Plaque assay.

BHK-21 cells (baby hamster kidney fibroblast cells; ATCC CCL-10) were a kind gift from the Dimopoulos laboratory (Johns Hopkins University), and they were grown to confluence as mentioned above for Vero E6 cells, seeded onto 24-well plates at a density of 5 × 10⁴ cells/well, and incubated at 37°C with 5% CO₂ until newly confluent. Midgut or body samples from day 7, 10, or 14 from either the DENV-4H or DENV-4L experimental groups were homogenized using a bullet blender (Nextadvance, NY) adjusted to speed setting 8 for 3 min. Samples were then immediately centrifuged at a relative centrifugal force (RCF) of 2,400 for 3 min. Each sample was then serially diluted 10-fold in reduced DMEM, and 100 μl of each dilution series was added to individual wells (Fig. S1). The 24-well plates were rocked at room temperature for 15 min and then incubated at 37°C with 5% CO₂ for 45 min. Afterwards, 500 μl of reduced DMEM with 0.8% (wt/vol) methylcellulose was added to each well, and the plates were incubated for 5 days. On the fifth day, the spent medium was removed from the 24-well plates, and a 1:1 methanol-acetone solution with 1% (wt/vol) crystal violet was added for 1 h to fix and stain the cells. Plaques were counted manually, and the titer in PFU per milliliter was determined.

Representative images of plaque assay plates with Aedes aegypti (ORL) midgut tissues. Midguts from day 7 were homogenized, serially diluted 10-fold, and added to individually numbered columns (one sample dilution series per column), with the highest concentration of sample at the top of the image (no dilution) and the lowest concentration of sample at the bottom of the image (1:1,000) for the DENV-4 Haiti (A) and DENV-4 laboratory (B) strains. Download FIG S1, PDF file, 1 MB.