Fig. 2. DENV3-CH53489 clubSPs contain RNA and remain infectious.

a The viral DENV3-CH53489 RNA genome is not extruded out of the particle after temperature-induced structural change at 25 and 37 °C. (Left panel) Viruses at 4 °C were transferred to different temperatures and incubated for 30 min. RNase is then added to digest away any RNA genome that was extruded outside the virus. RNase activity is inhibited and then the virus was lysed. Detection of a 260-nucleotide stretch of RNA genome using a primer set in qRT-PCR indicates intact viral genome. In all temperatures, the amount of genome with and without addition of RNase appeared the same, suggesting that viral RNA remains within the particle after temperature treatment and were thus protected from RNase treatment. The y-axis is expressed as difference in Cq values from virus-free reference. (Right panel) Control experiments to show when virus is lysed first before exposure to RNase, the RNases can successfully break down viral RNA genome. Results showed that when RNA genomes (due to lysis) are exposed, RNases digestion occurs and there are much lower amounts of intact RNA genome detected by qRT-PCR than when no RNase is added. b Attachment assay showing 29 and 37 °C pretreated viruses containing clubSPs have the same binding capacity to both C6/36 and BHK21 cells as the 4 °C sample. Data are presented as mean and error bars represent SD calculated from three individual experiments; each experiment was done in duplicate. The y-axis shows the relative DENV3-CH53489 RNA against housekeeping genes: beta-actin and GAPDH for C6/36 and BHK21 cells, respectively. Source data are provided in the source data file.