No abstract

Understanding how evolution promotes pathogen emergence would aid disease management, and prediction of future host shifts. Increased pathogen infectiousness of different hosts may occur through direct selection, or fortuitously via indirect selection. However, it is unclear which type of selection tends to produce host breadth promoting pathogen emergence. We predicted that direct selection for host breadth should foster emergence by causing higher population growth on new hosts, lower amongpopulation variance in growth on new hosts, and lower population variance in growth across new hosts. We tested the predictions using experimentally evolved vesicular stomatitis virus populations, containing groups of host-use specialists, directly selected generalists, and indirectly selected generalists. In novel-host challenges, viruses directly selected for generalism showed relatively higher or equivalent host growth, lower among-population variance in host growth, and lower population variance in growth across hosts. Thus, two of three outcomes supported our prediction that directly selected host breadth should favor host colonization. Also, we observed that indirectly selected generalists were advantaged over specialist viruses, indicating that fortuitous changes in host breadth may also promote emergence. We discuss evolution of phenotypic plasticity versus environmental robustness in viruses, virus avoidance of extinction, and surveillance of pathogen niche breadth to predict future likelihood of emergence.K E Y W O R D S : Experimental evolution, generalist, host shift, niche expansion, specialist, vesicular stomatitis virus.

Viruses and other pathogens can diverge in their evolved host-use strategies because of exposure to different host types and conflicts between within-host reproduction and between-host survival. Most host-pathogen studies have emphasized the role of intrahost reproduction in the evolution of pathogen virulence, whereas the role of extra-host survival has received less attention. Here, we examine the evolution of free-living virion survival in RNA virus populations differing in their histories of host use. To do so, we used lineages of vesicular stomatitis virus (VSV) that were experimentally evolved in laboratory tissue culture for 100 generations on cancer-derived cells, noncancerous cells, or alternating passages of the two host types. We observed that free-living survival improved when VSV populations specialized on human epithelial carcinoma (HeLa) cells, whereas this trait was not associated with selection on noncancer cells or combinations of the cell types. We attributed this finding to shorter-lived HeLa monolayers and/or rapid cell-to-cell spread of viruses on HeLa cells in tissue culture, both of which could select for enhanced virus stability between host-cell replenishment. We also showed evidence that increases in virion survival were associated with decreases in virulence, which suggests a trade-off between survival and virulence for the VSV populations on one cell type. Our results shed new light on the causes and consequences of "sit and wait" infection strategies in RNA viruses.

Parasites and hosts can experience oscillatory cycles, where the densities of these interacting species dynamically fluctuate through time. Viruses with different replication strategies can also interact to produce cyclical dynamics. Frequent cellular co-infection can select for defective-interfering particles (DIPs): “cheater” viruses with shortened genomes that interfere with intracellular replication of full-length (ordinary) viruses. DIPs are positively selected when rare because they out-replicate ordinary viruses during co-infection, but DIPs are negatively selected when common because ordinary viruses become unavailable for intracellular exploitation via cheating. Here, we tested whether oscillatory dynamics of ordinary viruses were similar across independently evolved populations of vesicular stomatitis virus (VSV). Results showed identical cyclical dynamics across populations in the first 10 experimental passages, which transitioned to repeatable dampened oscillations by passage 20. Genomic analyses revealed parallel molecular substitutions across populations, particularly novel mutations that became dominant by passage 10. Our study showed that oscillatory dynamics and molecular evolution of interacting viruses were highly repeatable in VSV populations passaged under frequent co-infection. Furthermore, our data suggested that frequent co-infection with DIPs caused lowered performance of full-length viruses, by reducing their population densities by orders of magnitude compared to reproduction of ordinary viruses during strictly clonal infections.

Although laboratory dependence is an acknowledged problem in microbiology, it is seldom intensively studied or discussed. We demonstrate that laboratory dependence is real and quantifiable even in the popular model Escherichia coli. Here laboratory effects alter the equilibrium composition of a simple community composed of two strains of E. coli. Our data rule out changes in the bacterial strains, chemical batches, and human handling but implicate differences in growth medium, especially the water component.Laboratory dependence, or variable performance of organisms when they are cultured at different locations, is an acknowledged phenomenon in microbiology and has the potential to cause major problems for microbial science (and for laboratory-based biology in general). However, laboratory dependence is rarely studied in detail, or even discussed. Underreporting may stem from the perception that the observed laboratory dependence is uninteresting (detracting from the impact of other results) or unbelievable (blamed on unidentified differences in experimental technique). It is crucial that microbiologists begin to understand which organisms are susceptible to laboratory dependence (perhaps all are), what factors in the laboratory environment commonly underlie this phenomenon, and what types of changes in microbial growth are to be expected. We begin to address these issues with a simple community composed of two genotypes of Escherichia coli. We hope that our study motivates closer investigation of laboratory dependence in other systems and promotes discussion of this important topic within the microbial ecology community.The strains used in this study, REL4397 (Lac ϩ ) and REL4398 (Lac Ϫ ), are previously described recombinant genotypes of E. coli B and E. coli K-12 that differ in lactose utilization (15,18). They evolved as a polymorphism in a single population propagated by 24-h serial culture. Under the conditions in which they evolved, the two strains coexisted stably at an equilibrium ratio of ϳ1:1 (18). The strains were isolated and stored at Ϫ80°C in the laboratory of R. E. Lenski at Michigan State University (lab I) in East Lansing, MI. Samples of the strains were shipped frozen to the laboratory of P. E. Turner at Yale University (lab II) in New Haven, CT.The coexistence of the Lac Ϫ and Lac ϩ strains in lab I was attributed to (i) the higher maximum growth rate (growth in abundant glucose) of the Lac Ϫ strain and (ii) the ability of the Lac ϩ strain to scavenge glucose at the end of the growth cycle and also to increase in cell number and relative fitness late in the growth cycle, long after glucose should have been exhausted from the medium (18). This late-cycle growth occurred only when the Lac ϩ strain was grown in the presence of the Lac Ϫ strain, which suggested a possible "cross-feeding" relationship (8, 14).We cultured the E. coli Lac ϩ and Lac Ϫ strains in lab II, where we observed a shift in the composition of this simple bacterial community in favor of the Lac ϩ strain. Below we describe labo...

RNA viruses may be particularly capable of contributing to the increasing biomedical problem of infectious disease emergence. Empirical studies and epidemiological models are informative for the understanding of evolutionary processes that promote pathogen emergence, but rarely are these approaches combined in the same study. Here, we used an epidemiology model containing observations of pathogen productivity in reservoirs, as a means to predict which pathogens should be most prone to emerge in a primary host such as humans. We employed as a model system a collection of vesicular stomatitis virus populations that had previously diverged in hostuse strategy: specialists, directly selected generalists and indirectly selected (fortuitous) generalists. Using data from experiments where these viral strategists were challenged to grow on unencountered novel hosts in vitro, logistic growth models determined that the directly selected generalist viruses tended to grow best on model reservoirs. Furthermore, when we used the growth data to estimate average reproductive rate across secondary reservoirs, we showed that the combined approach could be used to estimate relative success of the differing virus strategists when encountering a primary host. Our study suggests that synergistic approaches combining epidemiological modelling with empirical data from experimental evolution may be useful for developing efforts to predict which types of pathogens pose the greatest probability of emerging in the future.