Fig 5.

Fitness in human cells and sfRNA production of mosquito adapted DENV (A) Replication of parental (PT) and mutant (MS3) viruses in A549 cells monitored by immunofluorescence at 2 and 3-day post transfection. (B) Accumulation of sfRNAs in A549 cells transfected with PT or MS3 viral RNAs detected by Northern blot at 3-days post transfection. (C) Schematic representation of the key role of xrRNA2 for differential sfRNA accumulation in mosquito or human adapted viruses. Rope knots represent the xrRNA structures that impair Xrn1 movement and the gallows represents the terminal 3’SL structure. (D) Growth competition experiments highlight the fitness disadvantage of mosquito selected variant in human cells. A549 cells were infected with a mixture of PT: MS3 at a 1:99 ratio. At 30, 48 and 72 hpi, cells were collected for viral RNA purification,sequencing and sfRNA analysis. Relative abundance of each virus is represented in the plot shown on the right. (E) Viral RNA replication levels of WT and mosquito selected variants are shown together with a replication impaired control (mutant in the polymerase NS5, MNS5) in A549 cells. Mutations were incorporated in a replication and propagation competent reporter DENV carrying a luciferase gene. Normalized luciferase levels are shown in a logarithmic scale at 48h post-transfection. The values are the mean +/-SD, n = 3. (F) Quantitation of sfRNAs accumulation in A549 cells transfected with mutant RNAs. The values are the mean relative accumulation estimated with ImageJ Program from three independent experiments. (G) Plot of the mean relative amounts of different sfRNAs, shown in F, versus the replication capacity measured using the reporter virus shown in E.