Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines.

中文翻译：

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通过密码子优化进行的黄病毒基因组重新编码可在小鼠和蚊子中实现遗传稳定的体内减毒。

病毒基因组重新编码是一种减毒方法，通过重写具有大量沉默突变的病毒基因组来实现遗传稳定的减毒。之前的黄病毒基因组重新编码尝试利用了密码子去优化方法。然而，这些密码子去优化方法以物种依赖性方式起作用，并且无法在蚊子细胞或蚊子动物模型中赋予黄病毒减毒作用。为了克服这些限制，我们使用相反的密码子优化方法进行黄病毒基因组重新编码。2 型登革热病毒 (DENV2) 和寨卡病毒 (ZIKV) 等黄病毒的基因组含有调节病毒复制的功能性 RNA 元件。我们假设通过密码子优化进行黄病毒基因组重新编码会引入破坏这些 RNA 元件的沉默突变，导致复制效率降低和衰减。我们选择DENV2和ZIKV作为代表黄病毒，并通过密码子优化它们的基因组来重新编码它们以供人类表达。我们的研究证实，这种密码子优化的重新编码方法确实会降低哺乳动物、人类和蚊子细胞的复制效率，以及小鼠和蚊子的体内衰减。计算机模拟和 RNA SHAPE 分析证实 DENV2 重新编码导致基因组结构元件的广泛破坏。重新编码的DENV2的连续传代导致救援或适应突变的出现，但没有回复突变。这些挽救突变无法挽救重新编码的DENV2的延迟复制动力学和体内衰减，这表明重新编码赋予遗传稳定的衰减。因此，我们的重新编码方法是一种可靠的减毒方法，具有开发黄病毒疫苗的潜在应用。