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1.
Artigo em Português | LILACS | ID: biblio-1097211

RESUMO

Objetivo: Auxiliar no entendimento da COVID-19 em relação à origem do SARS-CoV-2, suas descobertas genômicas, patogenia, possíveis hospedeiros primários e intermediários, além da comparação com outros coronavírus. Metodos: foram utilizadas as bases de dados Scientific Eletronic Library Online e PubMed, com artigos de revisão e originais, em língua portuguesa e inglesa, pesquisados no período de 05 de março a 10 de abril de 2020, adotando os seguintes descritores: SARS-CoV, COVID-19, coronavirus, Wuhan, genome, structure, origin, transmission, evolution, zoonotic. Os artigos originais identificados foram incluídos nesta revisão, juntamente com artigos de suporte referenciados por estes. Resultados: As características genômicas descritas até o momento podem explicar, em parte, a infectividade e a transmissibilidade do SARS-CoV-2 em humanos. Devido aos notáveis recursos de SARS-CoV-2, incluindo o local otimizado do domínio de ligação ao receptor (RBD) e de clivagem polibásica, é pouco provável um cenário laboratorial para a origem do SARS-CoV-2. Conclusão: Para o presente, é de extrema importância obter mais dados genéticos e funcionais sobre o SARS-CoV-2, incluindo estudos em animais, sequenciamento do vírus em casos muito precoces e identificação dos parentes virais mais próximos do SARS-CoV-2 que circulam em animais.(AU)


Objective: To assist in the understanding of COVID-19 in relation to the origin of SARS-CoV-2, its genomic discoveries, pathogenesis, possible primary and intermediate hosts, in addition to comparison with other coronaviruses. Methods: the Scientific Electronic Library Online and PubMed databases were used, with review articles and originals, in Portuguese and English, researched from March 5 to April 10, 2020, adopting the following descriptors: SARS-CoV , COVID-19, coronavirus, Wuhan, genome, structure, origin, transmission, evolution, zoonotic. The original articles identified were included in this review, along with supporting articles referenced by them. Results: The genomic characteristics described so far may partly explain the infectivity and transmissibility of SARS-CoV-2 in humans. Due to the remarkable resources of SARS-CoV-2, including the optimized site of the receptor binding domain (RBD) and polybasic cleavage, a laboratory scenario for the origin of SARS-CoV-2 is unlikely. Conclusion: For the present, it is extremely important to obtain more genetic and functional data on SARS-CoV-2, including studies on animals, sequencing of the virus in very early cases and identification of the closest viral relatives of SARS-CoV-2 that circulate in animals.(AU)


Assuntos
Humanos , Genoma Viral , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/epidemiologia , Betacoronavirus/patogenicidade
2.
Braz. j. microbiol ; 49(4): 777-784, Oct.-Dec. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-974285

RESUMO

ABSTRACT The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time.


Assuntos
Animais , Gatos , Doenças do Gato/virologia , Calicivirus Felino/isolamento & purificação , Calicivirus Felino/genética , Infecções por Caliciviridae/veterinária , Animais de Estimação/virologia , Filogenia , Brasil , Fases de Leitura Aberta , Genoma Viral , Calicivirus Felino/classificação , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética
3.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 16(3): 6-12, dic. 2018. tab, ilus
Artigo em Espanhol | LILACS, BDNPAR | ID: biblio-998219

RESUMO

El cáncer de cuello uterino es el segundo cáncer femenino más común a nivel mundial. El agente causal es el virus de papiloma humano (VPH). Se han identificado 13 tipos de virus de papiloma humano de alto riesgo oncogénico (VPH-AR), entre los cuales el VPH 16 y VPH 18 son los más frecuentemente detectados en cáncer de cuello uterino, siendo en Paraguay detectados en el 70% de casos de cáncer invasor. Por ello, el objetivo fue estandarizar y determinar el límite de detección de una técnica de PCR convencional para la detección de VPH 16 y 18. Para la detección de ADN de VPH 16 y 18, se observaron mejores resultados con 2mM de MgCl2 y 60°C para la temperatura de alineamiento. El límite de detección para las PCR fue de 14,6x10-11ng/µL para VPH 16 y 21,7x10-12ng/µL para VPH 18. Este trabajo servirá de base a otros estudios de detección e identificación de estos tipos virales por PCR, con miras a identificar un grupo de mujeres positivas para VPH-AR que poseen mayor riesgo de desarrollo de lesión y cáncer de cuello uterino y precisan de un seguimiento más cercano(AU


