RESUMO
Mayaro virus (MAYV) is a neglected arthropod-borne virus found in the Americas. MAYV infection results in Mayaro fever, a non-lethal debilitating disease characterized by a strong inflammatory response affecting the joints and muscles. MAYV was once considered endemic to forested areas in Brazil but has managed to adapt and spread to urban regions using new vectors, such as Aedes aegypti, and has the potential to cause serious epidemics in the future. Currently, there are no vaccines or specific treatments against MAYV. In this study, the antiviral activity of a series of synthetic cyclic ketones were evaluated for the first time against MAYV. Twenty-four compounds were screened in a cell viability assay, and eight were selected for further evaluation. Effective concentration (EC50) and selectivity index (SI) were calculated and compound 9-(5-(4-chlorophenyl]furan-2-yl)-3,6-dimethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2))-dione (9) (EC50 = 21.5 µmol·L-1, SI = 15.8) was selected for mechanism of action assays. The substance was able to reduce viral activity by approximately 70% in both pre-treatment and post-treatment assays.
Assuntos
Infecções por Alphavirus/virologia , Alphavirus/efeitos dos fármacos , Antivirais/farmacologia , Cetonas/farmacologia , Aedes/virologia , Alphavirus/fisiologia , Infecções por Alphavirus/tratamento farmacológico , Infecções por Alphavirus/transmissão , Animais , Antivirais/química , Brasil , Avaliação Pré-Clínica de Medicamentos , Humanos , Cetonas/química , Mosquitos Vetores/virologiaRESUMO
To improve a DNA vaccine containing the truncated dengue virus serotype 2 (DENV-2) envelope (E) protein and evaluate the influence of precursor membrane (prM) glycoprotein polymorphism on E protein immunogenicity, two vaccine candidates have been constructed by upstream insertion of the DENV-2 and DENV-3 prM genes into the DENV-2 E gene, named pCID2EtD2prM and pCID2EtD3prM, respectively. Both constructs were able to induce antibody production, which were neutralizing against DENV-2 in a murine model. Splenocytes of immunized groups, when challenged with virus, demonstrated Th1 cytokine pattern and proliferation, in addition to the increase of specific T cells. Vaccine candidates pCID2EtD2prM and pCID2EtD3prM confer 70% and 90% protection against DENV-2, respectively. The pCID2EtD3prM plasmid conferred only 40% protection in the lethal challenge with DENV-2. The results demonstrate that DENV-3 prM has a greater influence on the immunogenicity of the E protein and, probably due to its role as a chaperone, these results may be related to the correct folding and, consequently, an increase in the presentation efficiency of produced transcripts.
Assuntos
Vacinas contra Dengue , Vírus da Dengue , Dengue , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Modelos Animais de Doenças , Glicoproteínas de Membrana , Camundongos , Proteínas do Envelope ViralRESUMO
The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development of therapeutics against this arboviral pathogen. In the present investigation, the screening of a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole (35), which inhibited the protease with IC50 of 6.86 µmol L-1. Enzyme kinetic assays showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular docking calculations predicted a recognition pattern involving the residues His51 and Ser135, which are members of the catalytic triad of the WNV NS2B-NS3 protease.
Assuntos
Antivirais/farmacologia , Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Vírus do Nilo Ocidental/enzimologia , Antivirais/química , Domínio Catalítico/efeitos dos fármacos , Descoberta de Drogas , Endopeptidases/química , Eugenol/química , Histidina/química , Histidina/metabolismo , Indanos/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Serina/química , Serina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
Oil refineries are known for the large volume of water used in their processes, as well as the amount of wastewater generated at the end of the production chain. Due to strict environmental regulations, the recycling of water has now become a viable alternative for refineries. Among the many methods available to treat wastewater for reuse, the use of membranes in reverse osmosis systems stands out due to several economic and environmental benefits. However, these systems are vulnerable to contamination and deposition of microorganisms, mainly because of the feedwater quality. In this study, the microbial diversity of feedwater and reverse osmosis membranes was investigated using a combination of culture-dependent and culture-independent methods in order to characterize the microorganisms colonizing and deteriorating the membranes. In total, 37 bacterial isolates, 17 filamentous fungi and approximately 400 clones were obtained and analyzed. Among the bacterial genera identified, the most represented were Sphingobium, Acidovorax, Microbacterium, Rhizobium and Shinella. The results revealed genera that acted as candidate key players in initial biofilm formation in membrane systems, and provided important information concerning the microbial ecology of oligotrophic aquatic systems.
