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

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.