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.