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.

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.

Figure 1

Topological arrangement of the flavivirus E stem-anchor region and its elements. The top panel (A) depicts the topology of part the polyprotein precursor (E-NS1) of YF virus, its insertion at the endoplasmic reticulum (ER) membrane, the expected proteolytic cleavage by the ER signal peptidase (blue arrow) and the flavivirus stem-anchor region with its different elements (H1 and H2; TM1 and TM2). The lower part of panel (A) illustrates the same region bearing the Enhanced Green Fluorescent Protein gene (EGFP). The EGFP protein is fused at its amino-terminus with nine amino acids of YF 17D NS1 protein and with the YF 17D E stem-anchor region at its carboxi-terminus. Blue arrows indicated ER signal peptidase cleavage sites Panel (B) presents the sequence alignment (Clustal W method) of the stem-anchor regions of flavivirus E proteins and the first nine amino acids of the NS1 protein amino-terminus (TBE; GenBank U27495; YF; GenBank U17066; JE; GenBank M18370; Den 2; GenBank M19197). Under the alignment, the following symbols denote the degree of conservation observed at each amino acid position: (*) identical in all sequences; (:) conserved substitutions and (.) semi-conserved substitutions.

Figure 2

Viral growth curves in Vero cells. Cells were infected with either the control YF 17DD (gray lozenges) and YF17D/E200T3 (black triangles) viruses or the recombinant YF17D/Esa/5.1glic virus (open circles) at MOI of 0.02. Each time-point represents the average titer obtained from three separate experiments with the respective standard deviations.

Figure 3

Analysis of the EGFP expression in YF 17D virus-infected Vero cells. (A) Flow citometry analysis at 72 h – post infection. Dot plots show the expression of YF antigens detected by intracellular staining with murine hyperimmune serum against YF virus (α-YF; y-axis) and of EGFP by direct detection of its fluorescence (EGFP; x-axis). The controls consisted of cells infected with no virus (control) and the parental virus (YF17D/E200T3). Cells infected by the recombinant virus were labeled (EGFP- α-YF) or (EGFP) only. The percentages of gated cell populations are indicated in each plot. (B) Immunoprecipitation profiles of protein extracts from supernatant and infected Vero cells with either YF 17DD or YF 17D/Esa/5.1glic viruses. These samples were immunoprecipitated with murine hyperimmune serum against yellow fever virus (α-YF) or rabbit polyclonal antiserum directed to EGFP (α-EGFP). Molecular weight markers are indicated on the left side of the figure whereas viral and recombinant proteins are identified on the right side.

Figure 4

Intracellular localization of the recombinant EGFP protein. (A) Co-localization of viral antigens and EGFP. Infected cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and processed for immunolabeling. The designation on the upper right corner indicates the localization of the heterologous protein (EGFP); (α-YF) corresponds to the same cells stained with a hyperimmune antiserum to YF virus proteins; (DAPI) represents DAPI-stained cell nuclei; (merge) co-localization assessed by spectral overlap (yellow in right down panel) of the images of this preparation. (B) Co-localization of EGFP and the ER compartment. Live infected cells were labeled with ER-Tracker Red (Molecular Probes) and fixed in 4% paraformaldehyde. (EGFP) localization of heterologous protein; (ER) cells labeled with ER marker; (DAPI) nuclei counterstained with DAPI; (merge) co-localization assessed by spectral overlap (yellow in right down panels) of the images of this preparation.

Figure 5

Viral genetic stability and artifactual DNA amplification of the EGFP gene. (A) Agarose gel electrophoresis of plasmid T3 DNA without and with the EGFP cassete (lanes 1 and 2, respectively); DNA amplification of plasmid T3 and the recombinant one (lanes 3 and 4, respectively); RT-PCR on RNA of YF17D/E200T3 and YF17D/Esa/5.1glic 2P viruses without and with the EGFP cassete (lanes 5 and 6, respectively). (B) Schematic representation of the amplification based on the correct annealing of the E protein gene (black bars) and the EGFP stem-anchor (white bars) domains from two different DNA strands yielding an amplicon of 2,030 bp. (C) and (D) schematic representation of the amplification based on the spurious alternative annealing possibilities of the E protein gene (black bars) and the EGFP stem-anchor (white bars) regions from two different DNA strands yielding amplicons of 1,001 bp (without the EGFP cassete and with a single stem-anchor domain, gray bars) or 3,059 bp (with the duplicated EGFP gene and an extra copy of stem-anchor region), respectively.

Figure 6

Analysis of recombinant virus genetic stability after serial passaging. (A) Schematics of viral regeneration and subsequent passages (10) of the YF 17D/Esa/5.1 glic virus obtained after RNA transfection. Two independent series of serial passages (at MOI of 0.02); P1 and P2 were analyzed by RT-PCR and flow citometry at passages 5 and 10 and are represented in all panels as 5P1, 10P1, 5P2 and 10P2. In these experiments the YF17D/E200-T3 virus was used as negative control for EGFP expression. (B) Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted of samples from the supernatant of cultures used to derive the citometry data (C) according the passage history (A). The length of the main RT-PCR bands are shown on the left side. (C) The rate of double gated cells (YF+, EGFP+) over the total YF+ gated cells (YF+, EGFP+ plus YF+, EGFP- gated cells) corresponds to the percentage of cells infected by YF 17D/Esa/5.1 glic virus stably expressing the EGFP protein. The respective columns indicate the values for each of the viral passages.

Figure 7

Molecular cloning of EGFP protein expression cassete in the chimeric YF17D/DEN4 virus genome. (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, 15^th and 20^th passages of the two independent passage lineages (5P1 and 5P2; 10P1 and 10P2, 15P1 and 15P2, 20P1 and 20P2, respectively).