RESUMO
The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.
Assuntos
Baculoviridae/química , Baculoviridae/metabolismo , Vírus da Hepatite A/química , Proteínas Virais/biossíntese , Baculoviridae/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.
Assuntos
Animais , DNA Intergênico/genética , Retículo Endoplasmático/virologia , Proteínas de Fluorescência Verde/genética , Mutagênese Insercional/genética , Vírus da Febre Amarela/genética , Chlorocebus aethiops , Proteínas de Membrana , Células VeroRESUMO
Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.
Assuntos
Humanos , Citocinas/biossíntese , Células Dendríticas/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia , Biomarcadores , Diferenciação Celular , Quimiocinas/biossíntese , Células Dendríticas , Vacinas contra Dengue/imunologia , Vírus da Dengue/fisiologia , Dengue , Interferon-alfa/imunologia , Interferon-alfa , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa , Replicação Viral , Vacina contra Febre Amarela/imunologia , Febre Amarela , Vírus da Febre Amarela/fisiologiaRESUMO
The dengue virus (DENV1-4) causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in tropical and subtropical areas. The aim of this study was to evaluate the circulating genotypes of DENV. This was accomplished by sequencing the PrM and E genes of Brazilian isolates of DENV2 and DENV3 that were obtained between 1991 and 2008 from various geographic regions. Phylogenetic analyses of DENV2 demonstrated that the genotype III (Southeast Asian/American), in spite of several nucleotide and amino acid changes, was the only one that circulated over the past 19 years. Since its introduction in 2000, the DENV3 isolates that have been analyzed have all grouped intogenotype III (Indian subcontinent) and there has been no evidence of DENV3 belonging to other genotypes in this study.
O vírus dengue (DENV1-4) causa a dengue clássica e a febre hemorrágica da dengue / síndrome de choque da dengue (FHD/SCD) em regiões tropicais e subtropicais. O objetivo deste estudo foi avaliar os genótipos circulantes de DENV2 e DENV3 obtidos em distintas regiões geográficas no período de 1991 a 2008. As análises filogenéticas de DENV2 demonstraram que o genótipo III (Sudeste da Ásia/América), apesar das diversas alterações nucleotídicas e de aminoácidos, foi o único a circular durante os últimos 19 anos. Desde a sua introdução no estudo, em 2000, todas as amostras isoladas de DENV3 analisadas foram agrupadas no genótipo III (subcontinente indiano). Não foram encontradas evidências de que o DENV3 pertença a outros genótipos investigados.
Assuntos
Masculino , Feminino , Humanos , Vírus da Dengue/isolamento & purificação , Flavivirus/isolamento & purificação , Dengue Grave/classificaçãoRESUMO
For the development of safe live attenuated flavivirus vaccines one of the main properties to be established is viral replication. We have used real-time reverse transcriptase-polymerase chain reaction and virus titration by plaque assay to determine the replication of yellow fever 17DD virus (YFV 17DD) and recombinant yellow fever 17D viruses expressing envelope proteins of dengue virus serotypes 2 and 4 (17D-DENV-2 and 17D-DENV-4). Serum samples from rhesus monkeys inoculated with YFV 17DD and 17D-DENV chimeras by intracerebral or subcutaneous route were used to determine and compare the viremia induced by these viruses. Viral load quantification in samples from monkeys inoculated by either route with YFV 17DD virus suggested a restricted capability of the virus to replicate reaching not more than 2.0 log10 PFU mL-1 or 3.29 log10 copies mL-1. Recombinant 17D-dengue viruses were shown by plaquing and real-time PCR to be as attenuated as YF 17DD virus with the highest mean peak titer of 1.97 log10 PFU mL-1 or 3.53 log10 copies mL-1. These data serve as a comparative basis for the characterization of other 17D-based live attenuated candidate vaccines against other diseases.
