive to stress because they experience a net reduction in available energy due to (1) rapid glycogen use, (2) an inability to access lipid energy stores, and (3) an overinduced transcriptional response to stress that further exacerbates energy demands. This suggests that G9a acts as a critical regulatory hub between the transcriptional and metabolic responses to OS. Our findings, together with recent studies that established a role for G9a in hypoxia resistance in cancer cell lines, suggest that G9a is of wide importance in controlling the cellular and organismal response to multiple types of stress.


Assuntos
Histona Metiltransferases/metabolismo , Animais , Antioxidantes/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Epigênese Genética/genética , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Histona Metiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Filogenia , Análise de Sequência de RNA
5.
J Virol ; 93(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31243123

RESUMO

Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses.IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.


Assuntos
Flavivirus/metabolismo , Mosquitos Vetores/virologia , Interferência Viral/fisiologia , Aedes/virologia , Animais , Arbovírus/metabolismo , Linhagem Celular , Dengue/virologia , Vírus da Dengue/isolamento & purificação , Vírus da Dengue/metabolismo , Flavivirus/genética , Flavivirus/isolamento & purificação , Humanos , Vírus de Insetos , Filogenia , Replicação Viral/fisiologia , Zika virus/isolamento & purificação , Zika virus/metabolismo , Infecção por Zika virus/virologia
6.
PLoS Pathog ; 11(4): e1004692, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25880195

RESUMO

Little is known about the tolerance mechanisms that reduce the negative effects of microbial infection on host fitness. Here, we demonstrate that the histone H3 lysine 9 methyltransferase G9a regulates tolerance to virus infection by shaping the response of the evolutionary conserved Jak-Stat pathway in Drosophila. G9a-deficient mutants are more sensitive to RNA virus infection and succumb faster to infection than wild-type controls, which was associated with strongly increased Jak-Stat dependent responses, but not with major differences in viral load. Genetic experiments indicate that hyperactivated Jak-Stat responses are associated with early lethality in virus-infected flies. Our results identify an essential epigenetic mechanism underlying tolerance to virus infection.


Assuntos
Drosophila melanogaster/virologia , Epigênese Genética , Regulação da Expressão Gênica/imunologia , Histona-Lisina N-Metiltransferase/imunologia , Tolerância Imunológica/imunologia , Infecções por Vírus de RNA/imunologia , Animais , Imunoprecipitação da Cromatina , Drosophila melanogaster/enzimologia , Drosophila melanogaster/imunologia , Vírus de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Curr Biol ; 30(18): 3495-3506.e6, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32679098

RESUMO

Endogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR-Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived P-element-induced wimpy testis-interacting RNAs (piRNAs). Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction.


Assuntos
Aedes/genética , Aedes/virologia , Flavivirus/genética , Genoma de Inseto , RNA Interferente Pequeno/genética , Replicação Viral , Animais , Feminino , Flavivirus/classificação , Flavivirus/isolamento & purificação , RNA Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
Science ; 369(6504): 718-724, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32661059

RESUMO

Coronavirus disease 2019 (COVID-19) is characterized by distinct patterns of disease progression that suggest diverse host immune responses. We performed an integrated immune analysis on a cohort of 50 COVID-19 patients with various disease severity. A distinct phenotype was observed in severe and critical patients, consisting of a highly impaired interferon (IFN) type I response (characterized by no IFN-ß and low IFN-α production and activity), which was associated with a persistent blood viral load and an exacerbated inflammatory response. Inflammation was partially driven by the transcriptional factor nuclear factor-κB and characterized by increased tumor necrosis factor-α and interleukin-6 production and signaling. These data suggest that type I IFN deficiency in the blood could be a hallmark of severe COVID-19 and provide a rationale for combined therapeutic approaches.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Interferon alfa-2/metabolismo , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Pneumonia Viral/imunologia , Adulto , Idoso , Betacoronavirus/fisiologia , COVID-19 , Infecções por Coronavirus/virologia , Estado Terminal , Estudos Transversais , Feminino , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2 , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Carga Viral
9.
Science ; 370(6519): 991-996, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33214283

RESUMO

The drivers and patterns of zoonotic virus emergence in the human population are poorly understood. The mosquito Aedes aegypti is a major arbovirus vector native to Africa that invaded most of the world's tropical belt over the past four centuries, after the evolution of a "domestic" form that specialized in biting humans and breeding in water storage containers. Here, we show that human specialization and subsequent spread of A. aegypti out of Africa were accompanied by an increase in its intrinsic ability to acquire and transmit the emerging human pathogen Zika virus. Thus, the recent evolution and global expansion of A. aegypti promoted arbovirus emergence not solely through increased vector-host contact but also as a result of enhanced vector susceptibility.


Assuntos
Aedes/virologia , Interações entre Hospedeiro e Microrganismos/genética , Mosquitos Vetores/virologia , Infecção por Zika virus/transmissão , Zika virus/fisiologia , Aedes/genética , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/genética
10.
Sci Rep ; 9(1): 2065, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765784

RESUMO

The fruit fly Drosophila melanogaster is a valuable model organism for the discovery and characterization of innate immune pathways, but host responses to virus infection remain incompletely understood. Here, we describe a novel player in host defense, Sgroppino (Sgp). Genetic depletion of Sgroppino causes hypersensitivity of adult flies to infections with the RNA viruses Drosophila C virus, cricket paralysis virus, and Flock House virus. Canonical antiviral immune pathways are functional in Sgroppino mutants, suggesting that Sgroppino exerts its activity via an as yet uncharacterized process. We demonstrate that Sgroppino localizes to peroxisomes, organelles involved in lipid metabolism. In accordance, Sgroppino-deficient flies show a defect in lipid metabolism, reflected by higher triglyceride levels, higher body mass, and thicker abdominal fat tissue. In addition, knock-down of Pex3, an essential peroxisome biogenesis factor, increases sensitivity to virus infection. Together, our results establish a genetic link between the peroxisomal protein Sgroppino, fat metabolism, and resistance to virus infection.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Metabolismo dos Lipídeos/genética , Peroxissomos/genética , Infecções por Vírus de RNA/genética , Vírus de RNA/patogenicidade , Animais , Dicistroviridae/patogenicidade , Drosophila melanogaster/virologia
11.
Cell Host Microbe ; 23(3): 353-365.e8, 2018 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-29503180

RESUMO

The RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. We find that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. We suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.