Cervical cancer is the second most common female cancer worldwide. It is caused by the human papilloma virus (HPV). Thirteen genotypes of high oncogenic risk human papilloma viruses (HPV-HR) have been identified, among which types 16 and 18 are the most frequently detected in cervical cancer. In Paraguay, they are detected in 70% of the invasive cancer cases. Therefore, the objective was to standardize and determine the detection limit of a conventional PCR technique for the detection of HPV 16 and 18. Better results were observed with 2mM MgCl2 and 60°C for the alignment temperature in detection of HPV 16 and 18 DNA. The limit of detection was 14.6x10-11ng/µL for HPV 16 and 21.7x10-12ng/µL for HPV 18. This work will help other studies for the detection and identification of these viral types by PCR in order to identify a group of HPV-HR positive women who have higher risk for the development of lesions and cervical cancer and need a closer follow-up(AU)


Assuntos
Humanos , Feminino , Neoplasias do Colo do Útero/virologia , Reação em Cadeia da Polimerase/métodos , Infecções por Papillomavirus/virologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Sequência de Bases , Genoma Viral , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Limite de Detecção
4.
Mem. Inst. Oswaldo Cruz ; 113(1): 38-44, Jan. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-894888

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.


Assuntos
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
5.
Braz. j. microbiol ; 49(supl.1): 260-261, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-974329

RESUMO

ABSTRACT Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.


Assuntos
Togaviridae/genética , Acanthamoeba/virologia , Genoma Viral , Virófagos/genética , Filogenia , Togaviridae/isolamento & purificação , Togaviridae/ultraestrutura , Brasil , Fases de Leitura Aberta , Microscopia Eletrônica de Transmissão , Virófagos/isolamento & purificação , Virófagos/ultraestrutura
6.
Mem. Inst. Oswaldo Cruz ; 113(5): e170385, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-894923

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.


Assuntos
Proteínas não Estruturais Virais/genética , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/virologia , Brasil/epidemiologia , Códon , Genoma Viral
7.
Braz. j. microbiol ; 49(supl.1): 262-268, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-974345

RESUMO

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 30-50 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 10−6 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.


Assuntos
Bacteriófagos/isolamento & purificação , Vírion/isolamento & purificação , Lagos/virologia , Especificidade de Hospedeiro , Bactérias/virologia , Bacteriófagos/classificação , Bacteriófagos/fisiologia , Bacteriófagos/genética , Vírion/classificação , Vírion/fisiologia , Cloreto de Sódio/análise , Lagos/análise , China , Genoma Viral
8.
Mem. Inst. Oswaldo Cruz ; 112(10): 728-731, Oct. 2017. tab
Artigo em Inglês | LILACS | ID: biblio-894837

RESUMO

The classification of human papillomavirus (HPV) intratypic lineages by complete genome sequencing is a determinant in understanding biological differences in association with this disease. In this work, we have characterised complete HPV genomes from southern Brazil. Fifteen cervicovaginal Pap smear negative samples previously categorised as HPV-positive were sequenced using ultradeep sequencing, and 18 complete genomes from 13 different HPV types were assembled. Phylogenetic and genetic distance analyses were performed to classify the HPV genomes into lineages and sublineages. This is the first report describing the distribution of HPV intratype lineages of high and low oncogenic risk in asymptomatic women from southern Brazil.


