Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses. The reporter viruses can infect known susceptible mammalian cell lines and primary CD14+ human monocytes. This design can accommodate several fluorescent protein genes, enabling the generation of multi-color DENV2-16681 reporter viruses with comparable replication capabilities, as demonstrated by their abilities to maintain their fluorescent intensities during co-infections and to exclude superinfections regardless of the fluorescent tags. The reported design of multi-color DENV2 should be useful for high-throughput analyses, single-cell analysis, and characterizations of interference and superinfection in animal models.

Reporter病毒是一种可视化和分析病毒感染的多功能工具。然而，对于黄病毒，很难将插入的报告基因维持在病毒基因组上，从而限制了其在需要均一病毒颗粒和几轮病毒复制的研究中的应用。在这里，我们显示了在两侧插入有跳跃核糖体2A序列的GFP基因的侧面，提高了登革病毒血清型2（DENV2）报告病毒的稳定性和荧光信号的一致性。该报告病毒可以感染已知的易感哺乳动物细胞系和原代CD14 +人单核细胞。这种设计可以容纳多个荧光蛋白基因，从而能够产生具有可比复制能力的多色DENV2-16681报告病毒，如其在共感染期间保持其荧光强度并排除超级感染的能力所证明的，而与荧光标签无关。报道的多色DENV2设计应可用于高通量分析，单细胞分析以及动物模型中干扰和超感染的表征。