Reliable estimates of Japanese encephalitis virus evolutionary rate supported by a formal test of temporal signal

The single-stranded RNA viruses exhibit stupendously high evolutionary rate spanned several orders of magnitude. The accurate and reliable estimates of the virus evolutionary rate are of importance for the robust interpretation of outbreak investigations and are critical for understanding short-term virus transmission patterns. In the previous studies, the substitution rate of the Japanese encephalitis virus (JEV) and time to the most recent common ancestor (MRCA) were reported, however, the estimates are substantially different between some of the studies. Importantly, temporal signal in data was not evaluated by a formal test. The purpose of our study is to estimate the substitution rate and the age of the JEV common clade (GI-GV) and of two JEV genotypes (GI and GIII) supported by a formal Bayesian analysis of temporal signal in the open reading frame (ORF) nucleotide data. Additionally, we assessed temporal signal in the data from the previous work to explain the observed discrepancy between the reported JEV evolutionary rate estimates. Results showed that the mean substitution rate of the JEV GI-GV was 2.41 × 10^- 4 nucleotide substitution per site per year (s/s/y) with 95% highest posterior density (HPD) interval of 1.67 × 10^- 4-3.14 × 10^- 4 or approximately 2.5 nucleotide substitutions per ORF (10,296 nucleotides) per year. That is one of the lowest substitution rates among the species closely related to JEV and other well-studied members of mosquito-borne flaviviruses. The mean substitution rate of GI and GIII was evaluated to be 4.13 × 10^- 4 s/s/y (95% HPD interval, 3.45 × 10^- 4- 4.82 × 10^- 4) and 6.17 × 10^- 5 s/s/y (95% HPD interval 3.84 × 10^- 5-8.62 × 10^- 5), accordingly, with the ages of clades of 153 (95% HPD, 87-237) and 216 (95% HPD, 139-317), respectively. The mean root height of JEV is 1234 years (95% HPD, 407-2333).