Assuntos
Bactérias/classificação , Biodiversidade , Fungos/classificação , Consórcios Microbianos , Indústria de Petróleo e Gás , Águas Residuárias/microbiologia , Purificação da Água/métodos , Bactérias/genética , Bactérias/isolamento & purificação , Fenômenos Fisiológicos Bacterianos , Biofilmes/crescimento & desenvolvimento , Brasil , Fungos/genética , Fungos/isolamento & purificação , Fungos/fisiologia , Metagenômica , OsmoseRESUMO
A simple and efficient Knoevenagel procedure for the synthesis of 2-arylidene indan-1,3-diones is herein reported. These compounds were prepared via ZrOCl2·8H2O catalyzed reactions of indan-1,3-dione with several aromatic aldehydes and using water as the solvent. The 2-arylidene indan-1,3-diones were obtained with 53%-95% yield within 10-45â¯min. The synthesized compounds were evaluated as inhibitors of the NS2B-NS3 protease of West Nile Virus (WNV). It was found that hydroxylated derivatives impaired enzyme activity with varying degrees of effectiveness. The most active hydroxylated derivatives, namely 2-(4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (14) and 2-(3,4-dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (17), were characterized as noncompetitive enzymes inhibitors, with IC50 values of 11⯵molâ¯L-1 and 3⯵molâ¯L-1, respectively. Docking and electrostatic potential surfaces investigations provided insight on the possible binding mode of the most active compounds within an allosteric site.
Assuntos
Inibidores de Proteases/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Vírus do Nilo Ocidental/enzimologia , Sítio Alostérico , Catálise , Hidroxilação , Indanos/síntese química , Indanos/farmacologia , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , ZircônioRESUMO
Dengue virus (DENV) is an arbovirus that belongs to the Flaviviridae family. Studies reveal that peptides secreted by amphibians have many functions, such as antiviral and antimicrobial activities. As there is no antiviral drug effective against the DENV, the antiviral activity of a synthetic peptide called HS-1, derived from the secretion of the anuran Hypsiboas semilineatus, has been evaluated. The assays of neutralization in the Vero cells show a complete inhibition of infection of the serotypes 2 and 3. Furthermore, the direct action of peptides on the viral particle can be observed through atomic force microscopy. In vivo tests display 80% protection against the dengue-2 virus due to the presence of HS-1, which reveals its potential as an antiviral against the DENV.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Peptídeos/farmacologia , Animais , Chlorocebus aethiops , Vírus da Dengue/classificação , Vírus da Dengue/fisiologia , Humanos , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H2O2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of â¼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H2O2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal.
Assuntos
Fatores Imunológicos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Sequência de Aminoácidos , Animais , Brassica , Bovinos , Células Cultivadas , Cromatografia de Afinidade , Cromatografia em Gel , Avaliação Pré-Clínica de Medicamentos , Cabras , Hemaglutinação , Cavalos , Humanos , Concentração de Íons de Hidrogênio , Fatores Imunológicos/química , Fatores Imunológicos/isolamento & purificação , Ativação de Macrófagos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificaçãoRESUMO
vB_EcoM-UFV13, a member of the T4virus genus, shows lytic activity against Escherichia coli and effectiveness in controlling the biofilm formed by Trueperella pyogenes, which qualifies it as a promising component of phage cocktails for mastitis and metritis control.
