Viruses were recovered a cDNA clone by transfection, seeded into fresh cells, passaged once in standard liquid culture at an moi of approximately 0.01, plaque-purified, and passaged twice plaque-to-plaque (1). Six plaques were picked, amplified by infecting liquid cultures and used for direct sequencing (i.e. without molecular cloning). It was estimated that one infection cycle was equivalent to 8 h of growth and, based on this, that the total number of cell infection cycles was c = 13. For the wild-type virus, three mutations were found after sequencing 120978 nucleotides in total. Hence, using Equation 5 we obtain min[m_s/n/c] = 3 / 120978 / 13 = 1.9 ´ 10^-6 whereas no max[m_s/n/c] can be obtained because mutation sampling was selective. To provide a more accurate estimate, we can use the selection correction method. Plaque-to-plaque passages constituted approximately 2 / 3 of the total passage time (c₁ = 13 ´ 2 / 3 = 8.7), although the exact fraction was not provided. Selection is typically relaxed under this passage regime, and assuming that all mutations except lethal ones accumulated neutrally, we have m_s/n/c = min[m_s/n/c] / (1 - p_L). This defines a correction factor a₁ = 1 - p_L for this phase. For the standard liquid culture phase (c₂ = 4.3 cycles), a correction factor a₂ with selective sampling assuming B = 600-700 (2). For p_L = 0.3 and E(s_v) = 0.12, a₂ » 0.26 (Figure 1). Using the weighted average to combine a₁ and a₂, we obtain a = (0.7 ´ 8.7 + 0.26 ´ 4.3) / (8.7 + 4.3) = 0.55. Therefore, the selection-corrected estimate of the mutation rate is m_s/n/c = 1.9 ´ 10^-6 / 0.55 = 3.5 ´ 10^-6. Notice, however, that empirical knowledge of mutational fitness effects is limited to viruses with genome sizes smaller than those of coronaviruses, implying that the parameters used in the exponential plus lethal model might not be appropriate for this virus. Also, there is some uncertainty in the number of cell infection cycles elapsed. For these reasons, the estimate should be taken with caution.

1. Eckerle, L. D., X. Lu, S. M. Sperry, L. Choi, and M. R. Denison. 2007. High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. J. Virol. 81:12135-12144.

2. Hirano, N., K. Fujiwara, and M. Matumoto. 1976. Mouse hepatitis virus (MHV-2). Plaque assay and propagation in mouse cell line DBT cells. Jpn. J Microbiol. 20:219-225.