Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

<p>Abstract</p> <p>Background</p> <p>The yellow fever virus, a member of the genus <it>Flavivirus</it>, 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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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 <it>in vivo </it>studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.</p

Molecular cloning of EGFP protein expression cassete in the chimeric YF17D/DEN4 virus genome

<p><b>Copyright information:</b></p><p>Taken from "Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes"</p><p>http://www.virologyj.com/content/4/1/115</p><p>Virology Journal 2007;4():115-115.</p><p>Published online 30 Oct 2007</p><p>PMCID:PMC2173888.</p><p></p> (A) Schematic representation of YF 17D/DEN4/Esa/EGFP/6 recombinant virus genome and the genetic elements fused to EGFP gene. (B) Growth of recombinant YF17D/DEN4 viruses in Vero cells. Three independent experiments were performed to measure viral spread in Vero cells after infection with an multiplicity of infection (MOI) of 0.02. Cell culture supernatant aliquots were taken at 24, 48, 72, 96, 120 and 140 hour post-infection (p.i.) and titrated by plaque formation on Vero cell monolayers. (C) Analysis of recombinant YF 17D/DEN4/Esa/6 virus genetic stability after serial passaging on Vero cell monolayers. Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted from samples of the supernatant of cultures according to the passage numbering indicated on top of each lane. The first lane corresponds to cDNA-derived YF17D/DEN4 virus RNA; the remaining lanes are RT-PCR profiles from YF17D/DEN4/Esa/6 virus RNA at different passage levels with lanes 2 and 3 corresponding to amplicons from RNAs of viral stocks (1P, transfection supernatant) or passage two (2P, first passage of transfection supernatant), respectively. Lanes 4 to 11 represent RT-PCR products, which were obtained from viral RNA in the fifth, tenth, 15and 20passages of the two independent passage lineages (5P1 and 5P2; 10P1 and 10P2, 15P1 and 15P2, 20P1 and 20P2, respectively)

Attenuation of Recombinant Yellow Fever 17D Viruses Expressing Foreign Protein Epitopes at the Surface

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