Evolution of subgenomic RNA shapes dengue virus adaptation and epidemiological fitness

Genetic changes in the dengue virus (DENV) genome affects viral fitness both clinically and epidemiologically. Even in the 3 untranslated region (3UTR), mutations could impact the formation of subgenomic flaviviral RNA (sfRNA) and the specificity of sfRNA in inhibiting host proteins necessary for successful viral replication. Indeed, we have recently shown that mutations in the 3UTR of DENV2 affected its ability to inhibit TRIM25 E3 ligase activity to reduce interferon (IFN) expression, which potentially contributed to the emergence of a new viral clade during the 1994 dengue epidemic in Puerto Rico. However, whether differences in 3UTRs shaped DENV evolution on a larger scale remains incompletely understood. Herein, we combined RNA phylogeny with phylogenetics to gain insights on sfRNA evolution. We found that sfRNA structures are under purifying selection and highly conserved despite sequence divergence. Interestingly, only the second flaviviral Nuclease-resistant RNA (fNR2) structure of DENV-2 has undergone strong positive selection. Epidemiological reports also suggest that nucleotide substitutions in fNR2 may drive DENV-2 epidemiological fitness, possibly through sfRNA-protein interactions. Collectively, our findings indicate that 3UTRs are important determinants of DENV fitness in human-mosquito cycles.\n\nHighlightsO_LIDengue viruses (DENV) preserve RNA elements in their 3 untranslated region (UTR).\nC_LIO_LISite-specific quantification of natural selection revealed positive selection on DENV2 sfRNA.\nC_LIO_LIFlaviviral nuclease-resistant RNA (fNR) structures in DENV 3UTRs contribute to DENV speciation.\nC_LIO_LIA highly evolving fNR structure appears to increase DENV-2 epidemiological fitness.\nC_LI