Both spike and background genes contribute to murine coronavirus neurovirulence

Abstract

Various strains of mouse hepatitis virus (MHV) exhibit different pathogenic phenotypes. Infection with the A59 strain of MHV induces both encephalitis and hepatitis, while the highly neurovirulent JHM strain induces a fatal encephalitis with little, if any, hepatitis. The pathogenic phenotype for each strain is determined by the genetic composition of the viral genome, as well as the host immune response. Using isogenic recombinant viruses with A59 background genes differing only in the spike gene, we have previously shown that high neurovirulence is associated with the JHM spike protein, the protein responsible for attachment to the host cell receptor (J. J. Phillips, M. M. Chua, G. F. Rall, and S. R. Weiss, Virology 301:109-120, 2002). Using another set of isogenic recombinant viruses with JHM background genes expressing either the JHM or A59 spike, we have further investigated the roles of viral genes in pathogenesis. Here, we demonstrate that the high neurovirulence of JHM is associated with accelerated spread through the brain and a heightened innate immune response that is characterized by high numbers of infiltrating neutrophils and macrophages, suggesting an immunopathogenic component to neurovirulence. While expression of the JHM spike is sufficient to confer a neurovirulent phenotype, as well as increased macrophage infiltration, background genes contribute to virulence as well, at least in part, by dictating the extent of the T-cell immune response.

FIG. 1.

The genomes of recombinant MHVs. The genomes of parental recombinant strains, RJHM and RA59, as well as those of chimeric viruses with exchanges of spike genes, SJHM/RA59 and SA59/RJHM, are all shown. LD₅₀ values, following intracranial inoculation, are provided to demonstrate the contribution of the viral spike protein to the level of neurovirulence.

FIG. 2.

Survival rates following intracranial infection. C57BL/6 mice (10 per virus) were inoculated intracranially with 10 PFU of RJHM (squares), SJHM/RA59 (diamonds), RA59 (circles), or SA59/RJHM (triangles); monitored daily for mortality; and plotted as percentages of mice surviving as a function of days postinfection. The values presented are representative of three independent experiments.

FIG. 3.

Viral spread within the brain. C57BL/6 mice were infected with 10 PFU of virus, RJHM (A), SA59/RJHM (B), RA59 (C), or SJHM/RA59 (D), intracranially and sacrificed 5 days postinfection. Sagittal brain sections were prepared and stained by immunohistochemistry using a monoclonal antibody directed against viral nucleocapsid. Representative images are shown at ×1 magnification. Region identities: 1, olfactory bulb; 2, cerebral cortex; 3, thalamus; 4, hypothalamus; 5, hippocampus; 6, midbrain; 7, pons; 8, cerebellum; 9, medulla.

FIG. 4.

Quantification of NK cells, macrophages, and neutrophils in the CNSs of infected mice. At day 5 p.i., mononuclear cells were isolated from the brains of B6 mice infected with RJHM, SA59/RJHM, SJHM/RA59, and RA59. Cells from five mice per virus were pooled and analyzed for surface antigen expression by staining them with cell-type-specific antibodies—NK1.1 for NK cells (open bars), F4/80 for macrophages (shaded bars), and Ly-6G for neutrophils (closed bars)—followed by FACS analysis. The level of each cell type is presented both as (A) a percentage of total gated cells following the collection of 10,000 FACS events and (B) the total number of each cell type per brain. The values represent the means (A and B) and standard deviations (A) of at least five individual experiments per virus.

FIG. 5.

Infection with RJHM induces a strong neutrophil response in the brain. Lymphocytes pooled from the brains of infected mice sacrificed at days 5 (A to D) and 7 (E to H) p.i. (five per day for each virus) were stained with antibodies against CD45 and Ly-6G and analyzed by FACS. The numbers shown in the upper right-hand corners show the percentages of neutrophils among CNS lymphocytes as measured by staining them with both CD45 and Ly-6G. The images are representative of at least five independent experiments per virus.

FIG. 6.

The depletion of neutrophils or inhibition of their respiratory burst via treatment of mice with aminoguanadine reduces apoptosis levels in the CNSs of RJHM-infected animals. RJHM-infected B6 mice were treated with either PBS (A, D, and G), aminoguanadine (B, E, and H), or the neutrophil-depleting antibody RB6-8C5 (C, F, and I) from day −1 throughout the infectious course until day 5 p.i., at which time the mice were sacrificed, all as described in Materials and Methods. Frozen sagittal brain sections were prepared and evaluated for viral antigen by immunofluorescent staining with polyclonal anti-viral antibody (A, B, and C), for infiltrating neutrophils by staining them with anti-Ly6G (D, E, and F), and for apoptosis by the degree of TUNEL staining, as described in Materials and Methods (G, H, and I). Staining for viral antigen and neutrophils was performed on the same section, while TUNEL staining was performed on an adjacent section. The sections shown are representative of two sections per animal from a total of three animals evaluated for each treatment.

FIG. 7.

Evaluation of the T-cell response following MHV infection. Total lymphocytes isolated from the brains of five infected animals per virus were pooled for analysis of surface antigen expression via FACS. The numbers of CD8⁺ (closed bars) and CD4⁺ (open bars) cells are presented as (A) percentages (plus standard deviations) of total gated cells following the collection of 10,000 FACS events and (B) the number of cells isolated per brain. The reported values are representative of at least five independent experiments per virus.

FIG. 8.

IFN-γ production by CNS-infiltrating CD8⁺ cells following infection with wild-type and chimeric viruses. Lymphocytes from the brains of seven mice infected with each virus were pooled and incubated with peptides representing viral S510 and S598 CD8⁺ T-cell epitopes, as indicated, followed by surface and intracellular staining for CD8 and IFN-γ (see Materials and Methods). (A) Representative panel demonstrating the detection of CD8⁺, IFN-γ-expressing cells by FACS analysis. (B) Total numbers of CD8⁺ cells per brain, as well as numbers of S510 and S598 epitope-specific CD8⁺ cells as measured by an IFN-γ secretion assay. The reported values are the averages of a minimum of three independent experiments per virus.