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

Vet Microbiol.2025 May:304:110503.doi: 10.1016/j.vetmic.2025.110503. Epub 2025 Apr 2.

Abstract

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.

Keywords: ANCHOR DNA labeling system; Envelope; Genome; Live-cell imaging; Pseudorabies virus.