Muller's ratchet is an important concept in population genetics. It predicts that when mutation rates are high and a significant proportion of mutations are deleterious, a kind of irreversible ratchet mechanism will gradually decrease the mean fitness of small populations of asexual organisms. In contrast, sexual recombination may stop or reverse this mutational ratchet by recombinational repair Muller (1) suggested that accumulation of deleterious mutations in asexual (2) organisms occurs by "a kind ofirreversible ratchet mechanism;" in contrast, sexual reproduction allows repair of genetic damage by recombination (2-4). Bell (5) used senescence in protozoa to provide experimental support for Muller's ratchet hypothesis, but some models of Muller's ratchet have been questioned recently (6). Chao (7) recently reported that 40 consecutive plaque-to-plaque (genetic bottleneck) transfers of the tripartite RNA bacteriophage 46 led to a significant decrease in mean fitness. Such clone-to-clone transfers of a virus repeatedly reduce virus population size to one infectious particle and should increase the probability for Muller's ratchet to become manifest if mutation rates are high and sexual recombination is absent. Chao (7) tested fitness of his repeatedly bottlenecked 46 phage clones in paired-growth experiments in which each transferred clone was mixed at a known ratio with a genetically marked parental phage clone. These mixtures were then diluted and plated on lawns of bacterial host cells to form independent plaques during each growth competition transfer. This precluded genetic reassortment of the three genome segments of this tripartite bacteriophage while allowing internally controlled comparisons ofthe relative replication rates of wild-type phage versus repeatedly bottlenecked clones. We have now quantitated the relative fitness of repeatedly bottlenecked clones of vesicular stomatitis virus (VSV) a single-stranded RNA virus of animals, humans, and insect vectors. VSV is the prototype model for negative-stranded animal RNA viruses (which include a large number of medically important human pathogens). Because VSV has only a single, nonsegmented genome, which does not undergo recombination at a detectable level, we are able to quantitate relative fitness directly in mixed-infection competition experiments. We can allow the parental VSV clone and the genetically marked monoclonal antibody (mAb)-resistant (MAR) clones to replicate and compete directly in liquid medium cell cultures (and even in the same cells late during each passage). We previously described quantitative relative fitness assay methods for determining virus fitness during direct replicative competition experiments (8, 9), and these are employed here.RNA viruses generally should be ideal for testing Muller's ratchet theory because most or all of them have extremely high mutation frequencies. RNA viruses apparently lack the mechanisms of proofreading and mismatch repair that are available for high-fidelity DNA replication. Their replic...
The great adaptability shown by RNA viruses is a consequence of their high mutation rates. Here we investigate the kinetics of virus fitness gains during repeated transfers of large virus populations in cell culture. Results always show that fitness increases exponentially. Low fitness clones exhibit regular increases observed as biphasic periods of exponential evolutionary improvement, while neutral clones show monophasic kinetics. These results are significant for RNA virus epidemiology, optimal handling of attenuated live virus vaccines, and routine laboratory procedures.RNA viruses are highly mutable and form complex quasispecies populations as defined by Eigen and colleagues (1-4). Quasispecies or "mutant swarms" of RNA viruses evolve thousands-to millionsfold faster than DNA-based organisms (5-9). This provides an invaluable tool to perform evolutionary studies that, for the latter, would take eons. Evolution of RNA viruses depends upon environmental selective forces and random drift (6,10,11). Examples are human immunodeficiency virus 1 (11), hepatitis C virus (12), and foot-and-mouth disease virus (13), all of which can replicate and evolve rapidly and continuously in infected individuals. Because the behavior of quasispecies populations is important for an understanding of RNA virus disease and epidemiology, quantitative studies of virus populations and population genetics are needed. We have developed a relative fitness assay to enable quantitative analysis of RNA virus population behavior (14). This employs genetically marked mutants that are mixed with wild-type virus (as an internal standard), and these mixed RNA virus quasispecies are allowed to compete during replication in a series of repeated transfers in cell culture. The changing ratios of genetically marked virus to wild-type virus allow determination of relative fitness vectors and relative fitness values (W) per passage. For the wild-type virus employed as the internal control, fitness is assigned a neutral value (W = 1.0) because it is the parental standard virus clone from which all of the genetically marked clones have been derived. The marked clones are monoclonal antibody-resistant mutants (MARMs), and their fitness is measured after replicative competition passages in a constant cell culture environment (14).It was observed (15-18) that whenever selection does not have the opportunity to act, as during repeated genetic bottleneck transfers of a MARM of vesicular stomatitis virus (VSV) or a marked mutant of an RNA bacteriophage, the high mutation rates lead to loss of virus fitness. Genetic bottleneck passages involve repeated transfers of only one or a few virions, and loss of fitness results from gradual stochastic accumulation of deleterious mutations in accord with Muller's ratchet theory (19,20). Muller (19) had predicted that when an asexual population is small and the mutation rate is high, the popu-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby ...
We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.
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Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32°C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10' to 106 PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.
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