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1.
Virol J ; 4: 115, 2007 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-17971212

RESUMO

BACKGROUND: The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor. RESULTS: YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 +/- 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus. CONCLUSION: This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.


Assuntos
Flavivirus/genética , Vetores Genéticos , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Animais , Quimera/genética , Quimera/imunologia , Chlorocebus aethiops , Flavivirus/imunologia , Engenharia Genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação , Células Vero , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
2.
J Virol ; 79(13): 8602-13, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15956601

RESUMO

The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines.


Assuntos
Vacinas Atenuadas/imunologia , Proteínas Virais/imunologia , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia , Animais , Clonagem Molecular , DNA Complementar , DNA Viral/genética , Epitopos/imunologia , Macaca mulatta , Modelos Moleculares , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Vacinas Atenuadas/química , Proteínas Virais/química , Vacina contra Febre Amarela/química
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