Abstract
Like other arthropod-borne viruses (arboviruses), mosquito-borne dengue virus (DENV) is maintained in an alternating cycle of replication in arthropod and vertebrate hosts. The trade-off hypothesis suggests that this alternation constrains DENV evolution because a fitness increase in one host usually diminishes fitness in the other. Moreover, the hypothesis predicts that releasing DENV from host alternation should facilitate adaptation. To test this prediction, DENV was serially passaged in either a single human cell line (Huh-7), a single mosquito cell line (C6/36), or in alternating passages between Huh-7 and C6/36 cells. After 10 passages, consensus mutations were identified and fitness was assayed by evaluating replication kinetics in both cell types as well as in a novel cell type (Vero) that was not utilized in any of the passage series. Viruses allowed to specialize in single host cell types exhibited fitness gains in the cell type in which they were passaged, but fitness losses in the bypassed cell type, and most alternating passages, exhibited fitness gains in both cell types. Interestingly, fitness gains were observed in the alternately passaged, cloned viruses, an observation that may be attributed to the acquisition of both host cell–specific and amphi-cell-specific adaptations or to recovery from the fitness losses due to the genetic bottleneck of biological cloning. Amino acid changes common to both passage series suggested convergent evolution to replication in cell culture via positive selection. However, intriguingly, mutations accumulated more rapidly in viruses passed in Huh-7 cells than in those passed in C6/36 cells or in alternation. These results support the hypothesis that releasing DENV from host alternation facilitates adaptation, but there is limited support for the hypothesis that such alternation necessitates a fitness trade-off. Moreover, these findings suggest that patterns of genetic evolution may differ between viruses replicating in mammalian and mosquito cells.
Highlights
Arthropod-borne RNA viruses pose an especially high risk of emergence from reservoir hosts into humans [1,2], due to the genetic plasticity of the viral RNA genome coupled with the dispersal potential of arthropod vectors and some amplification hosts
Recent studies suggest that the rate of dengue virus (DENV) evolution has accelerated, as well as the disease severity, most likely driven by the increased transmission and selection by the vector, Aedes aegypti, over the last 50 years
Despite the nearly global impact of DENV, little is known about the forces that influence its host range evolution, a deficiency confounded by the absence of a practical vertebrate model
Summary
Arthropod-borne RNA viruses (arboviruses) pose an especially high risk of emergence from reservoir hosts into humans [1,2], due to the genetic plasticity of the viral RNA genome coupled with the dispersal potential of arthropod vectors and some amplification hosts. Vector-borne RNA viruses undergo substantially slower rates of evolution (by a factor of ten) than many of their directly-transmitted counterparts [3,4,5] To explain this disparity, the trade-off hypothesis postulates that alternating replication in vertebrate and arthropod hosts constrains arbovirus evolution, because a fitness increase in one host usually diminishes fitness in the other. Ross River Virus, an alphavirus, showed phenotypic stability during alternating passages in Aedes aegypti mosquitoes and mice but increased in neurovirulence after serial passage in mouse brains, suggesting that host alternation restrains the evolution of virulence [6,7] In another alphavirus, Venezuelan equine encephalitis (VEEV), lineages passaged 10 times in Ae. aegypti exhibited an increase in mosquito infectivity relative to viruses passaged in alternation between rodents and mosquitoes, while rodent-specialized strains produced higher viremias in rodents relative to alternately-passaged virus. Both serially-passaged VEEV lines exhibited fitness declines in the bypassed host, while lineages passaged in mosquitoes and rodents in alternation demonstrated no detectable fitness gains, or losses, in either mosquitoes or vertebrates [8]
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