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1.
Int J Parasitol ; 34(7): 873-80, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15157770

RESUMO

14-3-3 proteins are highly conserved ubiquitous proteins found in all eukaryotic organisms. They are involved in various cellular processes including signal transduction, cell-cycle control, apoptosis, stress response and cytoskeleton organisation. We report here the cloning of two genes encoding 14-3-3 isoforms from the plant parasitic root-knot nematode Meloidogyne incognita, together with an analysis of their expression. Both genes were shown to be transcribed in unhatched second stage larvae, infective second stage larvae, adult males and females. The Mi-14-3-3-a gene was shown to be specifically transcribed in the germinal primordium of infective larvae, whereas Mi-14-3-3-b was transcribed in the dorsal oesophageal gland in larvae of this stage. The MI-14-3-3-B protein was identified by mass spectrometry in in vitro-induced stylet secretions from infective larvae. The stability and distribution of MI-14-3-3 proteins in host plant cells was assessed after stable expression of the corresponding genes in tobacco BY2 cells.


Assuntos
Proteínas 14-3-3/genética , Genes de Helmintos/genética , Proteínas de Helminto/genética , Tylenchoidea/genética , Proteínas 14-3-3/análise , Sequência de Aminoácidos , Animais , Clonagem Molecular/métodos , DNA Complementar/genética , DNA de Helmintos/genética , Inibidores Enzimáticos/análise , Feminino , Proteínas de Helminto/análise , Interações Hospedeiro-Parasita/genética , Larva/genética , Masculino , Espectrometria de Massas/métodos , Microscopia Confocal/métodos , Dados de Sequência Molecular , Raízes de Plantas/química , Raízes de Plantas/genética , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Alinhamento de Sequência , Transcrição Gênica/genética
2.
Virology ; 244(1): 59-65, 1998 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-9581778

RESUMO

The heterogeneous nature of the yellow fever (YF) 17D-204 vaccine virus population was exploited in this study to isolate virus variants able to escape neutralization by the 17D-204 vaccine-specific MAb 864. The conformational change on the virus surface that resulted in the loss of the MAb 864-defined epitope was effected in each variant by a single amino acid mutation in the envelope (E) protein at either position E-305 or E-325. Interestingly, both positions were mutated during attenuation of the 17D-204 vaccine substrain from the wildtype Asibi strain. The mutations in several of the variants represented reversion to the wildtype Asibi virus sequence consistent with loss of a 17D-204 substrain-specific epitope. The majority of the variant viruses were shown to have altered mouse neurovirulence phenotypes, ranging from complete avirulence through to increased virulence. The avirulent variants are the first flavivirus MAb-neutralization-resistant variants to be attenuated for neurovirulence in the adult mouse model. Overall, the results indicate that the E protein epitope recognized by MAb 864 defines a functionally important region that encodes major molecular determinants of YF virus pathogenesis in vivo.


Assuntos
Epitopos de Linfócito B/genética , Mutação Puntual , Proteínas do Envelope Viral/genética , Vacinas Virais , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/patogenicidade , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Imuno-Histoquímica , Camundongos , Conformação Proteica , Análise de Sequência de DNA , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência , Replicação Viral , Febre Amarela/patologia , Febre Amarela/virologia , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/fisiologia
3.
AIDS Res Hum Retroviruses ; 13(11): 913-22, 1997 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-9223407

RESUMO

The infection of macaque monkeys by attenuated simian immunodeficiency virus can vaccinate against pathogenic molecular clones and isolates of the same virus. The correlates of this potent protective immunity are not fully understood but may be the key to an effective AIDS vaccine for humans. Aiming to determine whether host immune responses to envelope glycoprotein are an essential component of the immunity to primate lentiviruses, we have tried to superinfect SIVmac-infected macaque monkeys with SHIVsbg, a chimeric primate lentivirus constructed from the SIVmac239 genome with the env, rev, tat, and vpu genes from HIV-1 Lai. After inoculation of a large dose of SHIVsbg, the chimeric virus was isolated by coculture of mononuclear blood cells from four of five SIV-infected monkeys, but three animals were protected from extracellular SHIV viremia and did not seroconvert to HIV-1 glycoproteins. In the two SIV-infected monkeys that did develop SHIV viremia, cell-associated viral load was reduced at least 100-fold. These data indicate that an antiviral response capable of effectively controlling primate lentivirus replication might not necessarily involve the envelope glycoprotein.


