RESUMO
Arboviruses are an important group of pathogens that cause diseases of medical and veterinary concern worldwide. The interactions of these viruses with their host cells are complex, and frequently, the coexistence of two different viruses in the same cell results in the inhibition of replication in one of the viruses, which is a phenomenon called viral interference. This phenomenon can be exploited to develop antiviral strategies. Insect cell lines persistently infected with arboviruses are useful models with which to study viral interference. In this work, a model of C6/36-HT cells (from Aedes albopictus mosquitoes) persistently infected with Dengue virus, serotype 2, was used. Viral interference was evaluated via plaque and flow cytometry assays. The presence of heterotypic interference against the other serotypes of the same virus and homologous interference against yellow fever virus was determined; however, this cell line did not display heterologous viral interference against Sindbis virus. The mechanisms responsible for viral interference have not been fully elucidated, but small RNAs could be involved. However, the silencing of Ago3, a key protein in the genome-derived P-element-induced wimpy testis pathway, did not alter the viral interference process, suggesting that viral interference occurs independent of this pathway.
RESUMO
The establishment of persistent dengue virus infection within the cells of the mosquito vector is an essential requirement for viral transmission to a new human host. The mechanisms involved in the establishment and maintenance of persistent infection are not well understood, but it has been suggested that both viral and cellular factors might play an important role. In the present work, we evaluated differential gene expression in Aedes albopictus cells acutely (C6/36-HT) and persistently infected (C6-L) with Dengue virus 2 by cDNA-AFLP. We observed that importin ß3 was upregulated in noninfected cells compared with C6-L cells. Using RT-qPCR and plaque assays, we observed that Dengue virus levels in C6-L cells essentially do not vary over time, and peak viral titers in acutely infected cells are observed at 72 and 120 h postinfection. The expression level of importin ß3 was higher in acutely infected cells than in persistently infected cells; this correlates with higher levels of NS5 in the nucleus of the cell. The differential pattern of importin ß3 expression between acute and persistent infection with Dengue virus 2 could be a mechanism to maintain viral infection over time, reducing the antiviral response of the cell and the viral replicative rate.
RESUMO
Dengue manifestations range from a mild form, dengue fever (DF), to more severe forms such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The ability of the host to present one of these clinical forms could be related to polymorphisms located in genes of the Toll-like receptors (TLRs) which activate the pro-inflammatory response. Therefore, the genotyping of single nucleotide genetic polymorphisms (SNPs) in TLR3 (rs3775291 and rs6552950), TLR4 (rs2737190, rs10759932, rs4986790, rs4986791, rs11536865, and rs10983755), TLR7 (rs179008 and rs3853839), and TLR8 (rs3764880, rs5741883, rs4830805, and rs1548731) was carried out in non-genetically related DHF patients, DF patients, and general population (GP) subjects. The SNPs were analyzed by real-time PCR by genotyping assays from Applied Biosystems®. The codominance model showed that dengue patients had a lower probability of presenting the TLR4-rs2737190-G/G genotype (odds ratio (OR) (95% CI) = 0.34 (0.14-0.8), p = 0.038). Dengue patients showed a lower probability of presenting TLR4-rs11536865-G/C genotype (OR (95% CI) = 0.19 (0.05-0.73), p = 0.0092) and had a high probability of presenting the TACG haplotype, but lower probability of presenting the TGCG haplotype in the TLR4 compared to GP individuals (OR (95% CI) = 0.55 (0.35-0.86), p = 0.0084). In conclusion, the TLR4-rs2737190-G/G and TLR4-rs11536865-G/C genotypes and TGCG haplotype were associated with protection from dengue.
Assuntos
Dengue/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/genética , Adulto , Idoso , Alelos , Estudos de Casos e Controles , Dengue/sangue , Dengue/epidemiologia , Epidemias , Feminino , Genótipo , Haplótipos , Humanos , Masculino , México/epidemiologia , Pessoa de Meia-IdadeRESUMO
The arthropod-borne flaviviruses are important human pathogens, and a deeper understanding of the virus-host cell interaction is required to identify cellular targets that can be used as therapeutic candidates. It is well reported that the flaviviruses hijack several cellular functions, such as exosome-mediated cell communication during infection, which is modulated by the delivery of the exosomal cargo of pro- or antiviral molecules to the receiving host cells. Therefore, to study the role of exosomes during flavivirus infections is essential, not only to understand its relevance in virus-host interaction, but also to identify molecular factors that may contribute to the development of new strategies to block these viral infections. This review explores the implications of exosomes in flavivirus dissemination and transmission from the vector to human host cells, as well as their involvement in the host immune response. The hypothesis about exosomes as a transplacental infection route of ZIKV and the paradox effect or the dual role of exosomes released during flavivirus infection are also discussed here. Although several studies have been performed in order to identify and characterize cellular and viral molecules released in exosomes, it is not clear how all of these components participate in viral pathogenesis. Further studies will determine the balance between protective and harmful exosomes secreted by flavivirus infected cells, the characteristics and components that distinguish them both, and how they could be a factor that determines the infection outcome.
