RESUMEN
Vectors of infectious disease include several species of Aedes mosquitoes. The life cycle of Aedes aegypti, the yellow fever mosquito, consists of a terrestrial adult and an aquatic larval life stage. Developing in coastal waters can expose larvae to fluctuating salinity, causing salt and water imbalance, which is addressed by two prime osmoregulatory organs - the Malpighian tubules (MTs) and anal papillae (AP). Voltage-gated ion channels (VGICs) have recently been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects. In the current study, we: (i) generated MT transcriptomes of freshwater-acclimated and brackish water-exposed larvae of Ae. aegypti, (ii) detected expression of several voltage-gated Ca2+, K+, Na+ and non-ion-selective ion channels in the MTs and AP using transcriptomics, PCR and gel electrophoresis, (iii) demonstrated that mRNA abundance of many altered significantly following brackish water exposure, and (iv) immunolocalized CaV1, NALCN, TRP/Painless and KCNH8 in the MTs and AP of larvae using custom-made antibodies. We found CaV1 to be expressed in the apical membrane of MTs of both larvae and adults, and its inhibition to alter membrane potentials of this osmoregulatory epithelium. Our data demonstrate that multiple VGICs are expressed in osmoregulatory epithelia of Ae. aegypti and may play an important role in the autonomous regulation of ion transport.
Asunto(s)
Aedes , Fiebre Amarilla , Animales , Aedes/fisiología , Agua/metabolismo , Túbulos de Malpighi/metabolismo , Fiebre Amarilla/metabolismo , Mosquitos Vectores , Cloruro de Sodio/metabolismo , Transporte Iónico , Canales Iónicos/genética , Larva/fisiologíaRESUMEN
Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8+ T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27-CD28-CD8+ T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27-CD28-CD8+ T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27-CD28-CD8+ T cells to acquire a broad-spectrum, innate-like killing activity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Senescencia Celular/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Proteínas Nucleares/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citotoxicidad Inmunológica , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Células Asesinas Naturales/metabolismo , Transducción de Señal , Fiebre Amarilla/genética , Fiebre Amarilla/inmunología , Fiebre Amarilla/metabolismo , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
Patients infected with the Dengue virus (DENV) often present with a massive generation of DENV-specific antibody-secreting cells (ASCs) in the blood. In some cases, these ASCs represent more than 50% of the circulating B cells, a higher magnitude than those induced by other infections, vaccinations, and plasma cell lymphomas. However, it remains unclear how the DENV infection elicits this colossal response. To address this issue, we utilised an in vitro strategy to induce human PBMCs of healthy individuals incubated with DENV particles (DENV4 TVP/360) to differentiate into ASCs. As controls, PBMCs were incubated with a mitogen cocktail or supernatants of uninfected C6/36 cells (mock). The ASC phenotype and function were increasingly detected in the DENV and mitogen-cultured PBMCs as compared to mock-treated cells. In contrast to the in vivo condition, secreted IgG derived from the PBMC-DENV culture was not DENV-specific. Lower ASC numbers were observed when inactivated viral particles or purified B cells were added to the cultures. The physical contact was essential between B cells and the remaining PBMCs for the DENV-mediated ASC response. Considering the evidence for the activation of the tryptophan metabolism detected in the serum of Dengue patients, we assessed its relevance in the DENV-mediated ASC differentiation. For this, tryptophan and its respective metabolites were quantified in the supernatants of cell cultures through mass spectrophotometry. Tryptophan depletion and kynurenine accumulation were found in the supernatants of PBMC-DENV cultures, which presented enhanced detection of indoleamine 2,3-dioxygenase 1 and 2 transcripts as compared to controls. In PBMC-DENV cultures, tryptophan and kynurenine levels strongly correlated to the respective ASC numbers, while the kynurenine levels were directly proportional to the secreted IgG titers. Contrastingly, PBMCs incubated with Zika or attenuated Yellow Fever viruses showed no correlation between their kynurenine concentrations and ASC numbers. Therefore, our data revealed the existence of distinct pathways for the DENV-mediated ASC differentiation and suggest the involvement of the tryptophan metabolism in this cellular process triggered by flavivirus infections.