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

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. Huh7 cells were infected with YFVs, treated with SHAPE chemical, 2-aminopyridine-3-carboxylic acid imidazolide (2A3), and subjected for selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP).

黄热病毒（Yellow fever virus, YFV）是黄病毒科（Flaviviridae）的典型成员，为小型有包膜单股正链RNA病毒，可引发嗜内脏性、常为致死性的疾病。1930年代，对强毒株YFV Asibi进行连续传代，获得了YFV17D毒株——该毒株至今仍是史上开发的最为有效的疫苗之一。值得注意的是，YFV17D与其强毒亲本基因组仅存在68个核苷酸差异，由此导致32个氨基酸位点的改变。然而，目前学界仍未明确这些序列差异中究竟哪些是病毒减毒的关键诱因。
本研究证实，尽管同义突变（synonymous mutation）在致病性黄热病毒中高度保守，但它们并未参与17D毒株或强毒株所引发的特征性宿主应答。本研究采用高度模块化的组合遗传学方法，在黄热病毒包膜蛋白与非结构蛋白2A（NS2A）中鉴定出了调控病毒扩散与宿主应答抑制能力的关键遗传元件。将这些突变引入其他黄热病毒基因组的感染性克隆后，可在体外与体内实现病毒减毒。综上，本研究明确了YFV17D减毒的遗传基础，并为开发其他致病性病毒的减毒活疫苗提供了通用策略。
研究人员将Huh7细胞感染黄热病毒后，使用SHAPE化学试剂2-氨基吡啶-3-羧酸咪唑化物（2A3）进行处理，并通过引物延伸突变谱分析（SHAPE-MaP）开展选择性2'-羟基酰化检测。