数字农科院2.0

Tracking of single virus: Dual fluorescent labeling of pseudorabies virus for observing entry and replication in the N2a cells

文献类型： 外文期刊

作者： Li, Mingzhi;Pan, Li;Ma, Caoyuan;Wu, Hongxia;Xiang, Guangtao;Li, Lian-Feng;Wang, Tao;Luo, Rui;Li, Yongfeng;Liu, Di;Zhai, Huanjie;Assad, Moon;Song, Xin;Wang, Yanjin;Gallardo, Franck;Qiu, Hua-Ji;Sun, Yuan

作者机构：

关键词： ANCHOR DNA labeling system;Envelope;Genome;Live-cell imaging;Pseudorabies virus

期刊名称： VETERINARY MICROBIOLOGY

ISSN： 0378-1135

年卷期： 2025 年 304 卷

页码：

收录情况： SCIE（2025版）

摘要： Pseudorabies virus (PRV) is a neurotropic herpesvirus. It is not easy to be track the whole replication progress of PRV, especially the nascent viral genome in the host cells. In this study, we developed a dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) with the viral genome and the envelope protein gM labeled by ANCHOR DNA labeling system and mCherry, respectively. Through single-virus tracking of rPRV-Anchor3-mCherry, we observed that PRV invaded mouse neuroblastoma Neuro-2a cells via both endocytosis and plasma membrane fusion pathway. During the replication stage, parental and progeny viral genome of rPRV-Anchor3-mCherry in the cell nuclei could be visible, and viral nucleocapsid appeared more specifically than traditional capsid protein labeled PRV particles (rPRV-VP26-EGFP). We found that numerous progeny viral particles were produced in the nuclear, causing the nucleus membrane to break using three-dimensional (3D) live-cell imaging and electron microscopy. Moreover, our findings confirmed that simultaneously targeting of the UL9 and UL54 genes using a CRISPR-Cas9 system led to the complete inhibition PRV replication. rPRV-Anchor3-mCherry can be used to research multiple steps of the viral cycle.

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