RESUMO
BACKGROUND: In recent times, studies have demonstrated that carbon nanotubes are good candidates for use as vehicles for transfection of exogenous material into the cells. However, there are few studies evaluating the behavior of carbon nanotubes as DNA vectors and few of these studies have used multi-walled carbon nanotubes (MWCNTs) or carboxylated MWCNTs. Thus, this study aims to assess the MWCNTs' (carboxylated or not) efficiency in the increase in expression of the tetravalent vaccine candidate (TVC) plasmid vector for dengue virus in vitro using Vero cells, and in vivo, through the intramuscular route, to evaluate the immunological response profile. RESULTS: Multi-walled carbon nanotubes internalized by Vero cells, have been found in the cytoplasm and nucleus associated with the plasmid. However, it was not efficient to increase the messenger ribonucleic acid (mRNA) compared to the pure vaccine candidate associated with Lipofectamine(®) 2000. The in vivo experiments showed that the use of intramuscular injection of the TVC in combination with MWCNTs reduced the immune response compared to pure TVC, in a general way, although an increase was observed in the population of the antibody-producing B cells, as compared to pure TVC. CONCLUSIONS: The results confirm the data found by other authors, which demonstrate the ability of nanotubes to penetrate target cells and reach both the cytoplasm and the cell nucleus. The cytotoxicity values are also in accordance with the literature, which range from 5 to 20 µg/mL. This has been found to be 10 µg/mL in this study. Although the expression levels are higher in cells that receive the pure TVC transfected using Lipofectamine(®) 2000, the nanotubes show an increase in B-cells producing antibodies.
Assuntos
Anticorpos Antivirais/biossíntese , Linfócitos B/efeitos dos fármacos , Vacinas contra Dengue/administração & dosagem , Dengue/prevenção & controle , Nanotubos de Carbono/química , Transfecção/métodos , Vacinação , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Chlorocebus aethiops , Dengue/imunologia , Dengue/virologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/imunologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Lipídeos/farmacologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/química , Plasmídeos/metabolismo , Células VeroRESUMO
The multidrug-resistant bacteria have become a serious problem to public health. In this scenery the antimicrobial peptides (AMPs) derived from animals and plants emerge as a novel therapeutic modality, substituting or in addition to the conventional antimicrobial. The anurans are one of the richest natural sources of AMPs. In this work several cycles of cDNA cloning of the skin of the Brazilian treefrog Hypsiboas semilineatus led to isolation of a precursor sequence that encodes a new AMP. The sequence comprises a 27 residue signal peptide, followed by an acidic intervening sequence that ends in the mature peptide at the carboxy terminal. The AMP, named Hs-1, has 20 amino acids residues, mostly arranged in an alpha helix and with a molecular weight of 2144.6 Da. The chemically synthesized Hs-1 showed an antimicrobial activity against all Gram-positive bacteria tested, with a range of 11-46 µM, but it did not show any effect against Gram-negative bacteria, which suggest that Hs-1 may have a selective action for Gram-positive bacteria. The effects of Hs-1 on bacterial cells were also demonstrated by transmission electron microscopy. Hs-1 is the first AMP to be described from H. semilineatus.
Assuntos
Proteínas de Anfíbios/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Anuros/metabolismo , Descoberta de Drogas , Bactérias Gram-Positivas/efeitos dos fármacos , Pele/química , Sequência de Aminoácidos , Proteínas de Anfíbios/efeitos adversos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Animais , Anti-Infecciosos/efeitos adversos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/efeitos adversos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sequência de Bases , Células Sanguíneas/efeitos dos fármacos , Brasil , DNA Complementar/química , Florestas , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/ultraestrutura , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Peso Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
KEY MESSAGE: Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.
Assuntos
Dengue/diagnóstico , Nicotiana/genética , Proteínas Recombinantes/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Aedes/virologia , Agrobacterium tumefaciens/genética , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Estudos de Casos e Controles , Vírus da Dengue/imunologia , Vírus da Dengue/patogenicidade , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Plantas Geneticamente Modificadas , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: Phages infecting spoilage microorganisms have been considered as alternative biocontrol agents, and the study of their genomes is essential to their safe use in foods. UFV-P2 is a new Pseudomonas fluorescens-specific phage that has been tested for its ability to inhibit milk proteolysis. RESULTS: The genome of the phage UFV-P2 is composed of bidirectional modules and presented 75 functionally predict ORFs, forming clusters of early and late transcription. Further genomic comparisons of Pseudomonas-specific phages showed that these viruses could be classified according to conserved segments that appear be free from genome rearrangements, called locally collinear blocks (LCBs). In addition, the genome organization of the phage UFV-P2 was shown to be similar to that of phages PaP3 and LUZ24 which have recently been classified as a Luz24likevirus. CONCLUSIONS: We have presented the functional annotation of UFV-P2, a new Pseudomonas fluorescens phage. Based on structural genomic comparison and phylogenetic clustering, we suggest the classification of UFV-P2 in the Luz24likevirus genus, and present a set of shared locally collinear blocks as the genomic signature for this genus.
