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1.
Braz. j. microbiol ; 46(4): 1265-1268, Oct.-Dec. 2015. graf
Artigo em Inglês | LILACS | ID: lil-769661

RESUMO

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.


Assuntos
Criança , Humanos , Centrifugação com Gradiente de Concentração/métodos , Cromatografia por Troca Iônica/métodos , Norovirus/genética , Proteínas Estruturais Virais/genética , Virossomos/isolamento & purificação , Brasil , Proteínas Estruturais Virais/metabolismo , Virossomos/genética , Virossomos/metabolismo
2.
Braz. j. med. biol. res ; 46(2): 121-127, 01/fev. 2013. graf
Artigo em Inglês | LILACS | ID: lil-668771

RESUMO

The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.


Assuntos
Animais , Humanos , Camundongos , Proteínas de Transporte/farmacocinética , Membrana Celular/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/farmacocinética , Proteínas Estruturais Virais/farmacocinética , Western Blotting , Polpa Dentária/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Estruturais Virais/genética
3.
Rio de Janeiro; s.n; 2009. xvi,100 p. ilus, tab, graf.
Tese em Português | LILACS | ID: lil-539530

RESUMO

Nas décadas de 80 e início de 90 os rotavírus A (RV-A) de genótipo G5, comum em suínos, equinos e bovinos eram detectados com frequência em amostras fecais de crianças brasileiras. Após 1996, deixou de circular em caráter endêmico, tornando-se apenas esporadicamente detectado, enquanto o genótipo G9 começou a ser detectado com frequência. Esta situação leva a crer que houve a substituição do genótipo G5 pelo G9. Tendo em vista a escassez de dados moleculares a respeito de amostras de genótipo G5 de RV-A, no presente estudo foi realizada a análise filogenética para os genes que codificam para as proteínas VP1, VP2, VP3, VP4 e VP7 de vinte e oito amostras de RV-A humano de genótipo G5P(8), coletadas em diferentes estados brasileiros entre 1986 e 2005. A análise filogenética do gene que codifica para a proteína VP7 demonstrou que as mesmas agrupam juntamente com amostras humanas brasileiras de genótipo G5 (IAL28, Br 1054 e Br H8), no entanto a análise do gene que codifica para a proteína VP4 demonstrou que circularam três linhagens do genótipo P(8) (P(8)-1, P(8)-2 e P(8)-3) no Brasil entre 1986 e 2005 em associação com G5. As análises filogenéticas para os genes que codificam para VP1, VP2 e VP3 demonstraram que os mesmos pertencem ao genogrupo Wa-Like, comum a humanos, o que sugere que estas amostras possam ter se originado de uma amostra de RV-A humano. As análises filogenéticas dos genes de VP1, VP2 e VP3 revelaram que todas as amostras foram classificadas dentro dos genótipos R1, M1 e C1, respectivamente. Os resultados do presente estudo enfatizam a importância do monitoramento contínuo e caracterização molecular das amostras de RV-A circulantes, principalmente para prever a possível emergência e/ou re-emergência de genótipos após a introdução de uma vacina contra RV-A nos diferentes continentes do mundo e para se melhor entender a dinâmica e o padrão de evolução dos RV-A de genótipo G5.


Assuntos
Humanos , Rotavirus , Rotavirus/classificação , Proteínas Estruturais Virais , Replicação Viral , Brasil/epidemiologia , Genótipo
4.
Mem. Inst. Oswaldo Cruz ; 101(7): 759-766, Nov. 2006. ilus, tab
Artigo em Inglês | LILACS | ID: lil-439460

RESUMO

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.


Assuntos
Humanos , Animais , Coelhos , Variação Genética , Vírus da Hepatite A/genética , Proteínas Estruturais Virais , Sequência de Aminoácidos , Sequência de Bases , Brasil , Escherichia coli/genética , Expressão Gênica , Genoma Viral , Vírus da Hepatite A/imunologia , Immunoblotting , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Viral
5.
Braz. j. med. biol. res ; 39(7): 873-881, July 2006. ilus, tab
Artigo em Inglês | LILACS | ID: lil-431558

RESUMO

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21 percent of the samples, respectively. Nucleotide sequencing was carried out in 46 percent of VP1/2A and in 53 percent of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0 percent. Phylogenetic analysis of the VP1/2A sequences showed that 65 percent belong to sub-genotype IA and 35 percent to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2 percent identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.


