Herpesvirus glycoproteins play dominant roles in the initiation of infection of target cells in culture and thus may also influence viral tropism in vivo. Whereas the relative contribution of several nonessential glycoproteins to neurovirulence and neurotropism of Pseudorabies virus (PrV), an alphaherpesvirus which causes Aujeszky's disease in pigs, has recently been uncovered in studies using viral deletion mutants, the importance of essential glycoproteins is more difficult to assess. We isolated an infectious PrV mutant, PrV-9112C2, which lacks the gene encoding the essential PrV glycoprotein B (gB) but stably carries in its genome and expresses the homologous gene of bovine herpesvirus 1 (BHV-1) (A. Kopp and T. C. Mettenleiter, J. Virol. 66:2754-2762, 1992). Apart from exhibiting a slight delay in penetration kinetics, PrV-9112C2 was similar in its growth characteristics in cell culture to wild-type PrV. To analyze the effect of the exchange of these homologous glycoproteins in PrV's natural host, swine, 4-week-old piglets were intranasally infected with 10⁶ PFU of either wild-type PrV strain Kaplan (PrV-Ka), PrV-9112C2, or PrV-9112C2R, in which the PrV gB gene was reinserted instead of the BHV-1 gB gene. Animals infected with PrV-Ka and PrV-9112C2R showed a similar course of disease, i.e., high fever, marked respiratory symptoms but minimal neurological disorders, and excretion of high amounts of virus. All animals survived the infection. In contrast, animals infected with PrV-9112C2 showed no respiratory symptoms and developed only mild fever. However, on day 5 after infection, all piglets developed severe central nervous system (CNS) symptoms leading to death within 48 to 72 h. Detailed histological analyses showed that PrV-9112C2R infected all regions of the nasal mucosa and subsequently spread to the CNS preferentially by the trigeminal route. In contrast, PrV-9112C2 primarily infected the olfactory epithelium and spread via the olfactory route. In the CNS, more viral antigen and significantly more pronounced histological changes resulting in more severe encephalitis were found after PrV-9112C2 infection. Thus, our results demonstrate that replacement of PrV gB by the homologous BHV-1 glycoprotein resulted in a dramatic increase in neurovirulence combined with an alteration in the route of neuroinvasion, indicating that the essential gB is involved in determining neurotropism and neurovirulence of PrV.