The molecular basis for the attenuation of the yellow fever 17D vaccine [wtYFV_RNAseq]

Yellow fever virus, a prototypical member of the Flaviviridae family, is a small single-stranded, positive sense enveloped RNA virus causing viscerotropic, frequently fatal disease. Serial passaging of the virulent YFV Asibi strain in the 1930s yielded the YFV17D strain which remains one of the most effective vaccines ever developed. Remarkably, YFV17D and the virulent parental genome differ only by 68 nucleotides leading to 32 amino acid changes. However, it remains largely unknown which of these sequence differences are responsible for attenuation. Here, we demonstrate that while synonymous mutations are highly conserved across pathogenic YFVs they do not contribute to the characteristic host responses elicited by 17D or virulent viruses. Using a highly modular combinatorial genetic approach we identified key genetic elements in the YFV envelope and non-structural 2A (NS2A) proteins that govern the virus’ ability to spread and suppress host responses.. Introducing these mutations into infectious clones of other YFV genomes results in viral attenuation in vitro and in vivo. Collectively, our results define the genetic basis for YFV17D attenuation and highlight a general approach for creating live-attenuated vaccines for other pathogenic viruses.