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/virología , Diferenciación Celular/inmunología , Virus del Dengue/inmunología , Dengue/metabolismo , Triptófano/metabolismo , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/inmunología , Infección por el Virus Zika/metabolismo , Virus Zika/inmunología , Donantes de Sangre , Células Cultivadas , Dengue/inmunología , Dengue/virología , Humanos , Quinurenina/metabolismo , Fiebre Amarilla/inmunología , Fiebre Amarilla/virología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Yellow fever virus (YFV) tropism is restricted to human and nonhuman primates. The nonstructural protein 5 (NS5) protein of YFV binds to primate signal transducer and activator of transcription 2 (STAT2) and antagonizes interferon (IFN) signaling. However, YFV NS5 is unable to bind mouse STAT2 and antagonize murine IFN signaling. A similar observation has been made with the NS5 protein of both dengue virus (DENV) and Zika virus (ZIKV). However, the key difference between the NS5 protein of YFV and those of DENV and ZIKV is that YFV NS5 binds human STAT2 in an IFN-dependent manner. In human cells, IFN-I treatment induces K63-linked ubiquitination on lysine (K) 6 of YFV NS5, which is required for binding human STAT2. This IFN-induced ubiquitination of YFV NS5 is absent in murine cells resulting in the lack of binding of YFV NS5 and human STAT2 in murine cells. This highlights the importance of YFV NS5 ubiquitination in determining the host cell range for YFV.
Asunto(s)
Interacciones Huésped-Patógeno , Factor de Transcripción STAT2/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/aislamiento & purificación , Animales , Antivirales/farmacología , Humanos , Interferón Tipo I/farmacología , Ratones , Ubiquitinación , Fiebre Amarilla/tratamiento farmacológico , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/efectos de los fármacosRESUMEN
Flaviviral infections result in a wide spectrum of clinical outcomes, ranging from asymptomatic infection to severe disease. Although the correlates of severe disease have been explored1-4, the pathophysiology that differentiates symptomatic from asymptomatic infection remains undefined. To understand the molecular underpinnings of symptomatic infection, the blood transcriptomic and metabolomic profiles of individuals were examined before and after inoculation with the live yellow fever viral vaccine (YF17D). It was found that individuals with adaptive endoplasmic reticulum (ER) stress and reduced tricarboxylic acid cycle activity at baseline showed increased susceptibility to symptomatic outcome. YF17D infection in these individuals induced maladaptive ER stress, triggering downstream proinflammatory responses that correlated with symptomatic outcome. The findings of the present study thus suggest that the ER stress response and immunometabolism underpin symptomatic yellow fever and possibly even other flaviviral infections. Modulating either ER stress or metabolism could be exploited for prophylaxis against symptomatic flaviviral infection outcome.
Asunto(s)
Estrés del Retículo Endoplásmico , Vacuna contra la Fiebre Amarilla/inmunología , Fiebre Amarilla/metabolismo , Adulto , Ciclo del Ácido Cítrico , Susceptibilidad a Enfermedades , Humanos , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismo , Vacunas Atenuadas/inmunología , Fiebre Amarilla/etiologíaRESUMEN
The genus Flavivirus comprises approximately 73 viruses, which share several common aspects, such as dimension, structure, nucleic acid properties, and shape in electronic microscopy. Global incidence of flavivirus infection increased dramatically over the last decades, causing large outbreaks in several areas of the world. These viruses are expanding from endemic tropical and subtropical areas to previously nonendemic areas, affecting and causing diseases in millions of individuals worldwide and posing a formidable challenge to public health in several countries. The majority of clinically significant flavivirus-associated infections are mosquito borne (arboviruses-acronym for ARthropod-BOrne VIRUSES), such as dengue, yellow fever, Japanese encephalitis, Zika, and West Nile fever. Most diseases caused by flaviviruses are asymptomatic or manifest as self-limited, mild, undifferentiated febrile diseases. In a limited number of cases, these diseases may evolve to severe inflammatory, multisystem diseases, causing high morbidity and mortality. Some flaviviruses have been consistently identified in kidney tissue and urine and have been clinically associated with kidney diseases. In this review, we will provide an overview of the epidemiology, risk factors, kidney pathology, etiopathogenesis, and outcomes of acute and chronic kidney syndromes associated with dengue, yellow fever, Zika, and West Nile virus disease.
