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1.
Pathogens ; 10(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34451447

RESUMO

Madariaga virus (MADV) is a member of the eastern equine encephalitis virus (EEEV) complex that circulates in Central and South America. It is a zoonotic, mosquito-borne pathogen, belonging to the family Togaviridae. Disturbances in the natural transmission cycle of this virus result in outbreaks in equines and humans, leading to high case fatality in the former and acute febrile illness or neurological disease in the latter. Although a considerable amount of knowledge exists on the eco-epidemiology of North American EEEV strains, little is known about MADV. In Brazil, the most recent isolations of MADV occurred in 2009 in the States of Paraíba and Ceará, northeast Brazil. Because of that, health authorities have recommended vaccination of animals in these regions. However, in 2019 an equine encephalitis outbreak was reported in a municipality in Ceará. Here, we present the isolation of MADV from two horses that died in this outbreak. The full-length genome of these viruses was sequenced, and phylogenetic analyses performed. Pathological findings from postmortem examination are also discussed. We conclude that MADV is actively circulating in northeast Brazil despite vaccination programs, and call attention to this arbovirus that likely represents an emerging pathogen in Latin America.

2.
Braz J Microbiol ; 52(2): 1021-1027, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33797731

RESUMO

Flaviviruses as West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilhéus virus (ILHV), and Rocio virus (ROCV) are previously reported in different Brazilian regions, but studies in Southern Brazil are still scarce. To improve the information regarding flaviviruses in Southern Brazil, horse serum samples were analyzed using RT-qPCR and a commercial ELISA-Ab against WNV followed by PRNT75. All 1000 samples analyzed by real-time RT-PCR resulted negative. The 465 subsampled samples were analyzed by a commercial ELISA-Ab against WNV, and the 18.5% (86/465) positive samples were further analyzed by PRNT75. In the PRNT75, 13/86 and 2/86 horses were positive for SLEV and WNV, respectively. It was observed that 5.8% (13/226) of the farms presented at least one positive animal for SLEV in PRNT75, whereas 0.9% (2/226) for WNV. Apart from the lower seroprevalences identified when compared to data previously reported in other Brazilian regions, our results suggest that public health professionals must be aware of the presence of these potential zoonotic pathogens.


Assuntos
Vírus da Encefalite de St. Louis/isolamento & purificação , Encefalite por Arbovirus/veterinária , Infecções por Flavivirus/veterinária , Doenças dos Cavalos/virologia , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Vírus da Encefalite de St. Louis/genética , Vírus da Encefalite de St. Louis/imunologia , Encefalite por Arbovirus/sangue , Encefalite por Arbovirus/epidemiologia , Encefalite por Arbovirus/virologia , Infecções por Flavivirus/sangue , Infecções por Flavivirus/epidemiologia , Infecções por Flavivirus/virologia , Geografia , Doenças dos Cavalos/sangue , Doenças dos Cavalos/epidemiologia , Cavalos , RNA Viral/genética , Estudos Soroepidemiológicos , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/imunologia
4.
Viruses ; 12(6)2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32486283

RESUMO

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Genes Reporter/fisiologia , Vírus de RNA/efeitos dos fármacos , Replicon/fisiologia , Animais , Antivirais/química , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Vírus de RNA/genética , Transfecção , Células Vero
5.
Acta Trop ; 188: 213-224, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30195666

RESUMO

Chikungunya fever is a mosquito-borne viral illness characterized by a sudden onset of fever associated with joint pains. It was first described in the 1950s during a Chikungunya virus (CHIKV) outbreak in southern Tanzania and has since (re-) emerged and spread to several other geographical areas, reaching large populations and causing massive epidemics. In recent years, CHIKV has gained considerable attention due to its quick spread to the Caribbean and then in the Americas, with many cases reported between 2014 and 2017. CHIKV has further garnered attention due to the clinical diagnostic difficulties when Zika (ZIKV) and dengue (DENV) viruses are simultaneously present. In this review, topical CHIKV-related issues, such as epidemiology and transmission, are examined. The different manifestations of infection (acute, chronic and atypical) are described and a particular focus is placed upon the diagnostic handling in the case of ZIKV and DENV co-circulating. Natural and synthetic compounds under evaluation for treatment of chikungunya disease, including drugs already licensed for other purposes, are also discussed. Finally, previous and current vaccine strategies, as well as the control of the CHIKV transmission through an integrated vector management, are reviewed in some detail.


