Pseudorabies virus expressing bovine herpesvirus 1 glycoprotein B exhibits altered neurotropism and increased neurovirulence

Abstract

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 10(6) 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.

FIG. 1

Clinical symptoms in pigs after infection with PrV-Ka and PrV-9112C2. Four piglets per group were infected intranasally with either PrV-Ka (⧫) or PrV-9112C2 (■). Every animal in each group was observed for symptoms of AD twice a day and assigned a clinical score correlating to five levels of severity as indicated in Materials and Methods. Average values and standard deviations of four pigs from each group are shown. (†) denotes death of animals.

FIG. 2

Virus excretion of pigs infected with PrV-Ka and PrV-9112C2. After intranasal infection of animals with PrV-Ka (⧫) or PrV-9112C2 (■), nasal swabs were taken daily and titrated on MDBK cells. Indicated are average titers and standard deviations in PFU per milliliter.

FIG. 3

Detection of PrV-infected cells in cryosections by indirect immunofluorescence with anti-gC MAb after infection with PrV-9112C2R (A to C) or PrV-9112C2 (D to F). (A) Regio cutanea, 3 dpi, PrV antigen-positive epithelial cells; magnification, ×240. (B) Ganglion trigeminale, 3 dpi, PrV antigen-positive perikarya, nerve fibers, and infiltrating cells; ×250. (C) Medulla oblongata, 2 dpi, small focus with PrV-positive perikarya and surrounding neuropil; ×375. (D) Regio cutanea, no PrV antigen detectable, 3 dpi; ×240. (E) Regio olfactoria, 3 dpi, PrV antigen-positive foci in the olfactory epithelium and logitudinal section of PrV-positive nerve fibers (arrow); ×250. (F) Bulbus olfactorius, 5 dpi, section of a prominent large focus within the granular layer containing PrV-positive perikarya, neuropil, and glial cells; ×240.

FIG. 4

Comparison of the number of encephalitic foci detected after infection with PrV-9112C2R (shaded columns) or PrV-9112C2 (black columns) between days 2 to 8 p.i. in different regions of the brain. Each column represents the total number of encephalitic lesions within 25 mm² per section and animal. M, medulla; B, bulbus; P, pedunculus.

FIG. 5

Histopathological alterations after infection with PrV-9112C2R (A to C) or PrV-9112C2 (D to F) visualized by hematoxylin-eosin staining. (A) Ganglion trigeminale, 2 dpi, ganglioneuritis, degeneration of perikarya, and focal infiltrations with macrophages and lymphocytes; magnification, ×200. (B) Medulla oblongata, 2 dpi, encephalitis, blood vessel with perivascular infiltrations (left) and prominent glia herds (right); ×200. (C) Bulbus olfactorius, 5 dpi, encephalitis, small vessels with perivascular infiltrates, and severe focal gliosis within the granular layer; ×100. (D) Ganglion trigeminale, 2 dpi, no inflammatory lesions at this time point; ×150. (E) B. olfactorius, 3 dpi, encephalitis, perivascular infiltrations within the glomerula olfactoria (bottom) and the molecular layer (top); ×240. (F) Pons, 6 dpi, encephalitis, severe perivascular infiltrates, and focal gliosis; ×100.