Assuntos
Comunicação Celular , Exossomos/metabolismo , Infecções por Flavivirus/metabolismo , Flavivirus/metabolismo , Interações Hospedeiro-Patógeno , Animais , Vetores Aracnídeos/virologia , Dengue/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Infecções por Flavivirus/transmissão , Humanos , Mosquitos Vetores/virologia , Carrapatos/virologia , Infecção por Zika virus/metabolismoRESUMO
Dengue virus (DENV) is an important flavivirus that is transmitted to humans by Aedes mosquitoes, where it can establish a persistent infection underlying vertical and horizontal transmission. However, the exact mechanism of persistent DENV infection is not well understood. Recently miR-927 was found to be upregulated in C6/36-HT cells at 57 weeks of persistent infection (C6-L57), suggesting its participation during this type of infection. The aim of this study was to determine the role of miR-927 during infection with DENV type 2. The results indicate an overexpression of miR-927 in C6-L57 cells and acutely infected cells according to the time of infection and the m.o.i. used. The downregulation of miR-927 in C6-L57 cells results in a reduction of both viral titre and viral genome copy number. The overexpression of miR-927 in C6-L40 and C6/36 cells infected at an m.o.i. of 0.1 causes an increase in both viral titre and viral genome copy number, suggesting a pro-viral activity of miR-927. In silico prediction analysis reveals target mRNAs for miR-927 are implicated in post-translational modifications (SUMO), translation factors (eIF-2B), the innate immune system (NKIRAS), exocytosis (EXOC-2), endocytosis (APM1) and the cytoskeleton (FLN). The expression levels of FLN were the most affected by both miR-927 overexpression and inhibition, and FLN was determined to be a direct target of miR-927 by a dual-luciferase gene reporter assay. FLN has been associated with the regulation of the Toll pathway and either overexpression or downregulation of miR-927 resulted in expression changes of antimicrobial peptides (Cecropins A and G, and Defensin D) involved in the Toll pathway response.
Assuntos
Aedes/genética , Aedes/virologia , Vírus da Dengue/genética , Dengue/virologia , MicroRNAs/genética , Animais , Linhagem Celular , Doenças Transmissíveis/genética , Doenças Transmissíveis/virologia , Genoma Viral/genética , Luciferases/genética , Replicação Viral/genéticaRESUMO
Exosomes are endocytic origin small-membrane vesicles secreted to the extracellular space by most cell types. Exosomes released from virus infected-cells can mediate the cell-to-cell communication to promote or modulate viral transmission. Dengue virus (DENV) is an arbovirus transmitted by Aedes mosquitoes bite to humans. Interestingly, the role of exosomes during the DENV infection in mammalian cells has already been described. However, little is known about exosomes derived from infected mosquito cells. Thus, the exosomes released from DENV-infected C6/36 cells were isolated, purified and analyzed using an antibody against the tetraspanin CD9 from human that showed cross-reactivity with the homologs to human CD9 found in Aedes albopictus (AalCD9). The exosomes from DENV infected cells were larger than the exosomes secreted from uninfected cells, contained virus-like particles, and they were able to infect naïve C6/36 cells, suggesting that exosomes are playing a role in virus dissemination.