Uma das principais propriedades a serem estabelecidas para o desenvolvimento de vacinas seguras e atenuadas de flavivirus,é a taxa de replicação viral. Neste trabalho, aplicamos a metodologia de amplificação pela reação em cadeia da polimerase em tempo real e titulação viral por plaqueamento para determinação da replicação do vírus 17DD (FA 17DD) e recombinantes, expressando proteínas do envelope de dengue sorotipos 2 e 4 (17D-DENV-2 e 17D-DENV-4). As amostras de soros de macacos inoculados por via intracerebral ou subcutânea com FA 17DD ou 17D-DENV foram usadas para determinar e comparar a viremia induzida por estes vírus. A quantificação da carga viral em amostras de macacos inoculados por ambas as vias com FA 17DD sugere restrita capacidade de replicação com taxa não superior a 2,0 log10 PFU mL-1 ou 3,29 log10 cópias/mL-1. Os vírus recombinantes 17D-DENV mostraram-se tão atenuados quanto o vírus 17DD, tanto porRT-PCR em tempo real quanto por plaqueamento, com título médio máximo de 1,97 log10 PFU mL-1 ou 3,53 log10 cópias/mL-1. Estes dados servem como base comparativapara caracterização de outros vírus vivos atenuados, derivados do vírus 17D, candidatos a vacinas contra outras doenças.
Assuntos
Animais , Anticorpos Antivirais , Vírus da Dengue/fisiologia , RNA Viral/imunologia , Replicação Viral , Viremia/imunologia , Vírus da Febre Amarela/fisiologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Macaca mulatta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Viral/sangue , Recombinação Genética/imunologia , Carga Viral , Vacinas Atenuadas/imunologia , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologiaRESUMO
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 ViralRESUMO
In order to investigate the pathogenicity of the virus strain GOI 4191 that was isolated from a fatal adverse event after yellow fever virus (YFV) vaccination, an experimental assay using hamsters (Mesocricetus auratus) as animal model and YFV 17DD vaccine strain as virus reference was accomplished. The two virus strains were inoculated by intracerebral, intrahepatic and subcutaneous routes. The levels of viremia, antibody response, and aminotransferases were determined in sera; while virus, antigen and histopathological changes were determined in the viscera. No viremia was detected for either strain following infection; the immune response was demonstrated to be more effective to strain GOI 4191; and no significant aminotransferase levels alterations were detected. Strain GOI 4191 was recovered only from the brain of animals inoculated by the IC route. Viral antigens were detected in liver and brain by immunohistochemical assay. Histothological changes in the viscera were characterized by inflammatory infiltrate, hepatocellular necrosis, and viral encephalitis. Histological alterations and detection of viral antigen were observed in the liver of animals inoculated by the intrahepatic route. These findings were similar for both strains used in the experiment; however, significant differences were observed from those results previously reported for wild type YFV strains.
Visando investigar a possível patogenicidade do vírus isolado (GOI 4191) de um evento adverso fatal pela vacinação antiamarílica, realizou-se um ensaio experimental em Syrian hamsters (Mesocricetus auratus), usando-se a cepa vacinal 17DD como parâmetro. As amostras virais foram inoculadas por via intracerebral, intra-hepática e subcutânea. Nos soros foram determinados níveis de viremia, resposta imune e aminotransferases, e nas vísceras a presença de vírus, antígeno e lesões teciduais. Não se detectou viremia para as duas amostras, a resposta imune foi maior para GOI 4191, e as aminotransferases não apresentaram alterações compatíveis com danos hepáticos. Nos animais inoculados por via intracerebral o vírus foi recuperado somente a partir do cérebro, sendo o antígeno viral detectado, por imuno-histoquímica, no cérebro e fígado. Infiltrado inflamatório e corpúsculos acidófilos foram observados no fígado e lesões tipo encefalite viral no sistema nervoso central. Alterações histológicas e antígeno viral foram observados, também, no fígado dos animais infectados por via intra-hepática, e ausentes naqueles inoculados por via subcutânea. Os resultados foram similares para as duas amostras testadas, entretanto distintos daqueles relatados na literatura para cepas silvestres do vírus amarílico.
