The resistance frequency to a monoclonal antibody was measured by plating clonal viral pools or viruses resuspended from plaques in the presence of the antibody (4). Mutations were assumed to be neutral. Virus titers averaged 4.2 ´ 10¹¹ pfu/mL and 3.7 ´ 10⁷ pfu/mL for clonal pools and resuspended plaques, respectively (2). Plating 0.1 mL of these stocks yielded f = 1.7 ´ 10^-4 and f = 2.3 ´ 10^-4, respectively. Sequencing showed that there were two possible G®A transitions conferring resistance (T = T_s = 2). Under conditions that restrict viral diffusion, B = 166 for this cell type (1), and B = 1250 in liquid medium under standard conditions (3). Hence, the formation of a plaque would require approximately log (3.7 ´ 10⁷ ´ 0.1) / log 166 = 3.0 cell infection cycles, an additional log (4.2 ´ 10¹¹ ´ 0.1) / log 1250 = 3.4 cycles would have taken place in clonal pools. Accordingly, m_s/n/c = 3 ´ 2.3 ´ 10^-4 / 3 / 2 = 1.2 ´ 10^-4 and m_s/n/c = 3 ´ 1.7 ´ 10^-4 / (3 + 3.4) / 2 = 4.0 ´ 10^-5 for the resuspended plaques and the clonal pools, respectively, giving a geometric mean of m_s/n/c = 6.9 ´ 10^-5. Since the only mutations scored were transitions, which are more likely than transversions, and since neutrality was not guaranteed, this value might be an overestimation.

1. Cuevas, J. M., A. Moya, and R. Sanjuán. 2005. Following the very initial growth of biological RNA viral clones. J Gen. Virol. 86:435-443.

2. Drake, J. W. 1993. Rates of spontaneous mutation among RNA viruses. Proc. Natl. Acad. Sci. USA 90:4171-4175.

3. Furió, V., A. Moya, and R. Sanjuan. 2005. The cost of replication fidelity in an RNA virus. Proc. Natl. Acad. Sci. USA 102:10233-10237.

4. Holland, J. J., J. C. de la Torre, D. A. Steinhauer, D. K. Clarke, E. A. Duarte, and E. Domingo. 1989. Virus mutation frequencies can be greatly understimated by monoclonal antibody neutralization of virions. J. Virol. 63:5030-5036.