Assuntos
Humanos , Masculino , Feminino , Adulto , /genética , Vírus da Hepatite A/genética , Hepatite A/virologia , RNA Viral/análise , Proteínas Estruturais Virais/genética , Sequência de Bases , Brasil , Genoma Viral , Genótipo , Vírus da Hepatite A/classificação , Vírus da Hepatite A/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
6.
Braz. j. med. biol. res ; 37(5): 691-695, May 2004. ilus
Artigo em Inglês | LILACS | ID: lil-357556

RESUMO

Hepatitis C virus (HCV) was first described in 1989 as the putative viral agent of non-A non-B hepatitis. It is a member of the Flaviviridae family and has been recognized as the major causative agent of chronic liver disease, including chronic active hepatitis, cirrhosis and hepatocellular carcinoma. HCV is a positive RNA virus with a genome containing approximately 9500 nucleotides. It has an open reading frame that encodes a large polyprotein of about 3000 amino acids and is characterized by extensive genetic diversity. HCV has been classified into at least 6 major genotypes with many subtypes and circulates within an infected individual as a number of closely related but distinct variants known as quasispecies. This article reviews aspects of the molecular biology of HCV and their clinical implication.


Assuntos
Humanos , Regiões 3' não Traduzidas , Genoma Viral , Genótipo , Variação Genética , Biologia Molecular , Proteínas não Estruturais Virais , Proteínas Estruturais Virais
7.
Rev. cuba. med. trop ; 55(3): 133-137, sep.-dic. 2003.
Artigo em Espanhol | LILACS | ID: lil-629309

RESUMO

Se describió la introducción de un método molecular para la identificación de los Enterovirus basado en la amplificación, secuenciación y análisis filogenético de la proteína VP1. Se demostró que este método reduce grandemente el tiempo requerido para la identificación de los Enterovirus aislados y resulta de gran utilidad en la caracterización de aislamientos difíciles de tipificar, con el empleo de los reactivos inmunológicos de rutina. Por la rapidez de ejecución de la técnica reviste vital importancia su uso durante epidemias, dada la rápida determinación del agente causal.


The introduction of a mollecular method to identify the Entoviruses based on the amplification, sequencing and phylogenetic analysis of protein VP1 was described. It was proved that this method reduces significantly the time required for the identification of the isolated Entoviruses and that it is very useful in the characterization of isolates which are difficult to typify by the routine immunoloigical reagents. As it is a very fast technqiue, its use is very important during epidemics to determine the causal agent rapidly.


Assuntos
Humanos , Enterovirus/genética , Enterovirus/isolamento & purificação , Proteínas Estruturais Virais/genética , Técnicas de Amplificação de Ácido Nucleico
8.
Mem. Inst. Oswaldo Cruz ; 98(3): 331-334, Apr. 2003. tab, graf
Artigo em Inglês | LILACS | ID: lil-340110

RESUMO

The protein profiles of the New Guinea "C" dengue virus type 2 (DENV-2)prototype and those of a Brazilian DENV-2 isolated in the State of Rio de Janeiro in 1995 were compared. SDS-PAGE analysis showed that the virus from Rio de Janeiro expresses NS5 (93.0 kDa), NS3 (66.8 kDa) E (62.4 kDa) and NS1 (41.2 kDa) proteins differently from the New Guinea "C" virus. The immunoblot revealed specificity and antigenicity for the NS3 protein from DENV-2 Rio de Janeiro mainly in primary infections, convalescent cases, and in secondary infections in both cases and only antigenicity for E and NS1 proteins for both viruses in primary and secondary infections


Assuntos
Animais , Humanos , Vírus da Dengue , Proteínas não Estruturais Virais , Proteínas Estruturais Virais , Western Blotting , Brasil , Vírus da Dengue , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos , Nova Guiné
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