Assuntos
Animais , Cricetinae , Masculino , Alanina Transaminase/sangue , Anticorpos Antivirais/sangue , Vacina contra Febre Amarela , Febre Amarela/virologia , Vírus da Febre Amarela/patogenicidade , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Imuno-Histoquímica , Fígado/patologia , Fígado/virologia , Mesocricetus , Fenótipo , Células Vero , Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/imunologiaRESUMO
The use of yellow fever (YF) virus 17D strain for vaccine production adapted in Brazil since its introduction in 1937 was reviewed. This was possible due to the availability of official records of vaccine production. The retrieved data highlight the simultaneous use of several serially passaged 17D substrain viruses for both inocula and vaccine preparation that allowed uninterrupted production. Substitution of these substrain viruses became possible with the experience gained during quality control and human vaccination. Post-vaccinal complications in humans and the failure of some viruses in quality control tests (neurovirulence for monkeys) indicated that variables needed to be reduced during vaccine production, leading to the development of the seed lot system. The 17DD substrain, still used today, was the most frequently used substrain and the most reliable in terms of safety and efficacy. For this reason, it is possible to derive an infectious cDNA clone of this substrain combined with production in cell culture that could be used to direct the expression of heterologous antigens and lead to the development of new live vaccines
Assuntos
Humanos , Animais , História do Século XX , Embrião de Galinha , Vacina contra Febre Amarela/história , BrasilRESUMO
The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF), dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain
Assuntos
Humanos , Flavivirus/imunologia , Vacinas Virais , Febre Amarela/imunologia , Flavivirus/genética , Genoma ViralRESUMO
The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members present in most continents. Among the most important are yellow fever (YF), dengue (DEN) with its 4 serotypes and Japanese escephalitis (JE) virus. A live attenuated virus is used as a cost-effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The development of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. The new approaches include the use of cDNAs encompassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated virues. The use of infectious cDNA as a carrier for heterologous antigens is a gaining importance since chimeric viruses are shown to be viable, immunogenic and less virulent in some cases as compared to the parental viruses. The use of DNA to overcome intrinsic mutation rates of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost of live attenuated vaccines.
Assuntos
Humanos , Flavivirus/imunologia , Infecções por Flaviviridae/prevenção & controle , Biologia Molecular , Vacinas Virais/imunologia , DNA Complementar/imunologia , DNA Recombinante/imunologiaRESUMO
Virus titration is an important step required on viral vaccines quality control. "Plaque assay", which emplosys several types of everlay media, is usually used on viral titrations. In this paper we describe the use of apioca as an overlay media. Firstly, the toxicity of Tapioca was tested on Vero cells inoculated or not with the Yellow Fever virus (YF) 17 DD vaccine strain. Secondly, different batches of the 17 DD virus using the Tapioca. Tapioca was also shown to be a suitable overlay to be used in thermostability and plaque reduction neutrabilization tests. Other systems could benefit from the use of Tapioca as an overlay, since it was possible to titer. Measles virus in Vero cells. Tapioca is a cheap Brazilian product, is locally available, easy to use, and reliable. Its use is suggested
Assuntos
Animais , Manihot , Titulometria , Células Cultivadas , Goma de Karaya , Macaca mulatta , Vírus do Sarampo , Testes de Neutralização , Ensaio de Placa Viral , Vírus da Febre AmarelaRESUMO
The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared world wide. As part of a broader approach to determine the genetic variability in YF l7D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purifed directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF l7D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot nybridization of virion RNAs of purified 17DD with two other strains of YF virus only fenome-sized molecules for all three viruses. These observations suggest that vaccine phenotype is primarily associated with the accumulation of mutations