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Id: lil-784965
Autor: Castro-Orozco, Raimundo; Castro-García, Lyda R; Gómez-Camargo, Doris E.
Título: Análisis filogenético de secuencias suramericanas del gen ns1 del serotipo dengue-2, relacionadas con sangrado clínico severo / Phylogenetic analysis of South American sequences of the nonstructural protein-1 (ns1) of dengue serotype 2 associated with severe clinical bleeding
Fonte: Rev. salud pública;18(3):1-1, mayo-jun. 2016. ilus, tab.
Idioma: es.
Resumo: Objetivo Comparar secuencias de nucleótidos y de aminoácidos de la proteína no estructural 1-NS1 de cepas DENV-2, aisladas de pacientes febriles de diferentes países suramericanos, que cursaron cuadros clínicos con severidad o sin ella. Materiales y Métodos El análisis filogenético fue realizado a partir de 28 secuencias moleculares completas (1 056 pb) del gen NS1 del serotipo DENV-2. Se realizó un análisis filogenético bayesiano utilizando el software MrBayes v.3.2.0, con el modelo SYM+G y un análisis filogenético con el método Neighbor-Joining con el modelo Jukes-Cantor. Además, las secuencias de aminoácidos fueron alineadas y comparadas entre sí, mediante el programa Clustal W incluido en el software MEGA v. 5.2. Resultados En las secuencias de aminoácidos asociadas a sangrado, la sustitución más frecuente fue isoleucina→ treonina, en la posición 93. Estas secuencias presentaron un mayor porcentaje (94,6 %) de homología de aminoácidos de la proteína NS1 en comparación con el porcentaje de homología (74 %) de los aislamientos DENV-2 no asociados a sangrado. Se identificaron cinco clados que agrupan la mayoría de las secuencias analizadas (19/24; 79,2 %) con valores de probabilidad posterior mayores o iguales al 58 %. Siete (87,5 %) secuencias asociadas a sangrado se relacionan filogenéticamente dentro de los clados 4 y 5, con valores de probabilidad posterior del 58 % y 97 %, respectivamente. Conclusión No se encontraron características filogenéticas ni tampoco diferencias entre las secuencias de aminoácidos de la proteína NS1-DENV-2 estudiadas, que pudieran ser relacionadas, de manera directa, con la severidad de la enfermedad.(AU)

Objective The objective of this in silico study was to compare nucleotide and amino acids DENV-2-NS1 sequences isolated from febrile patients, with and without disease severity, from different South American countries. Matherials and Methods A bayesian MCMC phylogenetic analysis was carried out using 28 complete sequences of the gene NS1 of the DENV-2 serotype (1 056 bp), using MrBayes v.3.2.0 software, with the model SYM+G (2.5 million generations). We also carried out a phylogenetic analysis with Neighbor-Joining method (Jukes-Cantor model). In addition, the amino acids sequences were aligned and compared with each other, using Clustal W included in MEGA v.5.2 software. Results In the amino acids sequences associated with bleeding, the most frequent substitution was isoleucine → threonine at posicion 93. These sequences showed a high percentage (94.6 %) of amino acid homology in comparison with the percentage of amino acids homology (74 %) of DENV-2 isolates not associated with bleeding. Five clades were identified that group the vast majority of the DENV-2-NS1 sequences analyzed (19/24; 79.2 %) with posterior probability values greater than or equal to 58 %. Seven sequences (87.5 %) associated with bleeding were phylogenetically related within clades 4 and 5, the posterior probability values were 58 % and 97 %, respectively. Conclusion Neither phylogenetic characteristics nor differences between amino acids of the DENV-2-NS1 sequences studied were found that could be associated directly with severity of the disease.(AU)
Descritores: Filogenia
Proteínas não Estruturais Virais/análise
Dengue Grave/diagnóstico
Vírus da Dengue/isolamento & purificação
-América do Sul
Simulação por Computador
Limites: Humanos
Tipo de Publ: Estudo Comparativo
Responsável: BR1.1 - BIREME


