RESUMO
Zika virus (ZIKV) has gained a lot of attention in the past few years due to its rapid spread worldwide and its close association to severe neurological outcomes, such as microcephaly and Guillain-Barre syndrome. In this study, the in vitro and in vivo anti-ZIKV activity of 7-deaza-7-fluoro-2'-C-methyl-adenosine (DFMA) was evaluated. In vitro, using primary mouse neuronal cells and human neural stem cells infected by ZIKV, treatment with DFMA resulted in impaired viral replication and protection against virus-induced cell death. In vivo, when administrated prior to infection, DFMA prevented lethality and markedly reduced viral loads and neuroinflammation, including microgliosis and overall brain damage. Additionally, as an early therapeutic treatment, DFMA increased survival rates in mice. Collectively, these findings demonstrate that the nucleoside analog DFMA inhibits ZIKV infection and viral-induced neuroinflammation in vitro and in vivo without apparent untoward effects, suggesting it may be useful in individuals infected with ZIKV.
Assuntos
Adenosina/análogos & derivados , Antivirais/farmacologia , Inflamação/virologia , Doenças do Sistema Nervoso/virologia , Infecção por Zika virus/complicações , Adenosina/farmacologia , Adenosina/uso terapêutico , Animais , Antivirais/uso terapêutico , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Culicidae/citologia , Humanos , Inflamação/tratamento farmacológico , Camundongos , Doenças do Sistema Nervoso/tratamento farmacológico , Células-Tronco Neurais , Células Vero , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Zika virus , Infecção por Zika virus/tratamento farmacológicoRESUMO
The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7days post infection in C6/36 cells (7×107 and 2×108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5×107PFU/ml at 72hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.
Assuntos
Virologia/normas , Cultura de Vírus/normas , Replicação Viral , Zika virus/crescimento & desenvolvimento , Zika virus/isolamento & purificação , Animais , Encéfalo/citologia , Linhagem Celular , Centrifugação , Chlorocebus aethiops , Culicidae/citologia , Células Endoteliais/virologia , Genoma Viral , Humanos , Metagenômica , Células Vero , Carga Viral/métodos , Virologia/métodos , Zika virus/genéticaRESUMO
In this study, 10 mosquito coils manufactured in China were obtained in Suriname, South America, where they are used extensively. The coils were analyzed for organics (allethrin, permethrin, and butylated hydroxytoluene) and heavy metals (Cr, Co, As, Cd, and Pb) by GC-MS and ICP-MS, respectively. Allethrin was the only target organic compound detected in all mosquito coils with concentrations ranging from ~1900 to ~4500 µg/g. The concentrations of heavy metals varied as follows (in µg/g): Cr: 2.9-9.4, Co: 0.1-1.2, Cu: 0.7-16.1, Se: 0.10-0.4, Ni: 2.1-5.8, As: 0.10-2.2, Cd: 0.10-0.2, and Pb: 1.1-3.6.
Assuntos
Culicidae/citologia , Repelentes de Insetos/análise , Repelentes de Insetos/química , Inseticidas/análise , Inseticidas/química , Metais Pesados/análise , Metais Pesados/toxicidade , Aletrinas/análise , Animais , China , Culicidae/química , Monitoramento Ambiental , Permetrina/análise , SurinameRESUMO
In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated 'Nhumirim virus'; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus's capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a 10(6)-fold and 10(4)-fold reduction in peak titres, respectively.
