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

Yellow fever virus, a prototypic member of the Flaviviridae family, is a small single-stranded, positive-sense, enveloped RNA virus that causes viscerotropic and frequently fatal disease. Serial passaging of the virulent YFV isolate Asibi in the 1930s yielded the YFV17D (17D) vaccine strain, which remains one of the most effective vaccines ever developed. Remarkably, 17D 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 contribute to virulence vs attenuation. Here, we demonstrate that these mutations do not induce major RNA structure changes and protein sequence mutations are mostly responsible for the drastic phenotypic differences between 17D and virulent YFV. Using a highly modular, combinatorial genetic approach, we identified key mutations in the envelope (E) and non-structural 2A (NS2A) proteins that increase 17D's ability to spread and enhance host antiviral responses. Introducing these mutations into infectious clones of virulent YFV genomes results in viral attenuation in vitro and in vivo. Collectively, our results define the genetic basis for 17D attenuation and highlight a general approach for creating live-attenuated vaccines for other pathogenic viruses.