Intrahost population dynamics of chikungunya virus in humans and naturally infected

RNA viruses exhibit a high mutation rate, contributing to their genetic diversity mainly because their RNA polymerase lacks proofreading ability. Arboviruses, which alternate between vertebrate and invertebrate hosts, are subjected to host-specific selective pressures and population bottlenecks, mainly within mosquito vectors. Although experimental studies have brought insights into their evolutionary dynamics, data from naturally infected vectors remain limited. Here, we investigated the intrahost genetic diversity of chikungunya virus (CHIKV-ECSA lineage) through whole-genome sequencing of 19 human- and 19 mosquito-derived genomes from the 2024 outbreak in São José do Rio Preto, Brazil. Our principal component analysis revealed a greater mutation number in mosquito-derived genomes, predominantly driven by low-frequency and unique variants. Overall, intrahost genetic diversity was significantly higher in mosquito-derived than in human-derived CHIKV genomes, and protein-coding regions showed host-specific patterns. We identified 303 mutations across all CHIKV genomes. Interestingly, shared mutations were predominantly classified as synonymous, whereas unique mutations were mainly nonsynonymous. Gene-wide selection analyses indicated that purifying selection predominates across CHIKV genomes from both humans and mosquitoes, suggesting that most mutations, particularly nonsynonymous ones, are deleterious and subject to purifying selection. However, in mosquito-derived CHIKV genomes, evidence of relaxed purifying selection and neutral evolution, in specific proteins, such as E3 and NSP3, respectively, was observed, in contrast to the stronger purifying selection observed in human-derived CHIKV sequences. Site-specific selection analyses corroborated these results, detecting negatively selected sites in human-derived genomes but not in mosquito-derived genomes for these specific proteins. Together, our results show that these host-specific differences enable mosquitoes to act as reservoirs of genetic diversity by maintaining nonsynonymous variants, likely driven by genetic drift. At the same time, human hosts may impose stronger selective pressures, contributing to preserving the genome stability. This dynamic balance between diversification in vector populations and selective constraints in vertebrate hosts likely drives CHIKV evolution and adaptation.