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Id: biblio-1122174
Autor: Leite, Beatriz Alcântara; Miyagi, Sueli Akemi Taniwaki; Brandão, Paulo Eduardo.
Título: Evolution of Avian coronavirus (AvCoV) in BHK-21 and VERO cells / Evolução de Coronavírus aviário (AvCoV) em células BHK-21 e VERO
Fonte: Braz. J. Vet. Res. Anim. Sci. (Online);57(2):e166086, mai. 2020. ilus, tab, graf.
Idioma: en.
Projeto: CNPq (Brazilian National Board for Scientific and Technological Development); . CAPES (Coordination for the Improvement of Higher Education Personnel, Brazil).
Resumo: Avian coronavirus (AvCoV) infects a range of tissues in chickens and several other avian species. Although the virus can be isolated in chicken embryos, only a few strains of the 6 genotypes/33 lineages can grow in cell lines, with the Beaudette strain (GI-1 lineage) being the most used for in vitro studies. Considering the differences between cell lines and chicken embryos as habitats for AvCoV, this study aimed to assess the diversity of the genes coding for the nonstructural protein 3 (nsp3) and spike envelope protein (S) after serial passages in BHK-21 and Vero cells. After 14 passages of an embryo-adapted Beaudette strain, the virus loads fluctuated in both cell lines, with the highest loads being 8.72 log genome copies/µL for Vero and 6.36 log genome copies/µL for BHK-21 cells. No polymorphisms were found for nsp3; regarding S, not only aa substitutions (Vero: 8th passage A150S, and 14th S150A; BHK-21: 4th S53F, 8th F53Y, and 8th S95R), but also minor variants could be detected on chromatograms with fluctuating intensities. As the regions of these aa substitutions are within the receptor-binding domain of S, it can be speculated that differences in cell receptors between Vero and BHK-21 cells and the speed of cell death led to the selection of different dominant strains, while the stability of nsp3 supports its function as a protease involved in AvCoV replication. In conclusion, AvCoV quasispecies evolution is influenced by the biological model under consideration, and a gradual transition is seen for minor and major variants.(AU)

O Coronavírus aviário AvCoV infecta uma variedade de tecidos de galinhas e de outras espécies aviárias. Apesar de este vírus poder ser isolado em ovos embrionados de galinha, apenas alguns dos 6 genótipos / 33 linhagens podem crescer em cultivo celular, sendo a cepa Beuadette (linhagem GI-11) a mais utilizada para estudos in vitro. Considerando as diferentes linhagens celulares e ovos embrionados como habitats para o AvCoV, este estudo teve por objetivo estudar a diversidade de genes que codificam para a proteína não-estrutural 3 (nsp3) e espícula (S) após passagens seriadas em células BHK-21 e VERO. Após 14 passagens, de uma amostra Beuadette adaptada a ovos embrionados, os títulos virais variaram em ambas as células, com os maiores títulos sendo de 8,72 log cópias genômicas/µL para Vero e 6,36 cópias genômicas/µL para BHK-21. Nenhum polimorfismo foi encontrando para nsp3. Considerando a proteína S, não somente foram encontradas substituições de aminoácidos (Vero: 8a passagem A150S e 14a passagem S150A; BHK-21: 4a passagem S53F, 8a passagem F53Y e S95R), mas também, variantes subconsensuais foram detectadas pelos cromatogramas com intensidades flutuantes. Uma vez que as regiões destes aa se encontram no domínio de ligação de receptor de S, pode-se especular que diferenças em receptores celulares entre Vero e BHK-21, além da velocidade da morte celular, levaram à seleção de diferentes cepas dominantes, enquanto que a estabilidade de nsp3 concorda com sua função como protease com papel na replicação de AvCoV. Como conclusão, a evolução de quase-espécies de AvCoV é influenciada pelo modelo biológico sob consideração e uma transição gradual é vista para variantes dominantes e subdominantes.(AU)
Descritores: Proteínas não Estruturais Virais
Infecções por Coronavirus/veterinária
Glicoproteína da Espícula de Coronavírus
Gammacoronavirus
Limites: Embrião de Galinha
Responsável: BR68.1 - Biblioteca Virginie Buff D'Ápice