Assuntos
Humanos , Feminino , Adulto , Papillomaviridae , Papillomaviridae/genética , Esfregaço Vaginal , DNA Viral , Doenças do Colo do Útero/virologia , Genoma Viral , Infecções por Papillomavirus/virologia , Fatores de Risco
9.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 15(1): 7-15, abr. 2017. ilus
Artigo em Espanhol | LILACS, BDNPAR | ID: biblio-1008720

RESUMO

Los flavivirus son responsables de una considerable morbi-mortalidad a nivel mundial. Entre ellos, el virus del dengue (DENV) es causante de graves problemas de salud pública en Paraguay. El objetivo del estudio fue detectar infecciones por flavivirus a través de una reacción de RT-nested PCR genérica para flavivirus en 195 muestras de individuos con sospecha de dengue, negativos por el test inmunocromatográfico (antígeno NS1 ­ DENV), provenientes del área metropolitana de Asunción entre 2011 y 2013. Las muestras positivas para flavivirus fueron sometidas a dos reacciones de RT-nested PCRs específicas para DENV. El límite de detección (LD) para flavivirus fue de 0,2 UFP/reacción. En total 43/195 muestras fueron positivas para flavivirus. De estas, 38/43 (88,4%) correspondieron a DENV (6 DENV-1, 30 DENV-2 y 2 DENV-3). Además, 5/43 casos (11,6%) positivos para flavivirus fueron negativos para DENV por ambas reacciones específicas, pudiendo deberse a infecciones por otros flavivirus. Los resultados sugieren que la utilización de una reacción genérica seguida de otras reacciones específicas para DENV en casos febriles negativos para NS1 por el método inmunocromatográfico permitiría detectar más casos de infecciones por DENV y además, podría contribuir a la identificación de casos debido a infecciones por otros flavivirus.


Flaviviruses are responsible for considerable worldwide morbidity and mortality. Among them, the dengue virus (DENV) causes serious public health problems in Paraguay. The objective of the study was to detect flavivirus infections using a generic RT-nested -PCR in 195 samples of individuals with suspected dengue and negative for the inmunochromatographic test (NS1 antigen ­ DENV), from the metropolitan area of Asuncion between 2011 and 2013. The flavivirus-positive samples were subjected to two reactions of DENV-specific RT-nested PCRs. The detection limit (DL) for flavivirus was 0.2 PFU / reaction. In total, 43/195 samples were positive for flavivirus. Of them, 38/43 (88,4%) corresponded to DENV (6 DENV-1, 30 DENV-2 and 2 DENV-3). In addition, 5/43 cases (11.6%) positive for flavivirus were negative for DENV by both specific reactions, and may be infections caused by other flaviviruses. The results suggest that the use of a generic reaction followed by other DENV specific reactions in febrile negative cases for NS1 by the immunochromatographic method would allow the detection of more cases of DENV infections and could contribute to the identification of cases due to infections by others flaviviruses.


Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Infecções por Flavivirus/diagnóstico , Vírus da Dengue/isolamento & purificação , Flavivirus/isolamento & purificação , Paraguai , Estudos Transversais , Genoma Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Febre , Flavivirus/genética , Antígenos Virais/isolamento & purificação
10.
Mem. Inst. Oswaldo Cruz ; 112(3): 175-181, Mar. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-841776

RESUMO

BACKGROUND Two novel viruses named circo-like virus-Brazil (CLV-BR) hs1 and hs2 were previously discovered in a Brazilian human fecal sample through metagenomics. CLV-BR hs1 and hs2 possess a small circular DNA genome encoding a replication initiator protein (Rep), and the two genomes exhibit 92% nucleotide identity with each other. Phylogenetic analysis based on the Rep protein showed that CLV-BRs do not cluster with circoviruses, nanoviruses, geminiviruses or cycloviruses. OBJECTIVE The aim of this study was to search for CLV-BR genomes in sewage and reclaimed water samples from the metropolitan area of São Paulo, Brazil, to verify whether the first detection of these viruses was an isolated finding. METHODS Sewage and reclaimed water samples collected concomitantly during the years 2005-2006 were purified and concentrated using methodologies designed for the study of viruses. A total of 177 treated reclaimed water samples were grouped into five pools, as were 177 treated raw sewage samples. Nucleic acid extraction, polymerase chain reaction (PCR) amplification and Sanger sequencing were then performed.e FINDINGS CLV-BR genomes were detected in two pools of sewage samples, p6 and p9. Approximately 28% and 51% of the CLV-BR genome was amplified from p6 and p9, respectively, including 76% of the Rep gene. The detected genomes are most likely related to CLV-BR hs1. Comparative analysis showed several synonymous substitutions within Rep-encoding sequences, suggesting purifying selection for this gene, as has been observed for other eukaryotic circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses. MAIN CONCLUSION The results therefore indicated that CLV-BR has continued to circulate in Brazil two and three years after first being detected.


