Evolution in spatially mixed host environments increases divergence for evolved fitness and intrapopulation genetic diversity in RNA viruses

Abstract

Virus populations may be challenged to evolve in spatially heterogeneous environments, such as mixtures of host cells that pose differing selection pressures. Spatial heterogeneity may select for evolved polymorphisms, where multiple virus subpopulations coexist by specializing on a narrow subset of the available hosts. Alternatively, spatial heterogeneity may select for evolved generalism, where a single genotype dominates the virus population by occupying a relatively broader host niche. In addition, the extent of spatial heterogeneity should influence the degree of divergence among virus populations encountering identical environmental challenges. Spatial heterogeneity creates environmental complexity that should increase the probability of differing adaptive phenotypic solutions, thus producing greater divergence among replicate virus populations, relative to counterparts evolving in strictly homogeneous host environments. Here, we tested these ideas using experimental evolution of RNA virus populations grown in laboratory tissue culture. We allowed vesicular stomatitis virus (VSV) lineages to evolve in replicated environments containing BHK-21 (baby hamster kidney) cells, HeLa (human epithelial) cells, or spatially heterogeneous host cell mixtures. Results showed that generalist phenotypes dominated in evolved virus populations across all treatments. Also, we observed greater variance in host-use performance (fitness) among VSV lineages evolved under spatial heterogeneity, relative to lineages evolved in homogeneous environments. Despite measurable differences in fitness, consensus Sanger sequencing revealed no fixed genetic differences separating the evolved lineages from their common ancestor. In contrast, deep sequencing of evolved VSV populations confirmed that the degree of divergence among replicate lineages was correlated with a larger number of minority variants. This correlation between divergence and the number of minority variants was significant only when we considered variants with a frequency of at least 10 per cent in the population. The number of lower-frequency minority variants per population did not significantly correlate with divergence.

Keywords: Vesicular stomatitis virus; adaptation; environmental heterogeneity; experimental evolution; niche breadth; quasispecies.

Figure 1.

Experimental design. The ancestral VSV strain was used to found three replicate virus lineages in each experimental treatment, which were then serially passaged for roughly 100 VSV generations. Experimental treatments varied in the composition of the host cell community. Here the relative frequency of each cell type is represented, with black representing HeLa cells and white representing BHK cells.

Figure 2.

Relative fitness (ln(W)) of evolved virus populations. Each point represents the fitness of an evolved population relative to the common competitor virus (mean of three trials). The grand mean (solid line) and 95 per cent CI are shown for each treatment. The dotted line represents the fitness of the ancestral virus relative to the common competitor. (A) Relative fitness on BHK cells. (B) Relative fitness on HeLa cells.

Figure 3.

Map of polymorphic loci. Polymorphic sites for which a minority allele was detected at a frequency of 1 per cent or greater are shown mapped onto the VSV genome. Allele frequency is determined as the mean of two technical replicates, and only sites with depth of coverage >30 were considered for this analysis. Each row represents one virus population, with the experimental treatment (ratio of BHK to HeLa cells) shown on the left. A reference map of the VSV genome is displayed above, with gene coding regions shaded, with overlaid gene labels. The frequency of minority alleles in each population is coded by color (see legend). The location of minority alleles that appear in any population >5 per cent are labeled below. Nonsynonymous changes are labeled in red, and the amino acid substitution is listed.