Examining the molecular clock hypothesis for the contemporary evolution of the rabies virus

The molecular clock is a method for measuring the rate of virus evolution but the key assumption that mutations accumulate on the genome at a constant rate over time does not always hold true. While modelling approaches exist to accommodate deviations from a strict molecular clock, assumptions about rate variation may not fully represent the underlying evolutionary processes and can affect the accuracy of clock calibration and divergence time estimates. There is considerable variability in rabies virus (RABV) incubation periods, ranging from days to over a year, during which viral replication may be reduced. This prompts the question of whether modelling RABV on a per infection generation basis might be more appropriate. We investigate how variable incubation periods affect root-to-tip divergence under per-unit time and per-generation models of mutation. Additionally, we assess how well these models represent root-to-tip divergence in time-stamped RABV sequences. We find that at low mutation rates (<1 substitution per genome per generation) divergence patterns between these models are difficult to distinguish, while above this threshold differences become apparent across a range of sampling rates. Using a Tanzanian dataset, we calculate the mean substitution rate to be 0.17 substitutions per genome per generation. At RABV's substitution rate, the per-generation substitution model is unlikely to represent rabies evolution substantially differently than the molecular clock model when examining contemporary outbreaks; over enough generations for any divergence to accumulate, extreme incubation periods average out. However, measuring substitution rates per-generation holds potential in applications such as inferring transmission trees and predicting lineage emergence. ### Competing Interest Statement The authors have declared no competing interest.