The 2014–2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

中文翻译：

---

单个氨基酸突变影响Zika病毒的体外复制和体内毒力。

2014-2016年在美洲的寨卡病毒（ZIKV）流行导致大量来自临床样品的下一代测序数据沉积。挖掘该资源是为了确定新出现的突变以及随着时间推移爆发的突变趋势。然后，基于宿主内突变体的传播动力学，患病率和持久性以及蛋白质上突变位置的信息，基于其影响ZIKV表型的能力，对544个已报告突变进行优先排序。使用此标准，使用2015年波多黎各流行病菌株PRVABC59作为亲本骨架，生成了6个突变体（代表自然发生的突变）作为合成传染性克隆。使用细胞培养和鼠模型系统检查了这些天然存在的变体的表型。突变体具有不同的表型，包括复制率变化，胚胎死亡和头部缩小。特别是，发现先前在恒河猴体内研究期间检测到的NS2B突变体在成年小鼠中引起致命感染，在雌性小鼠中流产，并在雌性小鼠的脑组织和血液中增加病毒基因组拷贝。此外，在复制过程中与NS5接触的NS3区域发生变化的突变体显示成年小鼠血液中复制的减少。这种分析途径结合了生物信息学和湿实验室实验，为理解自然发生的单一突变如何影响疾病结果提供了基础，并可用于预测未来ZIKV爆发的严重性。为了确定寨卡病毒流行基因型中的自然发生的个别突变是否在体外或体内影响病毒的毒力或复制率，我们生成了一个代表克隆波多黎各流行基因型的传染性克隆，2015年使用该克隆创建了六个突变体。改变基因组中的核苷酸以在编码的蛋白质中引起一到两个氨基酸取代。我们产生的六个突变体代表了将早期流行基因型与祖先或流行后期的基因型区分开的突变。我们测定了每个突变体的生长速率变化以及成年小鼠和妊娠小鼠的毒力。其中三个突变体造成了灾难性的胚胎影响，包括增加的胚胎死亡或头部直径的明显减少。在与复制相关的基因组区域中发生突变的其他三个突变体导致体外和体内复制速率发生变化。这些结果说明了病毒表型中个别突变的潜在影响。