Both viral and host factors contribute to neurovirulence of bovine herpesviruses 1 and 5 in interferon receptor-deficient mice

Abstract

Herpes simplex virus (HSV) type 1 and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5) can use the same cellular receptor for entry, but only HSV is known to cause disease in mice. We hypothesized that components of either the innate or the adaptive immune system, or a combination of both, were responsible for curbing replication of BHVs in mice. Therefore, wild-type mice as well as mice with various combined genetic deficiencies in the alpha/beta interferon receptor or gamma interferon receptor and in the ability to produce mature B and T lymphocytes (RAG-2 deletion) were infected with BHV-1 and BHV-5 and monitored clinically, serologically, histopathologically, and virologically. A functional immune system protected the mice from disease and death due to BHV infection, and the immune response was Th1 like. BHV-5 was transported to the central nervous system by the axonal pathway, whereas viremia was required for this outcome with BHV-1. The alpha/beta interferon system was able to obstruct quantitative spread of the viruses in the infected organism. The gamma interferon system had a protective effect against BHV-1, even in mice with the RAG-2 deletion. In contrast, the same mice succumbed to neurological disease and death upon infection with BHV-5. Productively infected neurons were detected only in BHV-5-infected mice with an intact gamma interferon system. We conclude that the alpha/beta interferon system had a protective effect, while an intact gamma interferon system was required for efficient replication of BHV-5 in mouse neurons and for the development of neurological disease.

FIG.1.

Serological immune response of mice inoculated with either BHV-5 (a to l) or BHV-1 (m to x). Individual ELISA titers of antibodies against BHV-1 are shown (y axis), each at three different occasions (x axis), i.e., at dpi 6, 16, and 43. Each row represents an experiment in which a specific mouse strain was infected with either BHV-5 or BHV-1. (a to d and m to p) Wild-type mice; (e to h and q to t) A129 mice; (i to l and u to x) AG129 mice. Each column compares the outcome of the different experimental settings with regard to isotype(s) of immunoglobulin measured. First column, total Ig; second column, IgG2a; third column, IgG2b; fourth column, IgG1. Note that the scaling of the y axis is identical in the first three columns but differs in the fourth.

FIG. 2.

Quantitative time course of viral DNA in blood, brain, and liver of AGR129 (A) and AR129 (B) mice inoculated with BHV-5. Viral DNA copy numbers per 1,000 cells (y axis) detected by quantitative real-time PCR in the various tissues collected at various time points after inoculation (x axis) are shown.

FIG. 3.

Quantitative time course of viral DNA in blood, brain, and liver of AGR129 (A) and AR129 (B) mice inoculated with BHV-1. Viral DNA copy numbers per 1,000 cells (y axis) detected by quantitative real-time PCR in the various tissues collected at various time points (x axis) after inoculation are shown.

FIG. 4.

Analysis of mouse brains from BHV-1- and BHV-5-infected mice by histology, in situ hybridization, and immunohistology. AR129 (A to F) and AGR129 (G to I) mice were inoculated with BHV-1 or BHV-5 as indicated. At intervals, groups of mice were euthanatized to be analyzed at various time points after infection. (A, D, and G) Examples of histological examination following hematoxylin and eosin (HE) staining at dpi 22 (A) or 16 (D and G), where no differences between various time points were recognized. (B, E, and H) Examples of results after in situ hybridization at dpi 22 (B), 16 (E) (arrows point to positive neuronal nuclei), or 11 (H). Viral DNA was not present at detectable levels in brain tissue collected from BHV-1-inoculated mice or in BHV-5-infected mice, before the onset of clinical symptoms (an example is shown in panel B). BHV-5 DNA detected by in situ hybridization is shown in panels E and H (arrows point to positive neuronal nuclei). (C, F, and I) Examples from immunohistological analyses at dpi 22 (C), 16 (F), and 11(I). Sections from BHV-1-inoculated mice and from BHV-5-infected mice, before the onset of clinical symptoms, reacted negatively (an example is shown in panel C). Structural glycoprotein C was detected by immunohistology following monoclonal antibody staining of brains from AR129 mice at dpi 16 after inoculation with BHV-5 (F) (the inset shows a positive neuron at a higher magnification) and from AGR129 mice at dpi 11 after BHV-5-infection (I) (the arrow points to a weakly positive cell that cannot be clearly identified as a neuron). Bars, 100 μm.