Assuntos
Humanos , Esgotos/virologia , DNA Viral/genética , Reação em Cadeia da Polimerase , Circovirus/isolamento & purificação , Circovirus/genética , Filogenia , Genoma Viral , Análise de Sequência
11.
Braz. j. microbiol ; 47(4): 993-999, Oct.-Dec. 2016. tab, graf
Artigo em Inglês | LILACS | ID: biblio-828184

RESUMO

Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.


Assuntos
Animais , Bovinos , Leveduras/genética , Genoma Viral , DNA Complementar , Vírus da Diarreia Viral Bovina/genética , Recombinação Homóloga , Replicação Viral , Leveduras/metabolismo , Linhagem Celular , Fases de Leitura Aberta , Análise de Sequência de DNA , Vírus da Diarreia Viral Bovina/fisiologia , Vírus da Diarreia Viral Bovina/ultraestrutura
12.
Braz. j. microbiol ; 47(3): 527-528, July-Sept. 2016.
Artigo em Inglês | LILACS | ID: lil-788968

RESUMO

ABSTRACT This communication reports full genome sequencing of the bluetongue virus-1 (BTV-1) isolate MKD20/08/Ind from goat in northern India. The total BTV-1 genome size was found to be 19,190 bp. A comparison study between the Indian isolate and other global isolates revealed that it belongs to the 'Eastern' BTV topotype. The full genome sequence of BTV-1 will provide vital information on its geographical origin and it will also be proved useful for comparing the Indian isolate with global isolates from other host species.


Assuntos
Animais , Cabras/virologia , Genoma Viral , Análise de Sequência de DNA , Vírus Bluetongue/genética , Filogenia , Vírus Bluetongue/isolamento & purificação , Vírus Bluetongue/classificação , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Sorogrupo , Índia
13.
Braz. j. microbiol ; 47(3): 529-530, July-Sept. 2016. tab
Artigo em Inglês | LILACS | ID: lil-788969

RESUMO

ABSTRACT Pseudomonas syringae pv. actinidifoliorum causes necrotic spots on the leaves of Actinidia deliciosa and Actinidia chinensis. P. syringae pv. actinidifoliorum has been detected in New Zealand, Australia, France and Spain. Four lineages were previously identified within the P. syringae pv. actinidifoliorum species group. Here, we report the draft genome sequences of five strains of P. syringae pv. actinidifoliorum representative of lineages 1, 2 and 4, isolated in France. The whole genomes of strains isolated in New Zealand, representative of P. syringae pv. actinidifoliorum lineages 1 and 3, were previously sequenced. The availability of supplementary P. syringae pv. actinidifoliorum genome sequences will be useful for developing molecular tools for pathogen detection and for performing comparative genomic analyses to study the relationship between P. syringae pv. actinidifoliorum and other kiwifruit pathogens, such as P. syringae pv. actinidiae.


Assuntos
Genoma Viral , Análise de Sequência de DNA , Pseudomonas syringae/classificação , Pseudomonas syringae/genética , Doenças das Plantas/microbiologia , Genômica/métodos , Pseudomonas syringae/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala
14.
Mem. Inst. Oswaldo Cruz ; 111(8): 532-534, Aug. 2016. graf
Artigo em Inglês | LILACS | ID: lil-788996

RESUMO

Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.