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Id: biblio-894923
Autor: Freire, Caio César de Melo; Palmisano, Giuseppe; Braconi, Carla T; Cugola, Fernanda R; Russo, Fabiele B; Beltrão-Braga, Patricia CB; Iamarino, Atila; Lima Neto, Daniel Ferreira de; Sall, Amadou Alpha; Rosa-Fernandes, Livia; Larsen, Martin R; Zanotto, Paolo Marinho de Andrade.
Título: NS1 codon usage adaptation to humans in pandemic Zika virus
Fonte: Mem. Inst. Oswaldo Cruz;113(5):e170385, 2018. tab, graf.
Idioma: en.
Projeto: FAPESP; . CNPq; . FAPESP; . FAPESP; . FAPESP; . FAPESP; . FAPESP.
Resumo: BACKGROUND Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. OBJECTIVES To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. FINDINGS The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. MAIN CONCLUSIONS Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.
Descritores: Proteínas não Estruturais Virais/genética
Infecção por Zika virus/epidemiologia
Infecção por Zika virus/virologia
-Brasil/epidemiologia
Códon
Genoma Viral
Responsável: BR1.1 - BIREME


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Brigido, Luís Fernando de Macedo
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Id: biblio-894888
Autor: Cabral, Gabriela Bastos; Ferreira, João Leandro de Paula; Souza, Renato Pereira de; Cunha, Mariana Sequetin; Luchs, Adriana; Figueiredo, Cristina Adelaide; Brígido, Luís Fernando de Macedo.
Título: Simple protocol for population (Sanger) sequencing for Zika virus genomic regions
Fonte: Mem. Inst. Oswaldo Cruz;113(1):38-44, Jan. 2018. tab, graf.
Idioma: en.
Projeto: FINEP; . FAPESP.
Resumo: BACKGROUND A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTIVE The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. METHODS Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. FINDINGS Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. MAIN CONCLUSIONS The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.
Descritores: RNA Viral/genética
Genoma Viral/genética
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Zika virus/genética
-Filogenia
Proteínas não Estruturais Virais
Sequenciamento de Nucleotídeos em Larga Escala
Responsável: BR1.1 - BIREME


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Id: biblio-1022119
Autor: Luchs, A; Timenetsky, M do C S T.
Título: Phylogenetic analysis of human group C rotavirus circulating in Brazil reveals a potential unique NSP4 genetic variant and high similarity with Asian strains
Fonte: Mol Genet Genomics;290(3):969-986, 2015.
Idioma: en.
Resumo: Group C rotaviruses (RVC) cause gastroenteritis in humans and animals worldwide, and the evidence for a possible zoonotic role has been recently provided. To gain information on the genetic diversity and relationships between human and animal RVC, we sequenced the VP4, VP7, and NSP4 genes of 12, 19, and 15 human strains, respectively, detected in São Paulo state during historical (1988 and 1993) and recent (2007 and 2008) Brazilian rotavirus surveillance. All RVC strains analyzed in the present study grouped into human genotype (G4-P[2]-E2), and did not show any evidence of animal ancestry. Phylogenetic analysis showed that RVC samples detected in 1988 and 1993 clustered together with strains from distinct continents, indicating that historical RVC strains circulating in São Paulo were closely related to those strains circulating worldwide. All three genes (VP7, VP4 and NSP4) of São Paulo RVC strains isolated in 2007-2008 exhibited close phylogenetic relationship with human RVC strains isolated in China and Japan, suggesting that they are genetically linked, and that a gene flow could be occurring between this Asian countries and Brazil. We identified two distinct clusters in the NSP4 phylogenetic tree. One cluster formed exclusively by human Brazilian strains detected in 1997 and 2003-2004 in Rio de Janeiro, Bahia, and Rio Grande do Sul states (Subgroup II) previously described in a different study, that displayed low sequence identities to other human strains formerly published, and to the Brazilian RVC strains (Subgroup I) characterized in the present study. These data suggests the circulation of two genetic profiles of the NSP4 gene in Brazil. High sequence diversity in NSP4 gene was previously reported in Asia, and additional diversity in NSP4 RVC strains spreading in the world should be expected. More in-depth molecular and epidemiological analysis of human RVC throughout the world will be needed to understand their diversity and clarify their evolution, as well as to develop classifications schemes.
Descritores:
Filogenia
Infecções por Rotavirus/virologia
Toxinas Biológicas/genética
Variação Genética
Brasil/epidemiologia
Humanos
RNA
RNA Viral/isolamento & purificação
Dados de Sequência Molecular
Sequência de Bases
Glicoproteínas
Glicoproteínas/genética
Glicoproteínas/química
Criança
Pré-Escolar
Demografia
Alinhamento de Sequência
Adolescente
Sequência de Aminoácidos
Proteínas não Estruturais Virais
Proteínas não Estruturais Virais/genética
Homologia de Sequência de Aminoácidos
Análise de Sequência de DNA
Rotavirus
Adulto
Proteínas do Capsídeo/química
Gastroenterite/virologia
Genótipo
Lactente
Animais
Pessoa de Meia-Idade
Antígenos Virais/genética
Responsável: BR91.2 - Centro de Documentação


