Conservation at synonymous sites in Japanese encephalitis and related viruses. Conservation at synonymous sites was calculated for an input alignment comprising the polyprotein CDSs from the seven JEV-group sequences listed in the caption to Figure 2. (A) Panels 1–3 show the positions of stop codons (blue triangles) in the three forward reading frames. The +0 frame is the polyprotein frame and is therefore devoid of stop codons. Alignment gaps are indicated in green. Panel 4 shows the difference between the expected number (assuming neutral evolution) and observed number of base substitutions at synonymous sites, summed over a phylogenetic tree, and averaged over a 25-codon sliding window. Panel 5 shows the estimated standard deviation for the statistic in panel 4 – major dips tend to correspond to alignment gaps (fewer pairwise sequence comparisons to sum over), while the rise at each end of the alignment corresponds to the shorter terminal windows over which statistics are averaged. Panel 6 is an approximation of the p-value corresponding to the statistic in panel 4, albeit subject to the assumption of normal distribution. Panel 7 shows a map of the JEV genome, indicating the position of the putative -1 ribosomal frameshift site, and the putative transframe protein which may equate to NS1'. (B) Zoom-in of the region corresponding to the conservation peak in the NS2A CDS with a 15-codon sliding window. Note the highest level of conservation corresponds precisely to the region covered by the predicted pseudoknot.