Assuntos
Humanos , Animais , Vírion/ultraestrutura , Zika virus/ultraestrutura , Técnicas de Cultura de Células , Chlorocebus aethiops , Genoma Viral , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Replicação Viral
15.
Rio de Janeiro; s.n; 2016. xvi, 112 p. ilus, graf, tab.
Tese em Português | LILACS | ID: biblio-971514

RESUMO

A infecção pelo vírus da hepatite B (HBV) representa um grave problema de saúde pública mundial. Dez genótipos (A a J) foram identificados e alguns deles foram ainda classificados em subgenótipos. O genótipo D (HBV/D) tem uma distribuição mundial e possui nove subgenótipos (D1 a D9) descritos. A história evolutiva do HBV/D nas Américas não é bem compreendida e poucas sequências de genoma completo HBV/D estão disponíveis. Oobjetivo deste estudo é analisar a proporção e a distribuição geográfica dos subgenótipos deD nas Américas, determinar as sequências genômicas completas do HBV/D isolados de diferentes regiões geográficas do Brasil e investigar a origem e a propagação dos subgenótipos de D nas Américas. Para identificar os subgenótipos circulantes nas Américas,foram obtidos do GenBank genomas completos e sequências dos genes pré-S/S e S (n =609). Foram detectados no continente os subgenótipos HBV/D1-D4 e HBV/D7. O HBV/D1 foi encontrado na Argentina (83%), Brasil (2%), Cuba (3%) e no Canadá (18%), enquanto que HBV/D2 foi detectado na Argentina (4%), Brasil (17%), Canadá (18%), Chile (75%), Cuba(5%) e EUA (90%). O subgenótipo HBV/D3 foi o mais frequente no Brasil (56%) e foi encontrado em todos os países americanos, exceto na Venezuela e Groelândia. O HBV/D4foi o subgenótipo mais frequente no Canadá (35%), Cuba (76%), Haiti (84%), Martinica(80%) e Venezuela (100%). O subgenótipo HBV/D7 foi observado apenas em Cuba (8%).A demais, amostras de soro HBsAg positivas, coletadas de todas as cinco regiões geográficas brasileiras e caracterizadas como HBV/D foram selecionadas para os equenciamento do genoma completo...


Hepatitis B virus (HBV) infection is a major global health problem. Ten genotypes (A to J)have been identified and some of them have been further divided into subgenotypes.Genotype D (HBV/D) has a worldwide distribution and nine subgenotypes (D1 to D9) have sofar been described. The evolutionary history of HBV/D in the Americas is not well understoodand few HBV/D complete genome sequences are available. The aim of this study is toexamine the proportion and geographical distribution of D subgenotypes in the Americas,determine the full-length genomic sequences of HBV/D isolates from different Brazilianregions and investigate the origin and spread of D subgenotypes in the Americas. To identifythe circulating subgenotypes, we downloaded American HBV/D complete and partial (pré-S/Sor S gene) available in GenBank (n=609). It was detected in the Americas the subgenotypesHBV/D1-D4 and HBV/D7. HBV/D1 was found in Argentina (83%), Brazil (2%), Cuba (3%) andCanada (18%), while HBV/D2 was detected in Argentina (4%), Brazil (17%), Canada (18%),Chile (75%), Cuba (5%) and USA (90%).The subgenotype HBV/D3 was the most prevalentsubgenotype in Brazil (56%) and was found in all American countries except Venezuela andGreenland. HBV/D4 was the most prevalent subgenotype in Canada (35%), Cuba (76%),Haiti (84%), Martinique (80%) and Venezuela (100%). HBV/D7 was observed only in Cuba(8%). In addition, HBsAg positive serum samples, collected from all five regions of Brazil andcharacterized as having HBV/D strains were selected for full genome sequencing...


Assuntos
Humanos , Vírus da Hepatite B , Genótipo , Genoma Viral , Filogeografia
16.
Mem. Inst. Oswaldo Cruz ; 110(6): 820-821, Sept. 2015. graf
Artigo em Inglês | LILACS | ID: lil-763091

RESUMO

Parvovirus B19 (B19V) infects individuals worldwide and is associated with an ample range of pathologies and clinical manifestations. B19V is classified into three distinct genotypes, all identified in Brazil. Here, we report a complete sequence of a B19V genotype 1A that was obtained by high-throughput metagenomic sequencing. This genome provides information that will contribute to the studies on B19V epidemiology and evolution.