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Id: biblio-894847
Autor: Delatorre, Edson; Mir, Daiana; Bello, Gonzalo.
Título: Tracing the origin of the NS1 A188V substitution responsible for recent enhancement of Zika virus Asian genotype infectivity
Fonte: Mem. Inst. Oswaldo Cruz;112(11):793-795, Nov. 2017. graf.
Idioma: en.
Resumo: A recent study showed that infectivity of Zika virus (ZIKV) Asian genotype was enhanced by an alanine-to-valine amino acid substitution at residue 188 of the NS1 protein, but the precise time and location of origin of this mutation were not formally estimated. Here, we applied a Bayesian coalescent-based framework to estimate the age and location of the ancestral viral strain carrying the A188V substitution. Our results support that the ancestral ZIKV strain carrying the A188V substitution arose in Southeastern Asia at the early 2000s and circulated in that region for some time (5-10 years) before being disseminated to Southern Pacific islands and the Americas.
Descritores: Proteínas/genética
Teorema de Bayes
Proteínas não Estruturais Virais/genética
Zika virus/genética
Mutação/genética
-Filogenia
Ásia
Genótipo
Limites: Humanos
Responsável: BR1.1 - BIREME


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Id: biblio-894924
Autor: Nunes, Allan RD; Alves, Brenda Elen B; Pereira, Hannaly WB; Nascimento, Yasmin M; Morais, Ingryd C; Fernandes, José Veríssimo; Araújo, Josélio MG; Lanza, Daniel CF.
Título: Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus
Fonte: Mem. Inst. Oswaldo Cruz;113(5):e170393, 2018. tab, graf.
Idioma: en.
Resumo: BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial. OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV. METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database. FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis. MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.
Descritores: Proteínas não Estruturais Virais
Vírus da Dengue/genética
Zika virus
-Primers do DNA/genética
Biologia Computacional/métodos
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Responsável: BR1.1 - BIREME


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Malaquias, Luiz Cosme Cotta
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Id: lil-775122
Autor: Drumond, Betania Paiva; Fagundes, Luiz Gustavo da Silva; Rocha, Raissa Prado; Fumagalli, Marcilio Jorge; Araki, Carlos Shigueru; Colombo, Tatiana Elisa; Nogueira, Mauricio Lacerda; Castilho, Thiago Elias; Silveira, Nelson José Freitas da; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil.
Título: Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil
Fonte: Braz. j. microbiol;47(1):251-258, Jan.-Mar. 2016. tab, graf.
Idioma: en.
Projeto: National Counsel of Technological and Scientific Development (CNPq).
Resumo: Abstract Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1–4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population.
Descritores: Vírus da Dengue/classificação
Vírus da Dengue/genética
Dengue/virologia
Variação Genética
Genótipo
Filogenia
-Substituição de Aminoácidos
Estruturas Animais/patologia
Brasil
Modelos Animais de Doenças
Vírus da Dengue/isolamento & purificação
Produtos do Gene env/química
Produtos do Gene env/genética
Histocitoquímica
Microscopia
Modelos Moleculares
Mutação Puntual
Conformação Proteica
Proteínas não Estruturais Virais/genética
Limites: Animais
Humanos
Camundongos
Responsável: BR1.1 - BIREME


