Evaluation of infectivity by a single-round infectious particle (SRIP) assay.

(A) Schematic representation of the reporter vector showing the position of the cytomegalovirus promoter (CMV), NanoLuc gene, 2A protein sequence of foot-and-mouth disease virus (FMDV2A) for self-excision, hepatitis delta virus ribozyme (HDV-RZ), and polyadenylation signal (pA). The structural protein-expressing plasmids (prME) used to generate SRIPs are also shown. Blue and red indicate the sequences derived from the Asian-I and Cosmopolitan viruses, respectively. (B, C) Luciferase activity of the SRIPs produced by the transfection of Lenti-X 293T cells with the reporter plasmid and structural protein-expressing plasmids as indicated. The multiple t test results with a statistically significant difference are indicated by asterisks (*:P < 0.05, **:P < 0.01). The means and standard deviation of triplicate samples are shown. Representative results of three independent experiments are shown.</p

Generation of DENV2 by the circular polymerase extension reaction (CPER) method.

(A) Schematic representation of the fragmentation of the whole dengue virus genome for the CPER method. In a previous publication by Setoh et al. (mSphere.2017; 2(3)) seven fragments were designed with an overlapping region of about 22 nt at the end. In the present study, five fragments were used, and each fragment was designed to have a 25-nt-overlapping region at the end. These fragments were mixed with a linker fragment containing the cytomegalovirus (CMV) promoter, hepatitis D virus ribozyme (HDVr), and a poly(A) tail (pA). (B) Comparison of the amounts of viral RNA in the culture supernatants at 6 days after the transfection of CPER products into BHK-21 cells. (C) Amounts of RNA in viruses propagated in C6/36 cells with the culture supernatants of transduced BHK-21 cells. Asian-I and Cosmopolitan viruses were infected at a multiplicity of infection of 0.01 copies/cell. We used NS5-C709A mutants as non-replicating negative controls.</p

Characteristics of the chimeric DENV2 between the Cosmopolitan and Asian-I genotypes.

(A) Schematic representation of 30 chimeric viruses with two different parental strains. The fragments derived from the Asian-I [A] genotype and Cosmopolitan [C] genotype are shown in blue and orange, respectively. AAAAA and CCCCC indicate the parental Asian-I and Cosmopolitan viruses, respectively. (B) The FFU of the generated recombinant viruses 3 days after the infection of Vero cells at a multiplicity of infection of 0.5 copies/cell. Viruses with titers equal to or lower than that of the parental Asian-I strain are shown in blue. Viruses with titers equal to or higher than that of the parental Cosmopolitan strain are shown in orange. Viruses with titers in between those of the two parental viruses are shown in green. The means and standard deviation of triplicate samples are shown. (C) Images of the foci of all recombinant viruses. The chimeric viruses are grouped according to the number of Cosmopolitan virus-derived fragments and whether the structural and NS1, NS2A, and NS2B proteins were derived from the Cosmopolitan or Asian-I viruses. Red CC and blue AA indicate the viruses with structural and NS1, NS2A, and NS2B proteins derived from the Cosmopolitan and Asian-I viruses, respectively.</p

Molecular modeling of the Asian-I and Cosmopolitan envelope and pr complex.

(A) The mutation sites in the pr and envelope proteins of the Asian-I and Cosmopolitan viruses are shown. The orange region is the pr protein, and the pink region is the envelope protein. The yellow spheres indicate mutation sites in the pr protein. The blue spheres indicate mutation sites in the envelope protein. (B) The mutation sites of position 52 of pr and position 71 of envelope are shown. Amino acid residues at positions 52 of pr and 71 of envelope are colored as follows: green indicates carbon atoms; gray indicates hydrogen atoms; blue indicates oxygen atoms; and red indicates nitrogen atoms. These figures were generated using PDB ID: 3J27 and 3C5X as templates for homology modeling.</p

Characteristics of the parental and chimeric DENV2 between the Cosmopolitan and Asian-I genotypes generated from an independent CPER reaction.

(A) Infectious titers of an independent lot of stock viruses. (B) Focus-forming unit of an independent lot of stock viruses 3 days after the infection of Vero cells at a multiplicity of infection of 0.5 copies/cell. The number of seeding Vero cells was 4.0 × 104 cells/well. These experiments were performed using the method described in the "Focus-forming assay" section of the Materials and Methods. (PDF)</p

Levels of viral RNA in the cell supernatants.

(A) Levels of viral RNA in the cell supernatants after the transfection of CPER products. (B) Levels of viral RNA in the cell supernatants after viral infection with the supernatants of the transfected cells. (PDF)</p

Characteristics of the DENV2 generated by the CPER method.

(A) Images of the foci of the recombinant DENV2 obtained by the ELISPOT reader. (B) Growth curves of the recombinant viruses in Vero cells infected with virus samples at a multiplicity of infection of 0.5 copies/cell. Culture supernatants were harvested every 24 h, and assayed for the level of viral RNA and the infectious titer. The means and standard deviation of triplicate samples are shown.</p

Replicon assay.

(A, B, C) Results of three independent experiments on Replicon assay. The fold increases in luciferase activity when compared to the activity at 24 h after the transfection of DENV2 replicons are shown as the means and standard deviation (SD) of triplicate samples. Statistical significance of the differences were tested using the Holm-Sidak method with alpha = 0.05. Each row was analyzed individually without assuming a consistent SD. (PDF)</p

Mutation sites in the non-structural proteins.

(A) The mutation sites in the Asian-I and Cosmopolitan viruses are shown on the 3D structure of the NS1 protein dimer (PDB ID: 4O6B). Both the orange and purple spheres indicate mutation sites in the NS1 protein. A region spanning amino acid positions 108 to 128, including mutation cites at positions 117 and 128, is not shown due to the lack of structural information in the template. (B, C) A topology model of the DENV2 NS2A and NS2B proteins on the endoplasmic reticulum membrane. The mutation sites in the NS2A and NS2B proteins (yellow points) of the Asian-I and Cosmopolitan viruses are shown. These topology models were drawn based on previous articles referred to in the text (reference numbers 41 to 43).</p

Evaluation of proliferation by the replicon assay.

(A) Structure of the CPER product used in the replicon assay. Schematic representation of the replicon construct showing the position of the Gaussia luciferase gene (Gluc) and foot-and-mouth disease virus 2A peptide (FMDV2A). (B) The fold increase in luciferase activity when compared to the activity at 24 h after the transfection of DENV2 replicons. Lenti-X 293T cells were transfected with the CPER product, and luciferase activity was monitored at the indicated time points. The means of triplicate samples were calculated in each of the three independent experiments and the averages and standard deviation (SD) of the means of the three independent experiments are shown. Statistical significance of the differences were tested using the Holm-Sidak method with alpha = 0.05. Each row was analyzed individually without assuming a consistent SD.</p