Asunto(s)
Lesión Renal Aguda/epidemiología , Dengue/epidemiología , Insuficiencia Renal Crónica/epidemiología , Fiebre del Nilo Occidental/epidemiología , Fiebre Amarilla/epidemiología , Infección por el Virus Zika/epidemiología , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/virología , Dengue/metabolismo , Dengue/patología , Flavivirus , Infecciones por Flavivirus/epidemiología , Infecciones por Flavivirus/patología , Humanos , Mosquitos Vectores , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/virología , Factores de Riesgo , Fiebre del Nilo Occidental/metabolismo , Fiebre del Nilo Occidental/patología , Fiebre Amarilla/metabolismo , Fiebre Amarilla/patología , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/patologíaRESUMEN
The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago2, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication.
Asunto(s)
Antivirales/farmacología , Retículo Endoplásmico/metabolismo , Interferón-alfa/farmacología , Proteínas Mitocondriales/metabolismo , Replicación Viral , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/efectos de los fármacos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Chaperón BiP del Retículo Endoplásmico , Técnicas de Inactivación de Genes , Estudio de Asociación del Genoma Completo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Mitocondriales/genética , Unión Proteica , Especificidad de la Especie , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/fisiologíaRESUMEN
Plasmacytoid dendritic cells (pDCs) are specialized in the production of interferons (IFNs) in response to viral infections. The Flaviviridae family comprises enveloped RNA viruses such as Hepatitis C virus (HCV) and Dengue virus (DENV). Cell-free flaviviridae virions poorly stimulate pDCs to produce IFN. By contrast, cells infected with HCV and DENV potently stimulate pDCs via short-range delivery of viral RNAs, which are either packaged within immature virions or secreted exosomes. We report that cells infected with Yellow fever virus (YFV), the prototypical flavivirus, stimulated pDCs to produce IFNs in a TLR7- and cell contact- dependent manner. Such stimulation was unaffected by the presence of YFV neutralizing antibodies. As reported for DENV, cells producing immature YFV particles were more potent at stimulating pDCs than cells releasing mature virions. Additionally, cells replicating a release-deficient YFV mutant or a YFV subgenomic RNA lacking structural protein-coding sequences participated in pDC stimulation. Thus, viral RNAs produced by YFV-infected cells reach pDCs via at least two mechanisms: within immature particles and as capsid-free RNAs. Our work highlights the ability of pDCs to respond to a variety of viral RNA-laden carriers generated from infected cells.
Asunto(s)
Cápside , Células Dendríticas/inmunología , Interferones/metabolismo , ARN Viral/metabolismo , Virión/inmunología , Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/inmunología , Adulto , Anciano , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/genética , Virión/metabolismo , Fiebre Amarilla/metabolismo , Fiebre Amarilla/virología , Adulto JovenRESUMEN
Outbreaks of Yellow Fever occur regularly in endemic areas of Africa and South America frequently leading to mass vaccination campaigns straining the availability of the attenuated Yellow Fever vaccine, YF-17D. The WHO has recently decided to discontinue regular booster-vaccinations since a single vaccination is deemed to confer life-long immune protection. Here, we have examined humoral (neutralizing antibody) and cellular (CD8 and CD4 T cell) immune responses in primary and booster vaccinees (the latter spanning 8 to 36 years after primary vaccination). After primary vaccination, we observed strong cellular immune responses with T cell activation peaking ≈2 weeks and subsiding to background levels ≈ 4 weeks post-vaccination. The number of antigen-specific CD8+ T cells declined over the following years. In >90% of vaccinees, in vitro expandable T cells could still be detected >10 years post-vaccination. Although most vaccinees responded to a booster vaccination, both the humoral and cellular immune responses observed following booster vaccination were strikingly reduced compared to primary responses. This suggests that pre-existing immunity efficiently controls booster inoculums of YF-17D. In a situation with epidemic outbreaks, one could argue that a more efficient use of a limited supply of the vaccine would be to focus on primary vaccinations.
