Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neurovirulence in the olfactory pathway of the rabbit

Abstract

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEDelta), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295-310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEDelta and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.

FIG. 1

BHV-5 genomic structure and schematic maps of gE recombinant plasmids. The genomic organization of BHV-5 depicted at the top consists of unique long (U_L) and short (U_S) regions and two repeat regions (I_R) and (T_R). Localization of gE gene is indicated (A and B), and the region encompassing the gE gene (NotI/XhoI) is enlarged (C). A restriction site map of the NotI and XhoI fragment was generated, and various restriction endonuclease subfragments, indicated as bold lines, were cloned and sequenced. The arrow represents the BHV-5 gE ORF, and the arrowhead shows the direction of transcription. The regions of interest are shown in the schematic structures of plasmids pBHV-5gEΔβ (D), pBHV-5gE1 (E), and pBHV-1gE5 (F).

FIG. 2

Comparison of the predicted amino acid sequence of the BHV-5 gE with the predicted amino acid sequences of BHV-1.1 and BHV-1.2 gEs (37, 49). The predicted amino acid sequences of BHV-1 and BHV-5 gEs were aligned by using the GCG Gap program. Eleven conserved cysteine residues are marked by boxes, and potential N-linked glycosylation sites are indicated (•••). The presumptive signal sequences and the transmembrane anchor sequences are shown by solid and broken lines, respectively. Peptide sequences selected for raising rabbit antibodies are indicated by rectangular boxes. The YXXL motif sequences are indicated (▾▾▾), and the acidic domain is highlighted.

FIG. 3

Immunoblotting analysis of recombinant viruses. Recombinants and the parental viruses were purified partially by ultracentrifugation through a 30% sucrose cushion as described earlier (15). SDS-PAGE and immunoblot analysis of mock-infected cell lysates and purified virion proteins was performed under reducing conditions as described earlier (12). (A) Coomassie blue-stained SDS-polyacrylamide gel containing lysates of cells infected with BHV-5, BHV-5gEΔ, BHV-5 gE revertant (BHV-5gE-R), BHV-1 gE5, BHV-5gE1, and BHV-1 and of mock-infected MDBK cells. (A′) Immunoblotting of the same proteins with BHV-5 gE-specific antipeptide rabbit polyclonal serum. (B) Coomassie blue-stained SDS-polyacrylamide gel containing lysates of cells infected with BHV-5, BHV-5gEΔ, BHV-5 gE-R, BHV-1 gE5, BHV-5gE1, BHV-1, and BHV-1 gEΔ (15) and of mock-infected MDBK cells. (B′) Immunoblotting of the same proteins with BHV-1 gE-specific antipeptide rabbit polyclonal serum.

FIG. 4

Immunoprecipitation, SDS-PAGE, and Western blot analyses of gE and gI complex formation in BHV-5 and BHV-1 gE recombinants. Mock- and virus-infected MDBK cells were labeled with [³⁵S]methionine-cysteine for 18 h. Detergent extracts of infected cells were prepared and immunoprecipitated with BHV-5 gE-specific (A), BHV-1 gE-specific (B), or BHV-1 gI-specific (D) polyclonal rabbit serum. After SDS-PAGE, the precipitated proteins were analyzed by immunoblotting with BHV-1 gI-specific (C) or BHV-1 gE-specific (E) rabbit sera. Panels A, B, and D show autoradiography of labeled proteins. The positions of molecular size markers, gE, and gI are indicated. White asterisks in panels C and E indicate heavy chain of rabbit immunoglobulin G.

FIG. 5

One-step growth curve of recombinants and parental viruses in MDBK cells. Confluent MDBK cells were infected with viruses at an MOI of 5 PFU per cell. After 1 h of adsorption at 4°C, residual input viruses were removed. The cultures were washed three times with phosphate-buffered saline, and 5 ml of medium was added to each flask before further incubation (37°C). At indicated time intervals, replicate cultures were frozen. Virus yields were determined by titration on MDBK cells. Each data point represents the average of duplicate samples obtained from separate infections.

FIG. 6

Plaque morphology of recombinant viruses in MDBK cell monolayer. BHV-1 (A), BHV-5 (B), BHV-1gE5 (C), BHV-5gEΔ (D), BHV-5gE1 (E), and BHV-5gE revertant (F) were inoculated onto MDBK cell monolayers, fixed after 30 h postinfection, and immunostained with bovine anti-BHV-5 serum (12).

FIG. 7

Localization of viral antigen in brain sections. Animals were inoculated intranasally with either wild-type BHV-5, BHV-5gEΔ, or BHV-5gE1 as described in Materials and Methods. The animals were euthanized on days 2, 4, 6, 8, 10, and 12 days postinfection or when they showed neurological signs, and their brains were processed for immunohistochemical analysis as described in Materials and Methods. Representative sections of the olfactory bulb (A), anterior olfactory nucleus (B), and piriform cortex (C) are pictured. In this assay, wild-type BHV-5 spread to the olfactory bulb at 4 to 6 dpi; however, labeling in the bulb was first observed at 8 and 6 dpi for the gE-deleted and gE-exchanged BHV-5, respectively. Wild-type BHV-5 spread to the anterior olfactory nucleus and piriform cortex at 8 dpi. gE-deleted and gE-exchanged BHV-5 took 10 to 12 dpi to spread to these areas. Bar in panel A, 100 μm; bar in panels B and C, 1,000 μm.

FIG. 8

Localization of viral antigen in representative sections showing the amygdala, hippocampus/dentate gyrus, and cingulate cortex. In this assay, wild-type BHV-5 spread to amygdala (A), hippocampus/dentate gyrus (B), and cingulate cortex (C). However, BHV-5gEΔ spread only up to piriform cortex (12 dpi) and was never found in these areas. In BHV-5gE1-infected animals, a few neurons in the amygdala and several neurons in the cingulate cortex were labeled at 12 dpi, but no labeling was found in the hippocampus/dentate gyrus.