Glycoprotein D of bovine herpesvirus 5 (BoHV-5) confers an extended host range to BoHV-1 but does not contribute to invasion of the brain

Abstract

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.

FIG. 1.

Phenotyping of parent, mutant, and rescue viruses by fluorescence microscopy. MDBK cells were infected with various viruses to be analyzed at 72 hpi. The top two rows show plaques of BAC-derived rBoHV-5 and its mutants, whereas the bottom two rows show BAC-derived rBoHV-1 and its mutants. The top row shows green fluorescent plaques of rBoHV-5 (A), rBoHV-5gD1HA (B), and rrBoHV-5gD5V5 (C). In the second row, the same dishes were stained with mcAbs and a red fluorescent Cy3 conjugate (mcAb against the HA tag [A′ and B′] and mcAb against the V5 tag [C′]). In the third row, the following fluorescent plaques are shown: green fluorescent rBoHV-1 (D), rBoHV-1gD5V5 (E), and yellow fluorescent rrBoHV-1gD1EYFP (F). In the fourth row (D′ and E′), the same viruses as those in the third row were stained with mcAb against V5 and a red fluorescent Cy3 conjugate.

FIG. 2.

Growth kinetics of recombinant BoHV-5 mutants and recombinant BoHV-1 mutants versus wt BoHV-5 and wt BoHV-1. MDBK cells were infected at an MOI of 0.01 with different viruses and harvested at various times postinoculation. The virus yields at each time point were determined by titration. (A and B) rBoHV-5 (full squares), rBoHV-5gD1 (open squares), rBoHV-1 (full circles), and rBoHV-1gD5 (open circles). (C and D) wt BoHV-5 (full squares), rrBoHV-5gD5 (open squares), wt BoHV-1 (full circles), and rrBoHV-1gD1 (open circles). Cell-free virus (supernatant) (A and C) and cell-associated virus (pellets) (B and D) were titrated separately. The x axis represents the time scale postinfection. Virus titers (y axis) are expressed as TCID₅₀/ml. Mean values with standard deviations (error bars) are shown.

FIG. 3.

Type-specific gD determines the host range of BoHV-1 and BoHV-5. J cells were mock infected (top row) or infected at an MOI of 5 with BoHV recombinants, all of which express the green fluorescent marker upon successful infection and replication. At 72 hpi, cells were fixed and stained with monoclonal antibodies and a secondary antibody carrying Cy3. (A and B) Mock-infected cells neither developed plaques (A) nor provided any green or red fluorescence (B). (C and D) rBoHV-1 neither gave rise to green fluorescence (C) nor provided red fluorescence (D) due to Cy3 conjugate binding to mcAb against V5. (E and F) rBoHV-5 caused the development of green fluorescent plaques (E) but did not bind mcAb against HA (F). (G and H) rBoHV-1gD5V5 caused green fluorescent plaque formation (G) and the binding of mcAb against V5 (H). (I and J) rBoHV-5gD1HA provided single green fluorescent cells (I), which also stained positive with mcAb against the HA tag (J).

FIG. 4.

Inhibition of infection by preincubation with soluble nectins. Relative infectivity rates are shown. Viruses were incubated with soluble proteins as described in Materials and Methods before being inoculated onto monolayers of J cells. Fluorescent cells were counted at 48 hpi. Fluorescent cell numbers emerging from virus incubated with 200 nM BSA were set as 100% (bars measured on the y axis). The identities of the proteins used for coincubation with virus stocks are indicated beneath each pair of bars. Each experiment was done at least in triplicate (mean values are shown, and the error bars represent the standard errors of the means).

FIG. 5.

Survival times of AR129 mice upon infection with different BoHVs. Groups of 7 to 10 mice were inoculated intraperitoneally with 10⁷ TCID₅₀ of virus and checked twice daily for their health status. The mice were euthanized upon the development of severe disease symptoms. The x axis gives the survival times postinoculation. The y axis gives the percentage of mice in each group at any given time point. The symbols used for each virus are given at the right.

FIG. 6.

DNA of BoHV-5 and BoHV-5gD1 in brain tissue. DNA was extracted from the brains of each animal and subjected to real-time PCR and quantification of the viral DNA load per cell as described in Materials and Methods. The viruses used for inoculation are listed on the x axis. Box plots refer to the y axis and show mean values, the 75th percentile (box), as well as the maximal and minimal values (whiskers).

FIG. 7.

Histology (HE) and immunohistology of brain sections. (A) HE staining of a section of mouse brain from an animal inoculated with BoHV-1. This picture is representative of all sections analyzed independent of the inoculated virus. (B to D) Immunohistology using a monoclonal antibody recognizing gCs of both BoHV-1 and BoHV-5. (B) Negative brain section of a mouse inoculated with BoHV-1. This picture is representative of all brain sections from mice inoculated with either BoHV-1, BoHV-1gD5, or rrBoHV-1. (C) Representative picture of a brain section from a mouse inoculated with rBoHV-5gD1. This picture is also representative of animals inoculated with BoHV-5. Neurons in the brain stem area show positive intracytoplasmic staining (red cytoplasmic staining). (D) Cutout of the section outlined in C. Bar, 100 μm.