Cherkashchenko Liubov, Gros Nathalie, Trausch Alice, Neyret Aymeric, Hénaut Mathilde, Dubois Gregor, Villeneuve Matthieu, Chable-Bessia Christine, Lyonnais Sébastien, Merits Andres, Muriaux Delphine
CEMIPAI UAR3725 CNRS, University of Montpellier, Montpellier, France.
Institute of Technology, University of Tartu, Tartu, Estonia.
Front Microbiol. 2023 Sep 15;14:1201640. doi: 10.3389/fmicb.2023.1201640. eCollection 2023.
Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated using viruses that easily express detectable marker proteins. Therefore, developing molecular tools, such as reporter-carrying versions of flaviviruses, for studying viral replication and screening antiviral compounds represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods that include the design and validation of infectious clones of Zika, Kunjin, and Dengue viruses harboring different reporter genes for infection, rescue, imaging, and morphology using super-resolution microscopy. It was observed that different flavivirus constructs with identical designs displayed strikingly different genetic stabilities, and corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase the stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies.
黄病毒已成为主要的节肢动物传播病原体，在全球范围内构成日益严重的公共卫生问题。使用易于表达可检测标记蛋白的病毒可促进高通量筛选。因此，开发分子工具，如携带报告基因的黄病毒版本，用于研究病毒复制和筛选抗病毒化合物是当务之急。然而，由于标记插入导致的遗传不稳定性，构建携带荧光或发光报告基因的黄病毒仍然具有挑战性；因此，需要新的方法来克服这些限制。在这里，我们描述了反向遗传方法，包括设计和验证寨卡病毒、库京病毒和登革病毒的感染性克隆，这些克隆携带不同的报告基因，用于使用超分辨率显微镜进行感染、拯救、成像和形态学研究。观察到具有相同设计的不同黄病毒构建体表现出显著不同的遗传稳定性，相应的病毒粒子在形状和大小上类似于野生型病毒颗粒。评估了一种成功的策略，以提高拯救的报告病毒的稳定性，并基于定量自动荧光显微镜和复制研究进行抗病毒药物筛选。