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Rácz, Maria Lúcia
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Id: lil-763094
Autor: Bertol, Jéssica Wildgrube; Fregolente, Maria Clara Duarte; Caruzo, Thabata Alessandra Ramos; Silva, Márcio José da; Munford, Veridiana; Sáfadi, Marco Aurélio Palazzi; Rácz, Maria Lucia; Gatti, Maria Silvia Viccari.
Título: Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in São Paulo, Brazil, from 1994 and 2006 to 2010
Fonte: Mem. Inst. Oswaldo Cruz;110(6):786-792, Sept. 2015. tab, graf.
Idioma: en.
Projeto: FAPESP; . FAPESP.
Resumo: Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.
Descritores: Antígenos Virais/isolamento & purificação
Glicoproteínas/genética
RNA Viral/genética
Rotavirus/genética
Toxinas Biológicas/genética
Proteínas não Estruturais Virais/genética
-Sequência de Aminoácidos
Sequência de Bases
Brasil
Fezes/virologia
Variação Genética
Genótipo
Ligação Genética/genética
Técnicas Imunoenzimáticas
Dados de Sequência Molecular
Filogenia
Reação em Cadeia da Polimerase Via Transcriptase Reversa
RNA Viral/isolamento & purificação
Rotavirus/classificação
Rotavirus/imunologia
Alinhamento de Sequência
Limites: Adulto
Criança
Humanos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME


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Id: lil-740332
Autor: Odreman-Macchioli, María; Vielma, Silvana; Atchley, Daniel; Comach, Guillermo; Ramirez, Alvaro; Pérez, Saberio; Téllez, Luis; Quintero, Beatriz; Hernández, Erick; Muñoz, Maritza; Mendoza, José.
Título: Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus / Análisis de las eficiencias de amplificación por PCR en tiempo real de tres regiones genómicas del virus dengue
Fonte: Invest. clín;54(1):5-19, mar. 2013. tab.
Idioma: en.
Resumo: Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3'-noncoding region (3'NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3'NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3'NC regions showed a weak (k= 0.109; CI 95%) and a moderate (k= 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan® format was much more sensitive than the C-prM/SYBR Green® I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.

El diagnóstico precoz de la infección por el virus dengue (DENV) constituye un elemento clave para la prevención de las complicaciones clínicas propias de la enfermedad. El objetivo del estudio fue evaluar la detección de DENV mediante un ensayo cuantitativo de PCR-tiempo real (qPCR), desarrollado localmente, utilizando la región no-estructural-5 (NS5), versus dos regiones tradicionalmente empleadas para la detección del virus, la región cápside/pre-membrana (C-prM), y la región noncodificante-3' (3'NC). Se recolectaron 167 muestras de suero de pacientes en fase aguda de la enfermedad. Las tres regiones génicas tuvieron perfiles de amplificación/coeficientes de correlación similares (0,987-0,999). Sin embargo, la región NS5 tuvo la eficiencia de amplificación más elevada para los cuatro serotipos (98-100%). Durante el proceso de validación, 41,1% (67/167) de las muestras de suero resultaron positivas para DENV al menos por dos de las regiones genómicas empleadas. Los valores de concordancia entre las regiones NS5/C-prM y NS5/3'NC fueron de 56,7% y 97%, respectivamente. La concordancia fue débil entre las regiones NS5/C-prM (k= 0,109; CI 95%), sin embargo, fue moderada entre las regiones NS5/3'NC (k= 0,489; CI 95%). El ensayo de tipificación uniplex en formato NS5/TaqMan® mostró alta sensibilidad (100%) que el protocolo C-prM/SYBRGreen®-I (76%). La validación externa del ensayo mostró una alta sensibilidad (100%), especificidad (78%) y acuerdo alto entre los ensayos utilizados. De acuerdo a los resultados obtenidos, la región NS5 ofrece la mayor opción para la detección y serotipificación del DENV en muestras clínicas.
Descritores: /genética
ABATTOIRS' UNTRANSLATED REGIONS/genética
Proteínas do Capsídeo/genética
Vírus da Dengue/genética
Dengue/virologia
Genoma Viral
Reação em Cadeia da Polimerase em Tempo Real
RNA Viral/análise
Proteínas não Estruturais Virais/genética
-Anticorpos Antivirais/sangue
Vírus da Dengue/classificação
Vírus da Dengue/imunologia
Vírus da Dengue/isolamento & purificação
Dengue/sangue
Diagnóstico Precoce
Ensaio de Imunoadsorção Enzimática
Imunoglobulina M/sangue
Compostos Orgânicos
Reprodutibilidade dos Testes
Reação em Cadeia da Polimerase em Tempo Real/métodos
Sensibilidade e Especificidade
Sorotipagem
Taq Polimerase
Cultura de Vírus
Limites: Humanos
Tipo de Publ: Estudo Comparativo
Estudo de Avaliação
Research Support, Non-U.S. Gov't
Estudo de Validação
Responsável: VE1.1 - Biblioteca Humberto Garcia Arocha



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