Assuntos
Criança , Humanos , Masculino , Genoma Viral/genética , /genética , Brasil , Evolução Fatal , Sequenciamento de Nucleotídeos em Larga Escala , /classificação , Análise de Sequência de DNA
17.
Pesqui. vet. bras ; 35(5): 403-408, May 2015. tab, ilus
Artigo em Inglês | LILACS | ID: lil-759379

RESUMO

Porcine teschovirus (PTV), porcine sapelovirus (PSV), and enterovirus G (EV-G) are infectious agents specific to pig host species that are endemically spread worldwide. This study aimed to investigate the natural infection by these porcine enteric picornaviruses in wild boars (Sus scrofa scrofa) of Paraná state, Brazil, and to evaluate peccaries (Pecari tajacu and Tayassu pecari) as alternative host species for these viruses. Fecal samples (n=36) from asymptomatic wild boars (n=22) with ages ranging from 2 to 7 months old (young, n=14) and 2 to 4 years old (adult, n=8) and from peccaries (6 to 8 months old, n=14) were collected from a farm and a zoo, respectively, both located in Paraná state. Reverse transcription-polymerase chain reaction (RT-PCR) and nested-PCR (n-PCR) assays targeting the 5'non-translated region of the virus genome were used for screening the viruses. Porcine enteric picornaviruses were detected in 12 out of the 22 wild boar fecal samples. According to each of the viruses, EV-G was most frequently (11/22, 50%) detected, followed by PTV (10/22, 45.5%) and PSV (4/22, 18.2%). Regarding the age groups, young wild boars were more frequently (9/14, 64.3%) infected with PTV, PSV, and EV-G than adult animals (3/8, 37.4%). One n-PCR amplified product for each of the viruses was submitted to sequencing analysis and the nucleotide sequences were compared with the related viruses, which showed similarities varying from 97.7% to 100% for PTV, 92.4% to 96.2% for PSV, and 87.1% to 100% for EV-G. Peccaries tested negative for the viruses and in this study they did not represent infection reservoirs. This study is the first to report the molecular detection of PTV, PSV, and EV-G from captive wild boars in a South American country and the first to screen peccaries as alternative host species for porcine enteric picornavirus.


Teschovírus suíno (PTV), sapelovírus suíno (PSV) e enterovírus G(EV-G) são agentes infecciosos específicos da espécie suína que estão endemicamente disseminados em todo o mundo. O objetivo deste estudo foi investigar a infecção natural por estes picornavírus entéricos suínos em javalis (Sus scrofa scrofa) do estado do Paraná, Brasil e avaliar pecaris (Pecari tajacu e Tayassu pecari) como hospedeiros alternativos para estes vírus. Amostras fecais (n=36) de javalis assintomáticos (n=22) com idades de 2 a 7 meses (jovens, n=14) e 2 a 4 anos (adultos, n=8) e de pecaris (6 a 8 meses de idade, n=14) foram coletadas em um cativeiro e zoológico, respectivamente, ambos localizados no estado do Paraná. A transcrição reversa seguida por reações da polimerase em cadeia (RT-PCR) e nested-PCR com alvo na região 5'-não traduzida do genoma viral foram utilizadas para a identificação dos vírus. Picornavírus entéricos suínos foram detectados em 12 das 22 amostras fecais de javalis. De acordo com cada um dos vírus, EV-G foi mais frequentemente (11/22, 50%) detectado, seguido pelo PTV (10/22; 45,5%) e PSV (4/22; 18,2%). Considerando os grupos de idade, javalis jovens foram mais frequentemente (9/14; 64,3%) infectados com PTV, PSV e EV-G do que os javalis adultos (3/8; 37,4%). Um produto amplificado na nested-PCR para cada um dos vírus foi submetido à análise de sequenciamento e as sequências de nucleotídeos foram comparadas com vírus relacionados, o que mostrou que as similaridades variaram entre 97,7% a 100% para o PTV, 92,4% a 96,2% para o PSV e 87,1% a 100% para o EV-G. Os pecaris foram negativos para as viroses investigadas e neste estudo não se apresentaram como hospedeiros alternativos para as infecções. Este estudo é o primeiro a relatar a detecção molecular de PTV, PSV e EV-G em javalis de cativeiro de um país da América Latina e o primeiro a avaliar pecaris como espécie hospedeira alternativa para picornavírus entéricos suínos.


