Genetic diversity enables a virus to colonize novel hosts, evade immunity, and evolve drug resistance. However, viral diversity is typically assessed at the population level. Given the existence of cell-to-cell variation, it is critical to understand viral genetic structure at the single-cell level. By combining single-cell isolation with ultra-deep sequencing, we characterized the genetic structure and diversity of a RNA virus shortly after single-cell bottlenecks. Full-length sequences from 881 viral plaques derived from 90 individual cells reveal that sequence variants pre-existing in different viral genomes can be co-transmitted within the same infectious unit to individual cells. Further, the rate of spontaneous virus mutation varies across individual cells, and early production of diversity depends on the viral yield of the very first infected cell. These results unravel genetic and structural features of a virus at the single-cell level, with implications for viral diversity and evolution.

It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10⁻⁶ to 10⁻⁴ substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10⁻⁵ s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1–21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature. RNA viruses show high rates of spontaneous mutation, a feature that profoundly influences viral evolution, disease emergence, the appearance of drug resistances, and vaccine efficacy. However, RNA virus mutation rates vary substantially and the factors determining this variability remain poorly understood. Here, we investigated the effects of host factors on viral replication fidelity by measuring the viral mutation rate in different cell types and under various culturing conditions. To carry out these experiments we chose the vesicular stomatitis virus (VSV), an insect-transmitted mammalian RNA virus with an extremely wide cellular and host tropism. We found that the VSV replication machinery was robust to changes in cellular physiology driven by cell immortalization or shifts in temperature and oxygen levels. In contrast, VSV mutated significantly more slowly in insect cells than in mammalian cells, a finding may help us to understand why arthropod-borne viruses tend to evolve more slowly than directly transmitted viruses in nature.

There is consensus that biodiversity losses will result in declining ecosystem functioning if species have different functional traits. Phylogenetic diversity has recently been suggested as a predictor of ecosystem functioning because it could approximate the functional complementarity among species. Here we describe an experiment that takes advantage of the rapid evolutionary response of bacteria to disentangle the role of phylogenetic and species diversity. We impose a strong selection regime on marine bacterial lineages and assemble the ancestral and evolved lines in microcosms of varying lineage and phylogenetic diversity. We find that the relationship between phylogenetic diversity and productivity is strong for the ancestral lineages but brakes down for the evolved lineages. Our results not only emphasize the potential of using phylogeny to evaluate ecosystem functioning, but also they warn against using phylogenetics as a proxy for functional diversity without good information on species evolutionary history.

Genome sizes and mutation rates covary across all domains of life. In unicellular organisms and DNA viruses, they show an inverse relationship known as Drake’s rule. However, it is still unclear whether a similar relationship exists between genome sizes and mutation rates in RNA genomes. Coronaviruses, the RNA viruses with the largest genomes (∼30 kb), encode a proofreading 3′ exonuclease that allows them to increase replication fidelity. However, it is unknown whether, conversely, the RNA viruses with the smallest genomes tend to show particularly high mutation rates. To test this, we measured the mutation rate of bacteriophage Qβ, a 4.2-kb levivirus. Amber reversion-based Luria–Delbrück fluctuation tests combined with mutant sequencing gave an estimate of 1.4 × 10⁻⁴ substitutions per nucleotide per round of copying, the highest mutation rate reported for any virus using this method. This estimate was confirmed using a direct plaque sequencing approach and after reanalysis of previously published estimates for this phage. Comparison with other riboviruses (all RNA viruses except retroviruses) provided statistical support for a negative correlation between mutation rates and genome sizes. We suggest that the mutation rates of RNA viruses might be optimized for maximal adaptability and that the value of this optimum may in turn depend inversely on genome size.

Biodiversity is thought to provide insurance for ecosystem functioning under heterogeneous environments; however, such insurance potential is under serious threat following unprecedented loss of biodiversity. One of the key mechanism underlying ecological insurance is that niche differentiation allows asynchronous responses to fluctuating environments, although the role of different ecological strategies (e.g., specialists vs. generalists) has yet to be formally evaluated. We present here a simple experimental study that illustrates how different ecological strategies (i.e., generalists vs. specialists) can shape the biodiversity-insurance relationship. We assembled microcosm of generalists and specialist bacteria over a gradient of salinity and found that, bacterial communities made up of generalists were more productive and more stable over time under environmental fluctuations. We discuss our results in context with simple theoretical predictions and propose future directions for biological insurance theory. We argue that beyond species richness itself, it is essential to incorporate the distribution of ecological strategies across relevant environmental gradients as predictors of the insurance potential of biodiversity in natural ecosystems.