Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
Institute of Anatomy, Faculty of Veterinary Medicine, Justus-Liebig University, Giessen, Germany.
Sci Rep. 2019 Apr 12;9(1):5972. doi: 10.1038/s41598-019-42540-z.
Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDV). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDV particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDV as a tool to study the dynamics of the whole life cycle of BVDV in real time.
病毒的荧光标记遗传允许实时研究它们的生命周期，而不需要固定或染色技术。在黄病毒科中，存在非结构蛋白的遗传标记选择。然而，到目前为止，还没有提出用于标记结构蛋白的系统。利用结构蛋白 E2 内的一个先前描述的位点，将荧光团引入细胞病变性（cpe）BVDV-1 病毒（BVDV）中。这种插入被很好地耐受，与亲本病毒相比，滴度下降了 2 倍，并且在超过 10 个传代中稳定地整合到基因组中。通过 Western blot 和荧光显微镜很容易检测到纯化病毒粒子中的荧光标记 E2 融合蛋白，并且通过冷冻电子显微镜确认了颗粒的完整性和形态。相同的整合位点也可用于标记相关的古典猪瘟病毒。此外，荧光标记的 CD46 表达细胞上结合的 BVDV 颗粒可以在荧光显微镜下分辨出来。这强调了 BVDV 作为一种工具，用于实时研究 BVDV 整个生命周期动态的适用性。
