title: Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular, de Flavivírus, Rio de Janeiro, Fundação Oswaldo Cruz, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900, Brazil. mbonaldo@ioc.fiocruz.br
authors.forename: Myrna C
authors.initials: MC
authors.lastname: Bonaldo
authors.forename: Samanta M
authors.initials: SM
authors.lastname: Mello
authors.forename: Gisela F
authors.initials: GF
authors.lastname: Trindade
authors.forename: Aymara A
authors.initials: AA
authors.lastname: Rangel
authors.forename: Adriana S
authors.initials: AS
authors.lastname: Duarte
authors.forename: Prisciliana J
authors.initials: PJ
authors.lastname: Oliveira
authors.forename: Marcos S
authors.initials: MS
authors.lastname: Freire
authors.forename: Claire F
authors.initials: CF
authors.lastname: Kubelka
authors.forename: Ricardo
authors.initials: R
authors.lastname: Galler
mesh_terms: D000818:Animals; D002678:Chimera / Q000235:genetics / Q000276:immunology; D002522:Chlorocebus aethiops; D005416:Flavivirus / Q000235:genetics* / Q000276:immunology; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D014614:Vaccines, Synthetic / Q000235:genetics / Q000302:isolation & purification; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014765:Viral Vaccines / Q000235:genetics / Q000302:isolation & purification
chemical_list: D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D014765:Viral Vaccines; C065696:glycoprotein E, Flavivirus; D049452:Green Fluorescent Proteins