Spatial and temporal mapping of early alphaherpesvirus invasion routes into the mouse central nervous system

Alphaherpesviruses such as Herpes Simplex Virus 1 (HSV-1) and Pseudorabies virus (PrV) invade the central nervous system (CNS) via peripheral nerves. While olfactory and trigeminal pathways are well-known, additional cranial routes remain underexplored. Using a PrV-∆UL21gfp/US3∆kin mutant in CD1 mice, we mapped early neuroinvasion (4-96 hpi) by immunofluorescence and RNA in situ hybridization. Viral antigen and lytic viral gene expression (UL19 RNA) were detected in the olfactory epithelium, vomeronasal organ, incisors, palate, olfactory bulb, and brainstem. These results indicate multineural CNS access involving olfactory (I), trigeminal (V), glossopharyngeal (IX), and hypoglossal (XII) nerves, highlighting this model's value for studying early alphaherpesvirus spread.