Assuntos
Produtos do Gene env/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Vírus Reordenados/imunologia , Vírus da Imunodeficiência Símia/imunologia , Superinfecção/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Antivirais/sangue , DNA Viral/sangue , Genes Virais , Anticorpos Anti-HIV/sangue , Infecções por HIV/prevenção & controle , HIV-1/genética , Humanos , Lentivirus de Primatas/isolamento & purificação , Leucócitos Mononucleares/virologia , Macaca fascicularis , Macaca mulatta , RNA Viral/sangue , Vírus Reordenados/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/genética , Carga Viral
4.
J Gen Virol ; 78 ( Pt 6): 1353-6, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9191929

RESUMO

Two monoclonal antibody neutralization resistant (MAbR) variants of the yellow fever (YF) 17D-204 vaccine virus strain were selected using YF type-specific MAb B39. These B39R variants were compared with the variant virus selected by Lobigs et al. (Virology 161, 474-478, 1987) using a second YF-type specific MAb (2E10) which mapped to amino acid position 71/72 in the envelope (E) protein. Neutralization assays with a panel of MAbs suggested that these two YF-type-specific epitopes are located in two discrete regions of the folded E protein. Each of the B39R variants had a single nucleotide mutation which encoded an amino acid substitution at either position E-155 or E-158. Thus, YF type-specific epitopes map to both domain I (B39) and II (2E10) of the YF virus E protein. The B39 defined epitope represents the first flavivirus neutralizing epitope localized to this region of domain I of the E protein.


Assuntos
Epitopos , Proteínas do Envelope Viral/imunologia , Vírus da Febre Amarela/imunologia , Sequência de Aminoácidos , Animais , Feminino , Camundongos , Dados de Sequência Molecular , Virulência , Vírus da Febre Amarela/patogenicidade
5.
Virology ; 230(2): 376-80, 1997 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-9143294

RESUMO

The live-attenuated yellow fever (YF) vaccine virus, strain 17D-204, has long been known to consist of a heterologous population of virions. Gould et al. (J. Gen. Virol. 70, 1889-1894 (1989)) previously demonstrated that variant viruses exhibiting a YF wild-type-specific envelope (E) protein epitope are present at low frequency in the vaccine pool and were able to isolate representative virus variants with and without this epitope, designated 17D(+wt) and 17D(-wt), respectively. These variants were employed here in an investigation of YF virus pathogenesis in the mouse model. Both the 17D-204 parent and the 17D(+wt) variant viruses were lethal for adult outbred mice by the intracerebral route of inoculation. However, the 17D(-wt) variant was significantly attenuated (18% mortality rate) and replicated to much lower titer in the brains of infected mice. A single amino acid substitution in the envelope (E) protein at E-240 (Ala-->Val) was identified as responsible for the restricted replication of the 17D(-wt) variant in vivo. The 17D(+wt) variant has an additional second-site mutation, believed to encode a reversion to the neurovirulence phenotype of the 17D-204 parent virus. The amino acid substitution in the E protein at E-173 (Thr-->Ile) of the 17D(+wt) variant which results in the appearance of the wild-type-specific epitope or nucleotide changes in the 5' and 3' noncoding regions of the virus are proposed as a candidates.


Assuntos
Variação Antigênica , Antígenos Virais/genética , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/patogenicidade , Animais , Encéfalo/patologia , Encéfalo/virologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , RNA Viral/análise , Proteínas do Envelope Viral/genética , Virulência , Febre Amarela/patologia , Vírus da Febre Amarela/imunologia
6.
J Virol ; 66(7): 4265-70, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1376368

RESUMO

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.


Assuntos
Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Vírus da Febre Amarela/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos Virais/imunologia , Células HeLa , Humanos , Camundongos , Testes de Neutralização , Inoculações Seriadas , Vacinas Atenuadas/imunologia , Febre Amarela/imunologia , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/patogenicidade
7.
Biologicals ; 20(2): 117-28, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1389107

RESUMO

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.


Assuntos
Anticorpos Monoclonais , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Vírus da Febre Amarela/imunologia , Testes de Inibição da Hemaglutinação , Testes de Hemaglutinação , Testes de Neutralização , Especificidade da Espécie , Vacinas Atenuadas/imunologia , Ensaio de Placa Viral , Vírus da Febre Amarela/isolamento & purificação
8.
Vaccine ; 10(10): 652-4, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1523874

RESUMO

Twenty-three yellow fever (YF) vaccine viruses and three wild-type YF viruses were propagated independently in human adenocarcinoma (SW13) cells and mosquito Aedes albopictus C6-36 cells. The three YF 17DD vaccine viruses were found to be slightly temperature sensitive (ts) at 39.5 degrees C versus 37.0 degrees C (efficiency of plaquing 0.04 to 0.1) following propagation in C6-36 but not in SW13 cells. A plaque-purified preparation of the 17DD vaccine manufactured in Brazil was ts when grown in C6-36 cells and remained ts when passaged back into the SW13 cell line.


