type, acquired after 61 consecutive transfers of large virus populations on BHK cells. Finally, MARM F is an extremely low fitness clone (0.00015 ± 0.00001) obtained after 20 plaque-to-plaque transfers from MARM X. MARM C and X originally were isolated from the wild-type virus by picking spontaneous I1 mAb-resistant clones (VandePol et al., 1986). Therefore, they were isogenic with wild type with the only exception of the above mutations responsible for the resistance.
The second step corresponds with the experiment itself (see next four sections), normally carried out with one of the four MARM clones, and in which viral populations experienced different demographic regimes and environmental conditions.
Third, fitness of the evolved viral populations was evaluated by competition assays with the ancestral wild-type clone in the following form. The evolved MARM population was mixed with a known amount of the wild-type clone. A differential quantitation of MARM clone, compared with the total virus, was done by parallel plating of the virus with and without I1 mAb. These virus mixtures then were used to initiate replicate serial competition passages. After each competition passage, the resulting virus mixture was 104-fold diluted and used to initiate the next competition transfer by infection of a fresh monolayer. The number of competition passages varied between two and a maximum of five, depending on the speed with which one competitor displaced the other. The antilogarithm of the slope of the regression ln is taken as an estimate of the mean fitness of the corresponding MARM population relative to the wild type, where pt and 1 - pt are the proportions at passage number t of MARM and wild type, respectively (Duarte et al., 1992; Clarke et al., 1993; Elena et al., 1996, 1998).
When finite populations with high mutation rates are considered, a significant proportion of the mutants should be deleterious. If populations are asexual and small in size, mutation-free individuals become rare and can be lost by random genetic drift. In that case a kind of irreversible ratchet mechanism gradually will decrease the mean fitness of the populations (Muller, 1964). Chao (1990) provided the first experimental evidence for the action of Muller's ratchet in RNA viruses. As can be observed in Table 1, there is a common pattern of fitness decline, but the magnitude of decline strongly depended on the virus studied. For instance, in the case of VSV (Duarte et al., 1992; Clarke et al., 1993; Duarte et al., 1993, 1994) we performed genetic bottleneck passages (plaque-to-plaque transfers) and quantified the relative fitness of bottlenecked clones