National Center for Genetic Engineering and Biotechnology, Klong Luang, Pathumthani, Thailand.
PLoS One. 2018 Mar 16;13(3):e0194399. doi: 10.1371/journal.pone.0194399. eCollection 2018.
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.
报告病毒是可视化和分析病毒感染的通用工具。然而，对于黄病毒，将插入的报告基因维持在病毒基因组上很困难，这限制了其在需要同质病毒颗粒和多个轮次病毒复制的几项研究中的应用。在这里，我们表明，在登革热病毒血清型 2（DENV2）报告病毒中，在插入的 GFP 基因两侧侧翼插入核糖体跳跃 2A 序列可提高其荧光信号的稳定性和一致性。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。该设计可以容纳多个荧光蛋白基因，从而能够生成具有可比复制能力的多色 DENV2-16681 报告病毒，正如它们在共感染期间维持荧光强度的能力以及无论荧光标记如何都能排除超感染所证明的那样。所报道的多色 DENV2 的设计应该对高通量分析、单细胞分析以及在动物模型中对干扰和超感染的特性分析有用。
