Improved consistency of cellular fluorescent distribution after separation of GFP genes from C25 by 2A ribosome skipping.

A) Design of FP reporter gene insertion in the DENV2 genome. P2A = 2A sequence of porcine technovirus-1. T2A = 2A sequence of Thoseasigna virus. 1x 2A denotes the reporter DENV2 design with one 2A sequence at the end of the reporter gene, while 2x 2A denotes the design with two 2A sequences flanking the reporter gene. B) Fluorescent images of Vero, Huh7, and BHK21 cells infected with DENV2-FPs with the 1X 2A design. C) Fluorescent images of Vero, Huh7, and BHK21 cells infected with DENV2-FPs with the 2X 2A design. D) Replication kinetics of DENV2-eGFP (right) and DENV2-clover2 (left) with 1x 2A and 2x 2A designs in Vero cells. Control DENV2 virus without a reporter gene and 2A sequences is denoted as 16681. Confluent Vero cells were infected with one virus at the multiplicity of infection (MOI) = 0.01. Infectious virus titer in the media was quantified by focus-forming assay. Each point is the average of three replicates with error bar representing the standard deviation. E) Focus images of the wild-type and the reporter DENV2-FPs (top panel) and the bar graph comparing the sizes of their foci (bottom panel). The size of each focus was quantified by the number of pixels that it occupied on a digitized image taken on ELISpot reader. Each bar represents the average of the measured focus sizes and error bars represent the standard deviation. At least 227 foci of each virus were used for foci-size quantification.