Assuntos
Antivirais/imunologia , DNA Viral/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/imunologia , RNA Helicases/metabolismo , Vírus de RNA/imunologia , Ribonuclease III/metabolismo , Animais , Arbovírus/imunologia , Arbovírus/patogenicidade , Culicidae/imunologia , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/genética , Genes Virais/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mutação Puntual , RNA Helicases/genética , Interferência de RNA/imunologia , Infecções por Vírus de RNA , Vírus de RNA/genética , Vírus de RNA/patogenicidade , RNA Interferente Pequeno/genética , RNA Viral/metabolismo , Ribonuclease III/genética , Carga Viral , Replicação Viral
12.
Nat Protoc ; 10(7): 1084-97, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26110714

RESUMO

Host defense to virus infection involves both resistance mechanisms that reduce viral burden and tolerance mechanisms that limit detrimental effects of infection. The fruit fly, Drosophila melanogaster, has emerged as a model for identifying and characterizing the genetic basis of resistance and tolerance. This protocol describes how to analyze host responses to virus infection in Drosophila, and it covers the preparation of virus stocks, experimental inoculation of flies and assessment of host survival and virus production, which are indicative of resistance or tolerance. It also provides guidance on how to account for recently identified confounding factors, including natural genetic variation in the pastrel locus and contamination of fly stocks with persistent viruses and the symbiotic bacterium Wolbachia. Our protocol aims to be accessible to newcomers to the field and, although optimized for virus research using Drosophila, some of the techniques could be adapted to other host organisms and/or other microbial pathogens. Preparation of fly stocks requires ∼1 month, virus stock preparation requires 17-20 d, virus injection and survival assays require 10-15 d and virus titration requires 14 d.


Assuntos
Drosophila melanogaster/virologia , Vírus de Insetos/patogenicidade , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Feminino , Genes de Insetos , Variação Genética , Interações Hospedeiro-Patógeno , Vírus de Insetos/genética , Masculino , Simbiose , Wolbachia/fisiologia
13.
Sci Rep ; 5: 12758, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26234525

RESUMO

Innate immunity is the first line of defence against pathogens and is essential for survival of the infected host. The fruit fly Drosophila melanogaster is an emerging model to study viral pathogenesis, yet antiviral defence responses remain poorly understood. Here, we describe the heat shock response, a cellular mechanism that prevents proteotoxicity, as a component of the antiviral immune response in Drosophila. Transcriptome analyses of Drosophila S2 cells and adult flies revealed strong induction of the heat shock response upon RNA virus infection. Dynamic induction patterns of heat shock pathway components were characterized in vitro and in vivo following infection with different classes of viruses. The heat shock transcription factor (Hsf), as well as active viral replication, were necessary for the induction of the response. Hsf-deficient adult flies were hypersensitive to virus infection, indicating a role of the heat shock response in antiviral defence. In accordance, transgenic activation of the heat shock response prolonged survival time after infection and enabled long-term control of virus replication to undetectable levels. Together, our results establish the heat shock response as an important constituent of innate antiviral immunity in Drosophila.


Assuntos
Drosophila/virologia , Resposta ao Choque Térmico/fisiologia , Interações Hospedeiro-Patógeno , Animais , Animais Geneticamente Modificados , Células Cultivadas , Vírus de DNA/patogenicidade , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/imunologia , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fatores de Transcrição de Choque Térmico , Masculino , Interferência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carga Viral , Viroses/etiologia , Viroses/veterinária
14.
J Insect Physiol ; 59(2): 159-70, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22824741

RESUMO

Virus transmission and spread by arthropods is a major economic and public health concern. The ongoing dissemination of arthropod-borne viruses by blood-feeding insects is an important incentive to study antiviral immunity in these animals. RNA interference is a major mechanism for antiviral defense in insects, including the fruit fly Drosophila melanogaster and several vector mosquitoes. However, recent data suggest that the evolutionary conserved Toll, Imd and Jak-Stat signaling pathways also contribute to antiviral immunity. Moreover, symbionts, such as the intracellular bacterium Wolbachia and the gut microflora, influence the course of virus infection in insects. These results add an additional level of complexity to antiviral immunity, but also provide novel opportunities to control the spread of arboviruses. In this review, we provide an overview of the current knowledge and recent developments in antiviral immunity in Dipteran insects, with a focus on non-RNAi mediated inducible responses.


Assuntos
Culicidae/imunologia , Drosophila melanogaster/imunologia , Imunidade Inata , Proteínas de Insetos/metabolismo , Animais , Arbovírus/fisiologia , Culicidae/genética , Culicidae/virologia , Drosophila melanogaster/genética , Drosophila melanogaster/virologia , Evolução Molecular , Proteínas de Insetos/genética , Interferência de RNA , Transdução de Sinais
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