Assuntos
Animais , Enterovirus Suínos/patogenicidade , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologia , RNA Viral , Sus scrofa/virologia , Teschovirus/patogenicidade , Genoma Viral , Transcrição Reversa , Reação em Cadeia da Polimerase em Tempo Real/veterinária
18.
Rev. panam. infectol ; 16(1): 17-24, 2014. tab, graf
Artigo em Português | LILACS, Sec. Est. Saúde SP | ID: biblio-1067134

RESUMO

O vírus de Epstein Barr (EBV) é o agente causador da mononucle¬ose infecciosa e está associado a várias desordens proliferativas malignas tais como: linfoma de Burkitt, linfoma de Hodgkin e lin¬fomas não Hodgkin. Objetivo: detectar o genoma do EBV mediante a identificação dos genes EBER1 e EBNA1 em casos de doença de Hodgkin. Métodos: um total de 65 casos de linfomas diagnosti¬cados no Hospital Ophir Loyola no período de 1996 e 2005 foram analisados no Instituto Evandro Chagas, Ananindeua, Brasil. Todos os espécimes parafinizados foram analisados por hibridização in situ (gene EBER1) e PCR em tempo real (EBNA1). Resultados: do total, 64,6% (42/65) dos pacientes eram do sexo masculino e 35,4% (23/65) do sexo feminino. O EBV foi identificado por HIS nas células Reed Sternberg e variantes em 76,9% (50/65) dos casos com idade média de 28,3 anos (variação 2-84 anos). Os subtipos histológicos de casos EBV-positivos foram os seguintes: esclerose nodular em 50% (25/50), celularidade mista em 28% (14/50), depleção linfocitária em 14% (7/50) e predominância linfocitária em 8% (4/50). O DNA do EBV foi detectado em 53% (26/49) dos casos de doença de Hodgkin com um coeficiente de regressão para a curva padrão de 0,99. Conclusão: este estudo foi a primeira descrição do vírus de Epstein Barr em casos de linfoma de Hodgkin na Amazônia Brasileira, reforçando a hipótese de que o EBV seja um co-fator no processo de transformação neoplásica em conjunto com a predisposição genética e imunidade do paciente


Introduction: EBV is the causative agent of infectious mononucleosis and is associated with several malignant proliferative disorders such as Burkitt’s lymphoma, Hodgkin’s lymphoma, some B and T cell non-Hodgkin’s lymphomas. Objective: The main objective of the study was to determine the prevalence of EBER 1 gene and EBNA1 gene in cases of Hodgkin’s disease. Material and Methods: A total of 65 cases of lymphomas diagnosed between 1996 and 2005 were obtained from “Instituto Ofir Loyola” and analyzed at the “Instituto Evandro Chagas” Ananindeua, Brazil. The EBV antigens using EBER 1 probe in situ hybridization (HIS) and real time quantitative PCR. Results: From the total obtained, 64.6% (42/65) were male and 35.4% (23/65) female. EBV was identified in the Reed- Sternberg cells and variants in 76.9% (50/65) of Hodgkin’s disease cases, the median age were 28.3 years (range 2-84). The histologic subtypes of EBV-positive cases were as follows: nodular sclerosis in 50% (25/50), mixed cellularity in 28% (14/50), lymphocyte depletion in 14% (7/50) and lymphocyte predominance in 8% (4/50). We detected EBV DNA in 53% (26/49) with a coefficient of regression for the standard curve of a minimum of 0.99. Conclusion: These results were the first demonstration of the role of Epstein Barr virus in cases of Hodgkin diseases in northern Brazil and are consistent with the hypothesis that the presence of EBV during neoplasic transformation could be an additional cofactor acting together with both genetic predisposition and immunity of the patient


Assuntos
Masculino , Feminino , Humanos , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/história , Genoma Viral , Hibridização In Situ , Reação em Cadeia da Polimerase em Tempo Real
19.
Rio de Janeiro; s.n; 2014. xii,79 p. ilus, graf, tab, mapas.
Tese em Português | LILACS | ID: lil-751006