Asunto(s)
Inmunidad Adaptativa , Inmunización Secundaria , Vacunación , Vacuna contra la Fiebre Amarilla/inmunología , Fiebre Amarilla/inmunología , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/inmunología , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antivirales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Dinamarca , Femenino , Voluntarios Sanos , Humanos , Activación de Linfocitos , Masculino , Ratones , Persona de Mediana Edad , Vigilancia en Salud Pública , Fiebre Amarilla/metabolismo , Adulto JovenRESUMEN
Positive-sense RNA viruses pose increasing health and economic concerns worldwide. Our limited understanding of how these viruses interact with their host and how these processes lead to virulence and disease seriously hampers the development of anti-viral strategies. Here, we demonstrate the tracking of (+) and (-) sense viral RNA at single-cell resolution within complex subsets of the human and murine immune system in different mouse models. Our results provide insights into how a prototypic flavivirus, yellow fever virus (YFV-17D), differentially interacts with murine and human hematopoietic cells in these mouse models and how these dynamics influence distinct outcomes of infection. We detect (-) YFV-17D RNA in specific secondary lymphoid compartments and cell subsets not previously recognized as permissive for YFV replication, and we highlight potential virus-host interaction events that could be pivotal in regulating flavivirus virulence and attenuation.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , ARN Viral/metabolismo , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/genética , Animales , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Femenino , Flavivirus/genética , Citometría de Flujo , Células HEK293 , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , Humanos , Masculino , Ratones , ARN Viral/inmunología , Análisis de la Célula Individual , Especificidad de la Especie , Trasplante Heterólogo , Fiebre Amarilla/inmunología , Fiebre Amarilla/virologíaRESUMEN
To successfully establish infection, flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits.
Asunto(s)
Interferón beta/metabolismo , Proteínas no Estructurales Virales/metabolismo , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Interacciones Huésped-Patógeno , Humanos , Fosforilación , Unión Proteica , Factor de Transcripción STAT1/química , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Transducción de Señal , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Fiebre Amarilla/genética , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/genéticaRESUMEN
Given the potency of interferon-α/ß, viral evasion of this pathway is crucial for infection. In this issue of Cell Host & Microbe, Laurent-Rolle et al. (2014) report that during yellow fever virus infection, interferon-α/ß stimulates the polyubiquitination of viral NS5, which binds to STAT2 and inhibits transcription of interferon-stimulated genes.
Asunto(s)
Interferón beta/metabolismo , Proteínas no Estructurales Virales/metabolismo , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/metabolismo , Animales , HumanosRESUMEN
Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled.
Asunto(s)
Ecdisteroides/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/genética , Fiebre Amarilla/transmisión , Aedes/genética , Aedes/metabolismo , Aedes/patogenicidad , Animales , Ecdisteroides/genética , Femenino , Oocitos/metabolismo , Ovario/metabolismo , Receptor de Insulina/genética , Fiebre Amarilla/metabolismoRESUMEN
Why cross-species transmissions of zoonotic viral infections to humans are frequently associated with severe disease when viruses responsible for many zoonotic diseases appear to cause only benign infections in their reservoir hosts is unclear. Sooty mangabeys (SMs), a reservoir host for SIV, do not develop disease following SIV infection, unlike nonnatural HIV-infected human or SIV-infected rhesus macaque (RM) hosts. SIV infections of SMs are characterized by an absence of chronic immune activation, in association with significantly reduced IFN-α production by plasmacytoid dendritic cells (pDCs) following exposure to SIV or other defined TLR7 or TLR9 ligands. In this study, we demonstrate that SM pDCs produce significantly less IFN-α following ex vivo exposure to the live attenuated yellow fever virus 17D strain vaccine, a virus that we show is also recognized by TLR7, than do RM or human pDCs. Furthermore, in contrast to RMs, SMs mount limited activation of innate immune responses and adaptive T cell proliferative responses, along with only transient antiviral Ab responses, following infection with yellow fever vaccine 17D strain. However, SMs do raise significant and durable cellular and humoral immune responses comparable to those seen in RMs when infected with modified vaccinia Ankara, a virus whose immunogenicity does not require TLR7/9 recognition. Hence, differences in the pattern of TLR7 signaling and type I IFN production by pDCs between primate species play an important role in determining their ability to mount and maintain innate and adaptive immune responses to specific viruses, and they may also contribute to determining whether disease follows infection.