Assuntos
Vírus da Dengue/fisiologia , Exossomos/metabolismo , Exossomos/virologia , Mosquitos Vetores/virologia , Aedes , Animais , Linhagem Celular , Dengue/metabolismo , Dengue/virologia , Difusão Dinâmica da Luz , Exossomos/imunologia , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mosquitos Vetores/classificação , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Filogenia , Tetraspaninas/química , Tetraspaninas/genética , Tetraspaninas/imunologia , Tetraspaninas/metabolismo , Replicação ViralRESUMO
Dengue viruses (DENV) are important arboviruses that can establish a persistent infection in its mosquito vector Aedes. Mosquitoes have a short lifetime in nature which makes trying to study the processes that take place during persistent viral infections in vivo. Therefore, C6/36 cells have been used to study this type of infection. C6/36 cells persistently infected with DENV 2 produce virions that cannot infect BHK -21 cells. We hypothesized that the following passages in mosquito cells have a deleterious impact on DENV fitness in vertebrate cells. Here, we demonstrated that the viral particles released from persistently infected cells were infectious to mosquito but not to vertebrate cells. This host restriction occurs at the replication level and is associated with several mutations in the DENV genome. In summary, our findings provide new information about viral replication fitness in a host-dependent manner.
Assuntos
Aedes/virologia , Vírus da Dengue/fisiologia , Especificidade de Hospedeiro , Mosquitos Vetores/virologia , Replicação Viral , Animais , Linhagem Celular , Dengue/virologia , Vírus da Dengue/genética , Genoma Viral , Mosquitos Vetores/crescimento & desenvolvimentoRESUMO
Here, we report for the first time the circulation of dengue virus type 1 (DENV-1) belonging to the lineage IV of genotype V (African American genotype) based on phylogenetic analysis of nucleotide sequences from 10 DENV-1-positive samples obtained in Mexico between 2012 and 2014. Our data revealed that the lineages III and IV of DENV-1 genotype V were found circulating during the same period, probably explaining the rise in the number of cases of severe dengue during that period.
Assuntos
Vírus da Dengue/genética , Genótipo , Filogenia , RNA Viral/genética , Dengue Grave/epidemiologia , Adolescente , Adulto , Criança , Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Evolução Molecular , Feminino , Efeito Fundador , Variação Genética , Humanos , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogeografia , Dengue Grave/diagnóstico , Dengue Grave/patologia , Dengue Grave/virologiaRESUMO
This study was designed to examine the effects of lyophilized red delicious apple peel (RDP) on the action potentials (APs) and the input resistance-threshold current relationship. The experiments were performed on isolated papillary heart muscles from healthy male rats, healthy male rats treated with RDP, diabetic male rats, and diabetic male rats treated with RDP. The preparation was superfused with oxygenated Tyrode's solution at 37°C. The stimulation and the recording of the APs, the input resistance, and the threshold current were made using conventional electrophysiological methods. The RDP presented no significant effect in normal rats. Equivalent doses in diabetic rats reduced the APD and ARP. The relationship between input resistance and threshold current established an inverse correlation. The results indicate the following: (1) The functional structure of the cardiac ventricular syncytium in healthy rats is heterogeneous, in terms of input resistance and threshold current. Diabetes further accentuates the heterogeneity. (2) As a consequence, conduction block occurs and increases the possibility of reentrant arrhythmias. (3) These modifications in the ventricular syncytium, coupled with the increase in the ARP, are the adequate substrate so that, with diabetes, the heart becomes more arrhythmogenic. (4) RDP decreases the APD, the ARP, and most syncytium irregularity caused by diabetes.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Coração/fisiopatologia , Malus/química , Extratos Vegetais/farmacologia , Potenciais de Ação , Animais , Arritmias Cardíacas/metabolismo , Glicemia/análise , Peso Corporal , Frutas/química , Hemoglobinas Glicadas/análise , Coração/efeitos dos fármacos , Soluções Isotônicas , Masculino , Músculos Papilares/metabolismo , Ratos , TemperaturaRESUMO
Mosquito-borne flaviviruses are important pathogens for humans, and the detection of two or more flaviviruses cocirculating in the same geographic area has often been reported. However, the epidemiological impact remains to be determined. Mosquito-borne flaviviruses are primarily transmitted through Aedes and Culex mosquitoes; these viruses establish a life-long or persistent infection without apparent pathological effects. This establishment requires a balance between virus replication and the antiviral host response. Viral interference is a phenomenon whereby one virus inhibits the replication of other viruses, and this condition is frequently associated with persistent infections. Viral interference and persistent infection are determined by several factors, such as defective interfering particles, competition for cellular factors required for translation/replication, and the host antiviral response. The interaction between two flaviviruses typically results in viral interference, indicating that these viruses share common features during the replicative cycle in the vector. The potential mechanisms involved in these processes are reviewed here.