Assuntos
Culicidae/citologia , Flavivirus/genética , Flavivirus/isolamento & purificação , Sequência de Aminoácidos , Animais , Brasil , Linhagem Celular , Regulação Viral da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Dengue viruses are the most important arthropod-borne viruses in terms of morbidity and mortality in the world. Since there is no dengue vaccine available for human use, we have set out to investigate the use of chloroquine as an antiviral drug against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of Vero and C6/36 cells infected with dengue virus type 2 (DENV-2). Real-time RT-PCR and plaque assays were used to quantify the DENV-2 load in infected Vero and C6/36 cells after chloroquine treatment. Our results showed that a dose of 50 µg/ml of chloroquine was not toxic to the cells and induced a statistically significant inhibition of virus production in infected Vero cells when compared to untreated cells. In C6/36 cells, chloroquine does not induce a statistically significant difference in viral replication when compared to untreated cells, showing that this virus uses an unlikely pathway of penetration in these cells, and results were also confirmed by the plaque assay (PFU). These data suggest that the inhibition of virus infection induced by chloroquine is due to interference with acidic vesicles in mammalian cells.
Assuntos
Antivirais/farmacologia , Cloroquina/farmacologia , Vírus da Dengue/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Células Cultivadas , Chlorocebus aethiops , Culicidae/citologia , Culicidae/virologia , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Relação Dose-Resposta a Droga , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero/virologiaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. It remains a naturally emerging disease as well as a highly developed biological weapon. VEEV is transmitted to humans in nature by mosquito vectors. Little is known about VEEV entry, especially in mosquito cells. Here, a novel luciferase-based virus entry assay is used to show that the entry of VEEV into mosquito cells requires acidification. Furthermore, mosquito homologs of key human proteins (Rab5 and Rab7) involved in endocytosis were isolated and characterized. Rab5 is found on early endosomes and Rab7 on late endosomes and both are important for VEEV entry in mammalian cells. Each was shown to have analogous function in mosquito cells to that seen in mammalian cells. The wild-type, dominant negative and constitutively active mutants were then used to demonstrate that VEEV requires passage through early and late endosomes before infection can take place. This work indicates that the infection mechanism in mosquitoes and mammals is through a common and ancient evolutionarily conserved pathway.
Assuntos
Culicidae/virologia , Vírus da Encefalite Equina Venezuelana/crescimento & desenvolvimento , Endossomos/virologia , Internalização do Vírus , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Culicidae/citologia , Endossomos/química , Genes Reporter , Humanos , Luciferases/biossíntese , Luciferases/genética , Microscopia Confocal , Dados de Sequência Molecular , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/análise , Proteínas rab5 de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Dengue virus envelope protein (E) contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses, while the site at position 67 is thought to be unique for dengue viruses. N-linked oligosaccharide side chains on flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here, we examined the relevance of each N-linked glycan on dengue virus E protein by removing each site in the context of infectious viral particles. Dengue viruses lacking Asn-67 were able to infect mammalian cells and translate and replicate the viral genome, but production of new infectious particles was abolished. In addition, dengue viruses lacking Asn-153 in the E showed reduced infectivity. In contrast, ablation of one or both glycosylation sites yielded viruses that replicate and propagate in mosquito cells. Furthermore, we found a differential requirement of N-linked glycans for E secretion in mammalian and mosquito cells. While secretion of E lacking Asn-67 was efficient in mosquito cells, secretion of the same protein expressed in mammalian cells was dramatically impaired. Finally, we found that viruses lacking the carbohydrate at position 67 showed reduced infection of immature dendritic cells, suggesting interaction between this glycan and the lectin DC-SIGN. Overall, our data defined different roles for the two glycans present at the E protein during dengue virus infection, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.
Assuntos
Vírus da Dengue/fisiologia , Dengue/metabolismo , Genoma Viral/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Asparagina/genética , Asparagina/metabolismo , Chlorocebus aethiops , Cricetinae , Culicidae/citologia , Culicidae/virologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Dengue/genética , Vírus da Dengue/patogenicidade , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional/genética , Células Vero , Proteínas do Envelope Viral/genética , Replicação Viral/genéticaRESUMO
The egg of Aedeomyia squamipennis (Lynch Arribalzaga) is described with the aid of scanning electron micrographs. This study allows separation of the eggs of Ad. squamipennis from the eggs of other mosquitoes inhabiting similar aquatic vegetation.