Regional and Temporal Patterns of Long-Term Pseudorabies Virus Detection and Neuropathology in the Murine CNS

Alphaherpesviruses, including Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PrV), establish lifelong latency in the nervous system and can cause recurrent disease. While latency has classically been attributed to peripheral sensory ganglia, accumulating evidence indicates that the central nervous system (CNS) may also serve as a site of long-term viral persistence and reactivation. Here, we investigated the CNS as a viral reservoir using the attenuated mutant PrV-∆UL21/US3∆kin, which preferentially targets mesiotemporal brain regions. Following intranasal inoculation, mice were analyzed at 11–14, 21, 28, 42, 105, and 190 days post-infection (dpi). To assess the reactivation potential, a subset of animals received cyclophosphamide/dexamethasone at 170 dpi. Viral transcripts were detected by RNAscope™ in situ hybridization and RT-qPCR targeting the lytic gene UL19 encoding the major capsid protein and the latency-associated transcript (LAT). Histopathology included hematoxylin and eosin staining and immunohistochemistry for CD3, Iba1, GFAP, cleaved caspase-3 and viral glycoprotein gB. UL19 RNA signals displayed marked regional and temporal heterogeneity, with prominent detection in mesiotemporal structures. In contrast, LAT RNA levels remained low overall, with a transient peak during the acute phase. RT-qPCR confirmed high UL19 and LAT transcript levels during early infection, while LAT transcription returned to baseline levels thereafter. Histopathology showed a transition from acute necrotizing meningoencephalitis to prolonged low-grade inflammation with glial activation and focal apoptosis. Notably, UL19 RNA signals strongly correlated with T-cell infiltration, particularly at 42 dpi. Together, these findings define regional and temporal patterns of long-term PrV transcriptional activity and associated neuropathology in the murine CNS.