Assuntos
Febre de Chikungunya/epidemiologia , Vírus Chikungunya , Dengue/diagnóstico , Infecção por Zika virus/diagnóstico , Febre de Chikungunya/complicações , Febre de Chikungunya/diagnóstico , Controle de Doenças Transmissíveis/métodos , Dengue/complicações , Dengue/epidemiologia , Diagnóstico Diferencial , Surtos de Doenças , Humanos , Infecção por Zika virus/complicações
6.
Acta Trop ; 182: 257-263, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29551394

RESUMO

Emerging and re-emerging viral infections transmitted by insect vectors (arthopode-borne viruses, arbovirus) are a serious threat to global public health. Among them, yellow fever (YFV), dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) viruses are particularly important in tropical and subtropical regions. Although vector control is one of the most used prophylactic measures against arboviruses, it often faces obstacles, such as vector diversity, uncontrolled urbanization and increasing resistance to insecticides. In this context, vaccines may be the best control strategy for arboviral diseases. Here, we provide a general overview about licensed vaccines and the most advanced vaccine candidates against YFV, DENV, CHIKV and ZIKV. In particular, we highlight vaccine difficulties, the current status of the most advanced strategies and discuss how the molecular characteristics of each virus can influence the choice of the different vaccine formulations.


Assuntos
Arbovírus/imunologia , Vírus Chikungunya/imunologia , Vírus da Dengue/imunologia , Vacinas Virais/imunologia , Zika virus/imunologia , Animais , Febre de Chikungunya/prevenção & controle , Febre de Chikungunya/transmissão , Dengue/prevenção & controle , Dengue/transmissão , Vacinas contra Dengue/imunologia , Descoberta de Drogas , Humanos , Insetos Vetores/imunologia , Febre Amarela/prevenção & controle , Febre Amarela/transmissão , Vacina contra Febre Amarela/imunologia , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/transmissão
7.
An Acad Bras Cienc ; 89(3 Suppl): 2119-2130, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28746549

RESUMO

Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.


Assuntos
Luciferases/genética , Vírus da Febre Amarela/genética , Animais , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Luciferases/análise , Replicação Viral
9.
J Proteome Res ; 16(4): 1542-1555, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28317380

RESUMO

Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. 59 proteins were found to be significantly increased when compared with a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher's exact test was significant for 58 proteins, and the p value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified 16 pathways, and this analysis indicated sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using sirolimus and cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.


Assuntos
Ciclofilina A/metabolismo , Proteínas/genética , Replicação Viral/genética , Vírus da Febre Amarela/genética , Ciclofilina A/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Biologia de Sistemas , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Vírus da Febre Amarela/patogenicidade
10.
An. acad. bras. ciênc ; 89(3,supl): 2119-2130, 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-886794

RESUMO

ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.


Assuntos
Animais , Vírus da Febre Amarela/genética , Luciferases/genética , Replicação Viral , Anticorpos Neutralizantes/análise , Luciferases/análise , Anticorpos Antivirais/análise
11.
J Infect Dis ; 214(12): 1897-1904, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27923950

RESUMO

BACKGROUND: Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates. METHODS: We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated. RESULTS: Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P < .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P < .0001). CONCLUSIONS: Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs in equivocal cases.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/líquido cefalorraquidiano , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Imunoglobulina M/sangue , Infecção por Zika virus/diagnóstico , Zika virus/imunologia , Adolescente , Adulto , Sangue/imunologia , Líquido Cefalorraquidiano/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Recém-Nascido , Masculino , Testes de Neutralização , Sensibilidade e Especificidade , Ensaio de Placa Viral , Adulto Jovem , Infecção por Zika virus/congênito
12.
F1000Res ; 5: 2523, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27909576