Assuntos
Bacteriófagos/classificação , Bacteriófagos/genética , Genoma Viral , Bacteriófago P2/genética , Análise por Conglomerados , Biologia Computacional , Fases de Leitura Aberta , Filogenia , Pseudomonas fluorescens/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Dengue is a major international public health concern. There is no drug to treat dengue virus infections and a vaccine is yet to be licensed. The laboratory diagnosis of dengue virus infection has been greatly improved during the last decade; therefore, the main limiting factor is the production of recombinant viral antigens on a large scale. Domain III of dengue virus envelope protein contains multiplex conformation-dependent neutralizing epitopes, making it an attractive diagnostic candidate. In this work, we have demonstrated the expression of dengue virus type 1 envelope domain III protein (EDIII-D1) in methylotrophic yeast, Pichia pastoris GS115. The recombinant secreted protein (sEDIII-D1) was purified by affinity chromatography and characterized by SDS-PAGE. Purified protein was recognized in immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue-infected human serum samples. In conclusion, secreted expressions of domain III protein can be obtained in P. pastoris by methanol induction. This product has the potential to be used for the diagnosis of dengue infections.
Assuntos
Antígenos Virais/genética , Vírus da Dengue/genética , Dengue/diagnóstico , Dengue/virologia , Pichia/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Sequência de Bases , Clonagem Molecular/métodos , Dengue/sangue , Dengue/imunologia , Vírus da Dengue/química , Vírus da Dengue/imunologia , Vírus da Dengue/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.
Assuntos
Biodegradação Ambiental , Biblioteca Gênica , Metagenômica , Petróleo , Fenol/metabolismo , Esgotos/microbiologia , Reatores Biológicos/microbiologia , Catecol 2,3-Dioxigenase/genética , Catecol 2,3-Dioxigenase/metabolismo , Clonagem Molecular , Mapeamento de Sequências Contíguas , DNA Bacteriano/genética , Genoma Bacteriano , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Águas Residuárias/microbiologiaRESUMO
Milk proteolysis caused by Pseudomonas fluorescens is a serious problem in the dairy industries as a result of its ability to grow under refrigeration. The use of phages to control contaminants in food has been considered an alternative to traditional methods; therefore, a thorough understanding of such organisms is vital for their use. In this study, we show the complete genome sequence and analysis of a P. fluorescens phage isolated from wastewater of a dairy industry in Brazil.
RESUMO
Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins in the gastrointestinal (GI) tract of ruminant and monogastric animals. In this study, viable (V) and heat-killed (HK) Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen) the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.
Assuntos
Trato Gastrointestinal/imunologia , Temperatura Alta , Infecções Estreptocócicas/imunologia , Streptococcus bovis/imunologia , Administração Oral , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Bacteriocinas/imunologia , Bacteriocinas/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-5/genética , Interleucina-5/imunologia , Interleucina-5/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Baço/metabolismo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/prevenção & controle , Streptococcus bovis/metabolismo , Streptococcus bovis/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Aumento de Peso/imunologiaRESUMO
Viruses belonging to the Flaviviridae family are found and distributed in most of the tropical and sub-tropical regions of the world. The genus has more than 56 members, most of which cause clinical symptoms in humans. The clinical diagnosis of dengue requires laboratory confirmation because of the similarity of symptoms with a series of other acute fevers and the primary use antibodies or antigens for detection. In this work, peptides E(1) and E(2) of the envelope protein (E) of the dengue virus were mapped using bioinformatics methods. These peptides were then expressed in a prokaryotic system and purified. An indirect ELISA for antibodies IgG and IgM from laboratory samples previously characterised was then used with the peptides to detect anti-dengue antibodies. For IgG using the peptide E(1), the sensitivity of the indirect ELISA was 88.3% and the specificity was 56%; using the peptide E(2), the sensitivity was 90% and the specificity was 59%; and using a combination of both peptides, the sensitivity was 93.3% and the specificity was 78%. For IgM using the peptide E(1), the sensitivity was 88% and the specificity was 66%; using the peptide E(2), the sensitivity was 88% and the specificity was 69%; and when used in combination, the peptides E(1)/E(2) demonstrated a sensitivity of 90% and a specificity of 86%. These results indicate that the use of the E(1) and E(2) peptides of the E protein are an alternative for serological diagnosis of dengue fever.