Villena-Gimenez, A.; Castiglioni, V. G.; Elena, S. F.

Background: Environmental conditions shape the evolutionary trajectories of RNA viruses, yet little is known about how complex physical stressors such as microgravity influence host-virus interactions and viral evolution. Here, we investigated the short-term evolutionary consequences of simulated microgravity on the Caenorhabditis elegans - Orsay virus (OrV) system. Methods: OrV was subjected to six serial passages in hosts acclimated to low-shear modeled microgravity, with parallel evolution under standard-gravity. Evolutionary outcomes were evaluated using virulence, transmission, and replication traits, all measured under standard-gravity conditions. Results: Viral load fluctuated across passages in both environments, with lower mean accumulation in microgravity-evolved lineages. After evolution, we detected no significant changes in virulence. Transmission increased in standard-gravity lineages but not in microgravity-evolved ones, while viral replication decreased in all lineages, with a stronger decline in those evolved under microgravity. However, the magnitude of phenotypic changes was generally modest. Discussion: These results indicate that evolution under microgravity can alter viral phenotypic trajectories over short timescales. However, because all traits were assayed under standard-gravity conditions, we cannot directly assess local adaptation to microgravity, and the observed differences may reflect environment-specific trade-offs rather than reduced fitness per se. Furthermore, the limited number of passages and the modest magnitude of phenotypic change suggest that evolutionary responses may still be in an early stage. Conclusion: Overall, our findings provide initial evidence that simulated microgravity can influence the evolutionary dynamics of an RNA virus, while highlighting the need for reciprocal fitness assays and longer-term experiments to fully characterize adaptation to altered gravitational environments.