Multi-platform profiling reveals host- and cell -type-specific pseudorabies virus gene expression.

Pseudorabies virus (PRV) is an alphaherpesvirus that follows a conserved immediate-early → early → late transcriptional cascade, yet how this program adapts to diverse cell types is unclear. We profiled PRV transcription in four permissive cell lines-two epithelial (porcine kidney PK-15 and rat kidney NRK), one glial (rat glioma C6), and one neuron-like (rat PC-12)-at six time points (1-12 h post-infection). Host-dependent differences peaked early in infection, particularly for the regulators ie180, ep0, and us1. ie180 showed strong species bias, with high expression in PK-15 but minimal in rodent lines, whereas ep0 and us1 varied quantitatively by cell type, with C6 and PC-12 showing marked early us1 activation. Combining long-read direct cDNA and direct RNA sequencing with 5'-capped CAGE-seq resolved viral transcription boundaries and identified 94 previously unannotated transcripts, including 5' UTR isoforms, polygenic RNAs, and noncoding transcripts. Differential transcript usage analysis revealed extensive isoform remodeling across conditions, with late infection showing shifts from long or polygenic isoforms toward shorter forms. Together, these results provide the first multi-host, isoform-resolved temporal atlas of PRV transcription and show that the canonical cascade is conserved yet quantitatively tuned by host species and cell-type background.