RESUMO

Background The recent epidemics of Zika virus (ZIKV) implicated it as the cause of serious and potentially lethal congenital conditions such microcephaly and other central nervous system defects, as well as the development of the Guillain-Barré syndrome in otherwise healthy patients. Recent findings showed that anti-Dengue antibodies are capable of amplifying ZIKV infection by a mechanism similar to antibody-dependent enhancement, increasing the severity of the disease. This scenario becomes potentially catastrophic when the global burden of Dengue and the advent of the newly approved anti-Dengue vaccines in the near future are taken into account. Thus, antiviral chemotherapy should be pursued as a priority strategy to control the spread of the virus and prevent the complications associated with Zika. Methods Here we describe a fast and reliable cell-based, high-content screening assay for discovery of anti-ZIKV compounds. This methodology has been used to screen the National Institute of Health Clinical Collection compound library, a small collection of FDA-approved drugs. Results and conclusion From 725 FDA-approved compounds triaged, 29 (4%) were found to have anti-Zika virus activity, of which 22 had confirmed (76% of confirmation) by dose-response curves. Five candidates presented selective activity against ZIKV infection and replication in a human cell line. These hits have abroad spectrum of chemotypes and therapeutic uses, offering valuable opportunities for selection of leads for antiviral drug discovery.

13.
Trop Med Int Health ; 20(12): 1707-10, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26426773

RESUMO

OBJECTIVE: To present results of virological surveillance and epidemiological aspects of dengue in the State of Rio Grande do Norte, Brazil. METHODS: A total of 1581 cases, reported from 2010 to 2012 at various health centres in the state, were analysed by viral isolation and/or RT-PCR for viral detection and typing. To identify whether different genotypes were circulating in the state during this period, sequencing of the complete E gene for DENV (1485 bp in length) was performed directly from patient serum samples. RESULTS: All four serotypes of dengue virus circulated in Rio Grande do Norte, with the introduction of DENV-4 in the state in 2011. In 2012, DENV-4 represented 100% of positive confirmed cases. 53.97% of cases occurred in Natal. Case numbers peaked in April (21%) and May (23%). Genetic characterisation of circulating strains confirmed the circulation of genotypes V, south-east Asian/American and II, respectively, for DENV-1, DENV-2 and DENV-4. CONCLUSIONS: This work furthers a better understanding of dengue viruses in the State of Rio Grande do Norte. Strengthening control efforts in the region is important considering the impact of dengue.


Assuntos
Vírus da Dengue/genética , Dengue/epidemiologia , Epidemias , Genótipo , Filogenia , Adolescente , Adulto , Brasil/epidemiologia , Criança , Pré-Escolar , Dengue/virologia , Surtos de Doenças , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Vigilância da População , Prevalência , Sorotipagem/métodos , Adulto Jovem
14.
Microb Ecol ; 67(4): 775-87, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24509705

RESUMO

We surveyed diversity patterns and engaged in bioprospecting for bioactive compounds of fungi associated with the endemic macroalgae, Monostroma hariotii and Pyropia endiviifolia, in Antarctica. A total of 239 fungal isolates were obtained, which were identified to represent 48 taxa and 18 genera using molecular methods. The fungal communities consisted of endemic, indigenous and cold-adapted cosmopolitan taxa, which displayed high diversity and richness, but low dominance indices. The extracts of endemic and cold-adapted fungi displayed biological activities and may represent sources of promising prototype molecules to develop drugs. Our results suggest that macroalgae along the marine Antarctic Peninsula provide additional niches where fungal taxa can survive and coexist with their host in the extreme conditions. We hypothesise that the dynamics of richness and dominance among endemic, indigenous and cold-adapted cosmopolitan fungal taxa might be used to understand and model the influence of climate change on the maritime Antarctic mycota.


