Viruses
from transfection of cDNA transcripts were passaged
three times at an moi = 1.0 (c » 3.0) and individual plaques were
isolated (1). The 5’ non-coding region
and capsid gene (L = 2821) were
sequenced directly from RT-PCR products (i.e. without molecular cloning). Thirteen mutations were observed in 18
plaque-derived viruses. For the
wild-type virus, 13 mutations were found after sequencing 50700 nucleotides in
total. Hence, using Equation 5 we obtain
min[ms/n/c]
= 13 / 50700 / 3 = 8.5 ´ 10-5. No
max[ms/n/c]
can be obtained since sampling was selective (i.e. the assumption that all
mutations are lethal is incompatible with plaque sequencing). The selection correction factor with
selective sampling and assuming the same burst size as above (B = 1694) is a = 0.28 for pL = 0.3 and E(sv)
= 0.12. Thus, the selection-corrected
estimate is ms/n/c =
min[ms/n/c]
/ a = 8.5 ´ 10-5 / 0.28 = 3.0 ´ 10-4.
1. Vignuzzi, M., J. K. Stone, J. J. Arnold, C. E.
Cameron, and R. Andino. 2006. Quasispecies
diversity determines pathogenesis through cooperative interactions in a viral
population. Nature 439:344-348.