RESUMO

A dengue é considerada a mais importante das doenças virais transmitida por artrópodes que acomete o homem. O vírus dengue (DENV) é mantido na natureza através de replicação cíclica em hospedeiros vertebrados e mosquitos Aedes, sendo o Aedes aegypti o principal vetor. O seqüenciamento completo de DENV-3 isolado de Ae. aegypti naturalmente infectado do Rio de Janeiro em 2001 e de um caso humano em 2002, demonstrou uma similaridade de 99 porcento com DENV-3 isolado de um caso fatal humano ocorrido no mesmo período. A análise da região 3´NC do genoma viral demonstrou uma mutação nesta região, sugerindo uma deleção de 8 nucleotídeos (nts) na inserção de 11nts, característica de DENV-3 isolados no Brasil. Neste estudo, avaliamos se as diferentes variantes de DENV-3 na interação vírus-vetor através da determinação da competência vetorial em Ae. aegypti. As cepas de DENV-3 BR74886 #5 (cepa representativa do vírus com inserção de 11nts na região 3’NC) e BR73356 #5 (cepa representativa do vírus com a deleção de 8 nts), apresentando títulos de 8 x 107 PFU/mL e 7,3 x 107 PFU/mL, respectivamente, mantiveram suas características na região 3’NC do genoma viral após cinco passagens em cultura celular e foram selecionadas para a infecção experimental. A estratégia de infecção consistiu na utilização de 2.925 fêmeas de Ae. aegypti, sendo que 2.340 da geração F1 da população de Tubiacanga (RJ) e 585 da cepa controle PaeaA população experimental se mostrou competente para transmitir as duas cepas virais de DENV-3, no entanto a disseminação viral no corpo do mosquito apresentou-se de forma heterogênea, sugerindo haver vantagens para a cepa com inserção de 11 nts, uma vez que disseminou-se mais rapidamente. Quando as fêmeas de Ae. aegypti foram alimentadas com ambas as cepas, a disseminação no vetor comportou-se de maneira semelhante à observada quando alimentadas com a cepa representativa da inserção de 11 nts...


Dengue is considered the most important arthropod-borne viral disease that affects humans. Dengue virus (DENV) is maintained in nature by a cyclic replication in vertebrate hosts and Aedes mosquitoes, with the Aedes aegypti as the main vector. The complete sequencing of a DENV-3 strain isolated from Ae. aegypti naturally infected in Rio de Janeiro in 2001 and from a human case occurred in 2002 demonstrated a similarity of 99 percent with a DENV-3 isolated from a human fatal case occurred in the same period. However, the analysis of the 3Untranslated Region (UTR) of the viral genome showed a mutation in thisregion, suggesting a deletion of 8 nucleotides (nts) within the 11 nucleotides insertion, characteristic of DENV-3 isolated in Brazil. In this study, we evaluated whether the distinct DENV-3 variants presenting those characteristics showeddifferences on the virus-vector interaction by determining the vectorcompetence of two populations of Ae. aegypti. The DENV-3 strain BR74886#5(with the 11nts insert in the region 3'UTR) and the strain BR73356#5 (with an 8 nts deletion), presented titers of 8 x 107 PFU/mL and 7.3 x 107PFU/mL,respectively, maintained its characteristics in the 3'UTR region of the viral genome after five passages in cell culture and were selected for experimental infection. The infection strategy consisted in the use of 2,925 female Ae. aegypti: 2,340 of a F1 generation from the Tubiacanga (RJ) population and 585 Paea control mosquitoes. The experimental population proved to be competentto transmit the two DENV-3 strains. However, the viral dissemination in thebody of the mosquito presented heterogeneously, suggesting that there are advantages for the strain with 11 nts insertion in the 3'UTR, once disseminated more rapidly...


Assuntos
Humanos , Aedes/classificação , Dengue/epidemiologia , Dengue/história , Dengue/transmissão , Genoma Viral , Vírus da Dengue/genética
20.
Invest. clín ; 54(1): 5-19, mar. 2013. tab
Artigo em Inglês | LILACS | ID: lil-740332

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.


Assuntos
Humanos , /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
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