New Insights into the Evolutionary Rate of Hepatitis B Virus at Different Biological Scales

The evolutionary rates of hepatitis B virus (HBV) estimated using contemporary sequences are 102 to 104 times higher than those derived from archaeological and genetic evidence. This discrepancy makes the origin of HBV and the time scale of its spread, both of which are critical for studying the burden of HBV pathogenicity, largely unresolved. To evaluate whether the dual de-mands (i.e., adaptation within hosts and colonization between hosts) of the viral life cycle affect this conundrum, the HBV quasi-species dynamics within and among hosts from a family consisting of a grandmother, her 5 children, and her 2 granddaughters, all of whom presumably acquired chronic HBV throughmother-to-infant transmission, were examined by PCR cloning and next-generation sequencing methods. We found that the evolutionary rate of HBV between hosts was considerably lower than that within hosts. Moreover, the between-host substitution rates of HBV decreased as transmission numbers between individu-als increased. Both observations were due primarily to changes at nonsynonymous rather than synonymous sites. There were significantly more multiple substitutions than expected for randommutation processes, and 97 % of substitutions were changed from common to rare amino acid residues in the database. Continual switching between colonization and adaptation resulted in a rapid accumulation of mutations at a limited number of positions, which quickly became saturated, whereas substitutions a