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Dengue/imunologia , Dengue/diagnóstico , Peptídeos , Proteínas do Envelope Viral , Vírus da Dengue/genética , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The aim of this study was to investigate HIV-1 molecular diversity and the epidemiological profile of HIV-1-infected patients from Ribeirão Preto, Brazil. A nested PCR followed by sequencing of a 302-base pair fragment of the env gene (C2-V3 region) was performed in samples from HIV-1-positive patients. A total of 45 sequences were aligned with final manual adjustments. The phylogenetic analyses showed a higher prevalence of HIV-1 subtype B in the studied population (97.8%) with only one sample yielding an F1 subtype. The viral genotyping prediction showed that CCR5 tropism was the most prevalent in the studied cohort. Geno2pheno analysis showed that R5 and CXCR4 prediction were 69% and 31%, respectively. There was no statistical significance, either in viral load or in CD4(+) T cell count when R5 and X4 prediction groups were compared. Moreover, the GPGR tetramer was the most common V3 loop core motif identified in the HIV-1 strains studied (34.1%) followed by GWGR, identified in 18.1% of the samples. The high level of B subtype in this Brazilian population reinforces the nature of the HIV epidemic in Brazil, and corroborates previous data obtained in the Brazilian HIV-infected population.
Assuntos
Linfócitos T CD4-Positivos/virologia , Regiões Determinantes de Complementaridade/genética , Genes env , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/epidemiologia , HIV-1/genética , Adolescente , Adulto , Sequência de Bases , Brasil/epidemiologia , Linfócitos T CD4-Positivos/imunologia , Estudos de Coortes , Regiões Determinantes de Complementaridade/imunologia , Epidemias , Feminino , Genótipo , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/imunologia , HIV-1/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores CXCR/genética , Receptores CXCR/imunologia , Carga ViralRESUMO
0 diagnóstico da infecção pelo vírus da dengue é baseado, em grande parte dos casos, apenas no exame clínico do paciente, já que somente alguns poucos grandes centros possuem laboratórios clinicos que disponibilizam testes diagnósticos para confirmar sintomas clinicos de infecção. Atualmente, o diagnóstico laboratorial da dengue pode ser feito por diferentes tipos de testes. Entre eles estao os métodos de pesquisa sorológica, de isolamento viral, de detecção de antigenos virais e do genoma viral. 0 desenvolvimento continuado de testes diagnósticos baratos, sensíveis, especificos e de fácil execucção, que sejam capazes de proporcionar diagnóstico precoce da infecção pelos virus da dengue, é ainda uma necessidade. Existem também outros obstáculos que nao são especificamente relacionados ao desenvolvimento tecnológico dos métodos diagnósticos. Por exemplo, a infra-estrutura dos laboratórios, o treinamento do pessoal técnico e a capacidade de pesquisa ainda são limitados em muitas partes do Brasil e do mundo, onde a dengue é endêmica. Laboratórios clinicos, principalmente os que atendem às regiões de maior incidência da dengue, devem se interar de todos os métodos diagnósticos disponíveis para a rotina atualmente e definir qual se adapta melhor às suas condições de trabalho e população atendida, com o intuito de salvarvidas.
The diagnosis of the infection caused by the dengue virus relies in most cases on the clinical examination of the patient, since only a few major centers have clinical laboratories providing diagnostic tests to confirm the clinical symptons of infection. Currently, routine laboratory diagnosis can be done by different kinds of testing. Among them the methods ofserological research, virus isolation, detection of viral antigens and of viral genome are included. The continued development of diagnostic tests that are cheap, sensitive, specific, easy to perform, and that are capable of giving early diagnosis of the dengue virus infection is still a need. There are also other obstacles not specifically related to the technological development ofdiagnostic methods. For instance, the laboratories' infrastructure, the workers training and the research capacity are still limited in many parts of Brazil and the world, where dengue is endemic. Clinical laboratories, specially those situated in regions with high incidence of dengue, should be aware of all the diagnostic methods available four routine nowadays, and choose the best one that fulfills their working conditions and populations served, in order to save lives.