Assuntos
Biodiversidade , Clorófitas/microbiologia , Fungos/fisiologia , Rodófitas/microbiologia , Regiões Antárticas , DNA Intergênico/genética , Fungos/genética , Fungos/isolamento & purificação , Fungos/metabolismo , Geografia , Dados de Sequência Molecular , Análise de Sequência de DNA
15.
An Acad Bras Cienc ; 86(4): 1749-59, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25590713

RESUMO

Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.


Assuntos
Vírus da Dengue/genética , Plasmídeos/genética , Transcrição Genética/genética , Replicação Viral , Brasil , Células Clonais , DNA Complementar/genética , Vírus da Dengue/classificação , RNA Viral/genética
16.
Hum Immunol ; 74(9): 1225-30, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23747994

RESUMO

Four genetic polymorphisms located at the promoter (C-257T) and coding regions of CFH gene (exon 2 G257A, exon 14 A2089G and exon 19 G2881T) were investigated in 121 dengue patients (DENV-3) in order to assess the relationship between allele/haplotypes variants and clinical outcomes. A statistical value was found between the CFH-257T allele (TT/TC genotypes) and reduced susceptibility to severe dengue (SD). Statistical associations indicate that individuals bearing a T allele presented significantly higher protein levels in plasma. The -257T variant is located within a NF-κB binding site, suggesting that this variant might have effect on the ability of the CFH gene to respond to signals via the NF-κB pathway. The G257A allelic variant showed significant protection against severe dengue. When CFH haplotypes effect was considered, the ancestral CG/CG promoter-exon 2 SNP genotype showed significant risk to SD either in a general comparison (ancestral × all variant genotypes), as well as in individual genotypes comparison (ancestral × each variant genotype), where the most prevalent effect was observed in the CG/CG × CA/TG comparison. These findings support the involvement of -257T, 257A allele variants and haplotypes on severe dengue phenotype protection, related with high basal CFH expression.


Assuntos
Fator H do Complemento/genética , Vírus da Dengue/imunologia , Dengue/genética , Adolescente , Adulto , Idoso , Brasil , Criança , Pré-Escolar , Dengue/imunologia , Progressão da Doença , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Polimorfismo Genético , Adulto Jovem
17.
An Acad Bras Cienc ; 85(1): 159-68, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23460439

RESUMO

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potential for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.


Assuntos
Clonagem Molecular , Genes Reporter/genética , Recombinação Genética/genética , Replicon/genética , Vírus da Febre Amarela/genética , Humanos , RNA Viral/genética , Replicação Viral , Vírus da Febre Amarela/fisiologia
18.
PLoS One ; 5(6): e11267, 2010 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-20585645

RESUMO

BACKGROUND: Symptomatic infection by dengue virus (DENV) can range from dengue fever (DF) to dengue haemorrhagic fever (DHF), however, the determinants of DF or DHF progression are not completely understood. It is hypothesised that host innate immune response factors are involved in modulating the disease outcome and the expression levels of genes involved in this response could be used as early prognostic markers for disease severity. METHODOLOGY/PRINCIPAL FINDINGS: mRNA expression levels of genes involved in DENV innate immune responses were measured using quantitative real time PCR (qPCR). Here, we present a novel application of the support vector machines (SVM) algorithm to analyze the expression pattern of 12 genes in peripheral blood mononuclear cells (PBMCs) of 28 dengue patients (13 DHF and 15 DF) during acute viral infection. The SVM model was trained using gene expression data of these genes and achieved the highest accuracy of approximately 85% with leave-one-out cross-validation. Through selective removal of gene expression data from the SVM model, we have identified seven genes (MYD88, TLR7, TLR3, MDA5, IRF3, IFN-alpha and CLEC5A) that may be central in differentiating DF patients from DHF, with MYD88 and TLR7 observed to be the most important. Though the individual removal of expression data of five other genes had no impact on the overall accuracy, a significant combined role was observed when the SVM model of the two main genes (MYD88 and TLR7) was re-trained to include the five genes, increasing the overall accuracy to approximately 96%. CONCLUSIONS/SIGNIFICANCE: Here, we present a novel use of the SVM algorithm to classify DF and DHF patients, as well as to elucidate the significance of the various genes involved. It was observed that seven genes are critical in classifying DF and DHF patients: TLR3, MDA5, IRF3, IFN-alpha, CLEC5A, and the two most important MYD88 and TLR7. While these preliminary results are promising, further experimental investigation is necessary to validate their specific roles in dengue disease.


