Figure 1.
Design of DENV3 infectious clone and chimeras.
Panel A provides a schematic representation of the DENV genome, divided into structural and nonstructural genes. Arrows indicate primer name and approximate primer locations and orientation on the genome. These primers were used to amplify the different cDNA fragments as well as adding appropriate, terminal restriction enzyme recognition sequences. The T7 promoter is located at the 5′end of primer DEN#1. Primer pairs that generate complete fragments are aligned opposite one another. The clone was propagated in in three circular and one linear plasmid. DENV fragments within each plasmid are highlighted in blue. The final fragments assembled to generate the clone are represented at the bottom of the figure as blue lines. Lengths and restriction site locations are approximate and supported by exact primer sequences, positions and PCR fragment sizes as noted in Text S1. Panel B illustrates the strategy used for E gene construct insertion. The top figures represent the DEN A and B fragments, which encode for the E protein. Arrows indicate approximate locations of primers EGENE+ and EGENE− used to silently introduce a BsaI recognition sequence into the 5′ end of fragment DEN B. The Bio Basic E construct in black represents the synthesized E gene, and the schematic below it illustrates approximate arrangement of E gene domains and restriction sites. Roman numerals indicate E domains; TM = transmembrane region. Arrows indicate primer names, approximate location and pair orientation used to introduce restriction enzyme recognition sequences Lengths and positions shown are approximate. The E construct is amplified for insertion into the DEN A fragment with primers pUC57 and with DEN2kb− and for the B fragment with primers DEN2kb+ and EGENE−. After sequence confirmation, constructs and DEN A and B fragments are digested with indicated enzymes, desired fragments gel purified and subjected to ligation to generate new DEN A and DEN B fragments containing E constructs. *Type IIS restriction endonuclease.
Figure 2.
Phylogenetic relationship of DENV-3 viruses.
The phylogenetic tree illustrates genetic relatedness of DENV-3 virus genotypes, including those viruses from which representative E genes were synthesized. This tree is meant to display DENV-3 diversity but does not include all 175 sequences used to evaluate the genetic variability of the DENV-3 E gene. Instead, representative sequences from each genotype were selected for inclusion in the tree. The tree was constructed using Maximum Likelihood method based on the Tamura-Nei model [66]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 37 nucleotide sequences. There were a total of 1479 positions in the final dataset. Evolutionary analyses were conducted in MEGA4 [67]. *Parent virus for E gene variants.
Table 1.
Mean focus size for parent virus and chimeric clones.
Figure 3.
Recombinant dengue virus growth kinetics in tissue culture.
Vero (figure 3A) and C6/36 (figure 3B) cells were inoculated at a multiplicity of infection (m.o.i) of 0.01 FFU. Cell culture supernatants were harvested at indicated times and the virus released from the infected cells was quantitated by immunofocus assay. Points show geometric mean titer (GMT) calculated from triplicate titrations. Error bars indicate 95% confidence intervals for each GMT. For Vero cells no focus forming units were observed at 0 hrs and for C6/36 cells no focus forming units were observed at 0 hrs and 24 hrs.
Table 2.
Summary of human primary homotypic, primary heterotypic and secondary sera used to in neutralization experiments.
Figure 4.
Mean FRNT50 values for homotypic primary and secondary sera.
Homotypic primary (anti DENV-3) sera (Figures 4A–4H) or secondary serum (Figure 4I) FRNT50 titers against each of the E variant isogenic clones. Each serum sample is identified above the graph. Serum histories are summarized in Table 2 and Text S1. Fold-dilution of serum is on the Y-axis and each clone is identified on the X-axis. Columns show GMT FRNT50 values calculated from FRNT done in triplicate. Error bars indicate 95% confidence intervals. Columns connected by horizontal lines indicate groups of clones that did not have statistically significantly different FRNT50 values (P<0.05). Connecting bars indicate individual or groups of clones that differed statistically (P<0.05) by Tukey's HSD. Sera 003 (Figure 4A), 005 (Figure 4B) and 103 (Figure 4E) did not have significantly different titers against the recombinant viruses.
Figure 5.
Mean FRNT50 values for heterotypic primary sera.
Each serum sample is identified above the graph. Serum 001(Figure 5A) is from a primary DENV-2 infection in; Serum 006 (Figure 5B) is from a primary DENV-1 infection; Serum 102 (Figure 5C) is from a primary DENV-4 infection. Serum histories are summarized in Table 2 and Text S1. Fold-dilution of serum is on the Y-axis and each clone is identified on the X-axis. Columns show GMT FRNT50 values calculated from FRNT done in triplicate.