Asunto(s)
Inmunidad Adaptativa/inmunología , Inmunidad Innata/inmunología , Interferón-alfa/inmunología , Receptor Toll-Like 7/inmunología , Virus de la Fiebre Amarilla/inmunología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Células Cultivadas , Cercocebus atys , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Reservorios de Enfermedades/virología , Citometría de Flujo , Humanos , Interferón-alfa/metabolismo , Cinética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Macaca mulatta , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismo , Virus Vaccinia/inmunología , Fiebre Amarilla/inmunología , Fiebre Amarilla/metabolismo , Fiebre Amarilla/prevención & control , Vacuna contra la Fiebre Amarilla/administración & dosificación , Vacuna contra la Fiebre Amarilla/inmunologíaRESUMEN
Interferon (IFN) is an innate immune response protein that is involved in the antiviral response during viral infection. Treatment of acute viral infections with exogenous interferon may be effective but is generally not feasible for clinical use due to many factors, including cost, stability, and availability. To overcome these limitations, an adenovirus type 5-vectored consensus alpha IFN, termed DEF201, was constructed as a potential way to deliver sustained therapeutic levels of systemic IFN. To demonstrate the efficacy of DEF201 against acute flaviviral disease, various concentrations of the construct were administered as a single intranasal dose prior to virus infection, which resulted in a dose-responsive, protective effect in a hamster model of yellow fever virus (YFV) disease. A DEF201 dose of 5×10(7) PFU/animal administered intranasally just prior to YFV challenge protected 100% of the animals, while a 10-fold lower DEF201 dose exhibited lower, although significant, levels of protection. Virus titers in the liver and serum and levels of serum alanine aminotransferase were all significantly reduced as a result of DEF201 administration at all doses tested. No toxicity, as indicated by weight loss or gross morbidity, was observed in non-YFV-infected animals treated with DEF201. Protection of YFV-infected animals was observed when DEF201 was delivered as early as 7 days prior to virus challenge and as late as 2 days after virus challenge, demonstrating effective prophylaxis and therapy in a hamster model of disease. Overall, it appears that DEF201 is effective in the treatment of YFV in a hamster model.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/genética , Interferones/metabolismo , Fiebre Amarilla/metabolismo , Fiebre Amarilla/terapia , Virus de la Fiebre Amarilla/patogenicidad , Animales , Chlorocebus aethiops , Cricetinae , Femenino , Interferones/genética , Mesocricetus , Células VeroRESUMEN
BACKGROUND: The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti. METHODOLOGY/PRINCIPAL FINDINGS: Using a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti. Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis. CONCLUSIONS/SIGNIFICANCE: Our results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies.
Asunto(s)
Acuaporinas/genética , Fiebre Amarilla/metabolismo , Aedes , Animales , Transporte Biológico , Biología Computacional/métodos , Drosophila melanogaster , Evolución Molecular , Femenino , Glicerol/química , Túbulos de Malpighi/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Interferencia de ARN , Agua/químicaRESUMEN
Yellow fever virus (YFV) encodes two envelope proteins, pre-membrane (prM) and envelope (E), that accumulate in the endoplasmic reticulum (ER). The C termini of prM and E form two antiparallel transmembrane alpha-helices that contain ER-retention signals. To understand further the ER retention of the prME heterodimer, we characterized the subcellular localization of chimeric proteins made of a reporter protein fused to the transmembrane segments of YFV envelope proteins. We showed that at least three of the transmembrane segments of the prME heterodimer are ER-retention signals. Interestingly, increasing the length of these alpha-helices led to the export of the chimeric proteins out of the ER. Furthermore, adding a diacidic export signal at the C terminus of the first transmembrane segment of the E protein also induced export to the cell surface. However, adding this export signal at the C terminus of the first transmembrane segment of E in the context of prME did not change the subcellular localization of the prME heterodimer, suggesting the presence of a stronger ER-retention signal outside the first transmembrane segment of E. Importantly, the diacidic export motif added to the C terminus of the first transmembrane segment of the prM protein was not sufficient to export a chimeric protein out of the ER, indicating that this sequence is a dominant ER-retention signal. Together, these data indicate that a combination of several signals of different strengths contributes to the ER retention of the YFV envelope protein heterodimer.