Assuntos
Dengue/classificação , Expressão Gênica , Dengue/genética , Dengue/imunologia , Humanos , Imunidade Inata/genética , RNA Mensageiro/genética
19.
An. acad. bras. ciênc ; 81(4): 671-677, Dec. 2009. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-529928

RESUMO

The management of acute dengue patients during outbreaks is a challenging problem. Most of the dengue fever cases are benign, but some cases develop into a severe and possibly lethal vasculopathy, known as dengue hemorrhagic fever. Early symptoms of dengue and hemorrhagic fever are very similar. An early differential diagnosis is needed to predict which of these two clinical presentations is crucial to proper patient care and public health management. This study evaluates the predictive potential of specific mRNA expression markers of dengue hemorrhagic fever using quantitative real-time PCR assays. Six candidate "dengue hemorrhagic fever specific signature genes" were evaluated and all showed good correlation among their transcription levels at early days of infection and the later development of severe vasculopathy. The markers selected were able to indicate, at early stages of infection, the evolution of a dengue-infected patient to the severe form of the illness. Despite the fact that these results grant further validation studies, the panel of candidate prognostic markers obtained demonstrated the potential to be useful for clinical use in the form of a fast assay based in blood samples.


O manejo de pacientes infectados pelo dengue ainda é um problema desafiador. A maioria dos casos de dengue é benigna mas parte desses casos pode evoluir para o desenvolvimento de vasculopatia severa conhecida como dengue hemorrágica, que pode ser letal. Os sintomas iniciais da dengue e sua forma hemorrágica são bastante similares. O desenvolvimento de um teste diagnóstico que seja rápido e capaz de diferenciar as duas formas clínicas da dengue é crucial para o cuidado adequado de pacientes. O presente estudo avalia, através da PCR quantitativa em tempo real, o potencial preditivo dos níveis de expressão de RNAm candidatos a marcadores da dengue hemorrágica, previamente identificados por estudos genômicos funcionais. Um conjunto de seis marcadores moleculares para a dengue hemorrágica foi avaliado e apresentou correlação entre seus níveis de transcrição e o posterior desenvolvimento da vasculopatia severa. Os marcadores selecionados foram capazes de indicar, nos momentos iniciais dos sintomas, a evolução de um paciente infectado pelo dengue para a forma severa da doença. O painel de candidatos a marcadores de prognóstico obtido demonstrou um bom potencial para uso clínico na forma de um ensaios rápido baseado em amostras de sangue.


Assuntos
Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dengue Grave/diagnóstico , Vírus da Dengue/genética , Estudos de Coortes , DNA Viral/análise , Dengue Grave/virologia , Diagnóstico Precoce , Marcadores Genéticos , Análise em Microsséries , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Viral/análise
20.
PLoS One ; 4(11): e7892, 2009 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-19936257

RESUMO

BACKGROUND: We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity: classic dengue fever or dengue hemorrhagic fever (DHF) samples were compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2-3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels. CONCLUSIONS/SIGNIFICANCE: Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection.


Assuntos
Dengue/diagnóstico , Dengue/genética , Febre/diagnóstico , Regulação da Expressão Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores/metabolismo , Brasil , Estudos de Coortes , Dengue/metabolismo , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Feminino , Febre/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos , Resultado do Tratamento
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