Dating the time of viral subtype divergence

Precise dating of viral subtype divergence enables researchers to correlate divergence with geographic and demographic occurrences. When historical data are absent (that is, the overwhelming majority), viral sequence sampling on a time scale commensurate with the rate of substitution permits the inference of the times of subtype divergence. Currently, researchers use two strategies to approach this task, both requiring strong conditions on the molecular clock assumption of substitution rate. As the underlying structure of the substitution rate process at the time of subtype divergence is not understood and likely highly variable, we present a simple method that estimates rates of substitution, and from there, times of divergence, without use of an assumed molecular clock. We accomplish this by blending estimates of the substitution rate for triplets of dated sequences where each sequence draws from a distinct viral subtype, providing a zeroth-order approximation for the rate between subtypes. As an example, we calculate the time of divergence for three genes among influenza subtypes A-H3N2 and B using subtype C as an outgroup. We show a time of divergence approximately 100 years ago, substantially more recent than previous estimates which range from 250 to 3800 years ago.