Assuntos
Aedes/microbiologia , Temperatura Alta , Vacinas Virais/análise , Vírus da Febre Amarela/crescimento & desenvolvimento , Animais , Linhagem Celular , Humanos , Fenótipo , Células Tumorais Cultivadas , Vírus da Febre Amarela/genética
9.
J Gen Virol ; 71 ( Pt 10): 2301-6, 1990 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-2230735

RESUMO

During the 1960s three different research groups reported that passage of wild-type yellow fever (YF) virus [strain Asibi (YF-Asibi)] in HeLa cells resulted in attenuation of the virus for monkeys so that the virus no longer caused viscerotropic disease. We have repeated and extended this observation to analyse the process of attenuation of YF virus during cell culture passage. A large plaque (LP) variant of YF-Asibi virus became attenuated for both monkeys and mice following six serial subcultures in HeLa cells (YF-Asibi-LP HeLa p6). Thus, attenuation was probably due to a genetic change in the virus population rather than to selective enrichment of a pre-existing variant of YF-Asibi-LP virus. No evidence was obtained to implicate defective interfering particles in the attenuation process. Comparison of the YF-Asibi-LP viruses before and after passage in HeLa cells, using a panel of envelope protein-reactive monoclonal antibodies (MAbs), showed that MAbs which specifically neutralize YF-Asibi-LP virus, and not YF 17D-204 vaccine virus, also neutralized YF-Asibi-LP HeLa p6. This indicated that the epitopes involved in the biological process of neutralization were not altered during attenuation. However, two MAbs that recognize envelope protein epitopes did distinguish between HeLa- and non-HeLa-passaged YF-Asibi-LP virus. One of these (MAb 117) which is YF wild-type-specific, recognized YF-Asibi-LP virus but not YF-Asibi-LP HeLa p6 virus, whereas the other (MAb411), which is YF vaccine-specific, recognized YF-Asibi-LP HeLa p6 virus but not YF-Asibi-LP virus. These results suggest that antigenic changes in the viral envelope protein may determine the relative virulence or attenuation of YF virus.


Assuntos
Vírus da Febre Amarela/patogenicidade , Animais , Anticorpos Monoclonais , Células HeLa , Humanos , Macaca fascicularis , Camundongos , Testes de Neutralização , Vacinas Atenuadas , Proteínas do Envelope Viral/imunologia , Ensaio de Placa Viral , Vírus da Febre Amarela/imunologia
10.
J Gen Virol ; 71 ( Pt 1): 13-8, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1689367

RESUMO

Eight monoclonal antibodies (MAbs) prepared against the flaviviruses Saint Louis encephalitis, dengue 2 and dengue 3 viruses all recognized epitopes on the envelope protein of the prototype flavivirus, yellow fever (YF) virus. Three of these MAbs with flavivirus group-common specificity and two MAbs with a flavivirus-subgroup specificity were found to distinguish wild-type YF viruses from YF 17D-204 vaccine virus, but not from the closely related 17DD vaccine virus, nor from the French neurotropic vaccine virus. This pattern of reactivity was seen only with viruses grown in Aedes albopictus C6/36 cells and not with viruses grown in vertebrate cells (SW13 and Vero cells), where all five MAbs recognized epitopes on both wild-type and 17D-204 viruses. Examination of adult A. aegypti mosquitoes infected with the same YF viruses as above gave a different pattern of results to those in C6/36 cells. Thus, epitope expression differs between mammalian and arthropod cells and between arthropod cells in vitro and in vivo. Neutralization tests showed that all five MAbs would neutralize wild-type Asibi virus grown in SW13 cells, but not Asibi virus grown in C6/36 cells, nor 17D-204 vaccine virus grown in either cell type. Therefore, it is concluded that when YF virus is grown in mosquito cells, wild-type virus is antigenically and biologically distinct from the 17D-204 vaccine virus.


Assuntos
Anticorpos Monoclonais/imunologia , Flavivirus/imunologia , Vírus da Febre Amarela/imunologia , Adenocarcinoma , Aedes , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Reações Cruzadas , Epitopos/imunologia , Imunofluorescência , Humanos , Testes de Neutralização , Células Tumorais Cultivadas , Células Vero , Vacinas Virais/imunologia , Vírus da Febre Amarela/isolamento & purificação
11.
Vaccine ; 7(4): 333-6, 1989 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2479185

RESUMO

Two panels of envelope glycoprotein reactive monoclonal antibodies (mAbs) were prepared against yellow fever (YF) 17D vaccine viruses. Five mAbs were prepared against the World Health Organization 17D-204 avian leukosis virus-free secondary seed virus and eight mAbs against 17DD vaccine manufactured in Brazil. The majority of these mAbs were type-specific and displayed differing reactions in neutralization tests. One, B14, would only neutralize YF vaccine virus grown in invertebrate cells. Others would differentiate 17D-204 and 17DD vaccines, from different manufacturers, in neutralization tests when the viruses were grown in vertebrate cells. The data indicate that heterogeneity exists between the epitopes that elicit neutralizing antibody on YF vaccine from different manufacturers.


Assuntos
Anticorpos Monoclonais , Epitopos/análise , Glicoproteínas/imunologia , Vacinas/imunologia , Proteínas do Envelope Viral/imunologia , Vírus da Febre Amarela/imunologia , Animais , Linhagem Celular , Feminino , Imunofluorescência , Imunização Passiva , Camundongos , Camundongos Endogâmicos , Testes de Neutralização , Ensaio de Placa Viral , Vírus da Febre Amarela/crescimento & desenvolvimento
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