Asunto(s)
Retículo Endoplásmico/virología , Señales de Clasificación de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/metabolismo , Secuencia de Aminoácidos , Dimerización , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Transporte de Proteínas , Proteínas del Envoltorio Viral/genética , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/química , Virus de la Fiebre Amarilla/genéticaRESUMEN
Yellow fever (YF) virus is currently found in tropical Africa and South America, and is responsible for a febrile to severe illness characterized by organ failure and shock. The attenuated YF 17D strain, used in YF vaccine, was derived from the wild-type strain Asibi. Although studies have been done on genetic markers of YF virulence, differentiation of the two strains in terms of host-cell interaction during infection remains elusive. As YF wild-type strains are hepatotropic, we chose a hepatic cell line (HepG2) to study YF virus-host cell interaction. HepG2 cells rapidly produced high titres of infectious viral particles for 17D and Asibi YF strains. However, HepG2 cells were more susceptible to the attenuated 17D virus infection, and only this virus strain induced early apoptosis in these cells. Molecular markers specific for the 17D virus were identified by microarray analysis and confirmed by quantitative RT-PCR analysis. As early as 1h postinfection, three genes, (IEX-1, IRF-1, DEC-1) all implicated in apoptosis pathways, were upregulated. Later in infection (48 h) two other genes (HSP70-1A and 1B), expressed in cases of cellular stress, were highly upregulated in 17D-infected HepG2 cells. The early specific upregulation of these cellular genes in HepG2 cells may be considered markers of the 17D virus. This study on the YF attenuated strain gives a new approach to the analysis of the factors involved in virus attenuation.
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Neoplasias Hepáticas/virología , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/fisiología , Animales , Antígenos Virales/aislamiento & purificación , Apoptosis/fisiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica , Humanos , Etiquetado Corte-Fin in Situ , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Viral/química , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Vero , Replicación Viral/fisiología , Fiebre Amarilla/genética , Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/patogenicidadRESUMEN
This report describes the clinical laboratory findings in golden hamsters experimentally infected with yellow fever (YF) virus. An accompanying paper describes the pathologic findings. Following intraperitoneal inoculation of a virulent strain of YF virus, hamsters developed a high-titered viremia (up to 109/mL) lasting 5--6 days and abnormal liver function tests. YF hemagglutination-inhibiting antibodies appeared 4 or 5 days after infection, often while viremia was still present. The mortality rate in YF-infected hamsters was variable, depending on the virus strain and the age of the animals. Clinical and pathologic changes in the infected hamsters were very similar to those described in experimentally infected macaques and in fatal human cases of YF, which indicates that the golden hamster may be an excellent alternative animal model, in place of nonhuman primates, for research on the pathogenesis and treatment of YF and other viscerotropic flavivirus diseases.
Asunto(s)
Modelos Animales de Enfermedad , Mesocricetus , Fiebre Amarilla , Animales , Anticuerpos Antivirales/biosíntesis , Cricetinae , Femenino , Hematócrito , Recuento de Leucocitos , Pruebas de Función Hepática , Tasa de Supervivencia , Viremia , Fiebre Amarilla/inmunología , Fiebre Amarilla/metabolismo , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/aislamiento & purificaciónRESUMEN
When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both Aedes aegypti and Aedes albopictus. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an A. aegypti transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3' end exactly matched a cDNA encoding a polypeptide that is up-regulated when A. aegypti encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994) Exp. Parasitol. 79, 312-321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron.