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. 2005 Jul;79(14):9108-18.
doi: 10.1128/JVI.79.14.9108-9118.2005.

Contributions of the viral genetic background and a single amino acid substitution in an immunodominant CD8+ T-cell epitope to murine coronavirus neurovirulence

Katherine C MacNamara  1 , Ming Ming Chua, Joanna J Phillips, Susan R Weiss
Affiliations

Contributions of the viral genetic background and a single amino acid substitution in an immunodominant CD8+ T-cell epitope to murine coronavirus neurovirulence

Katherine C MacNamara et al. J Virol. 2005 Jul.

Abstract

The immunodominant CD8+ T-cell epitope of a highly neurovirulent strain of mouse hepatitis virus (MHV), JHM, is thought to be essential for protection against virus persistence within the central nervous system. To test whether abrogation of this H-2Db-restricted epitope, located within the spike glycoprotein at residues S510 to 518 (S510), resulted in delayed virus clearance and/or virus persistence we selected isogenic recombinants which express either the wild-type JHM spike protein (RJHM) or spike containing the N514S mutation (RJHM(N514S)), which abrogates the response to S510. In contrast to observations in suckling mice in which viruses encoding inactivating mutations within the S510 epitope (epitope escape mutants) were associated with persistent virus and increased neurovirulence (Pewe et al., J Virol. 72:5912-5918, 1998), RJHM(N514S) was not more virulent than the parental, RJHM, in 4-week-old C57BL/6 (H-2b) mice after intracranial injection. Recombinant viruses expressing the JHM spike, wild type or encoding the N514S substitution, were also selected in which background genes were derived from the neuroattenuated A59 strain of MHV. Whereas recombinants expressing the wild-type JHM spike (SJHM/RA59) were highly neurovirulent, A59 recombinants containing the N514S mutation (SJHM(N514S)/RA59) were attenuated, replicated less efficiently, and exhibited reduced virus spread in the brain at 5 days postinfection (peak of infectious virus titers in the central nervous system) compared to parental virus encoding wild-type spike. Virulence assays in BALB/c mice (H-2d), which do not recognize the S510 epitope, revealed that attenuation of the epitope escape mutants was not due to the loss of a pathogenic immune response directed against the S510 epitope. Thus, an intact immunodominant S510 epitope is not essential for virus clearance from the CNS, the S510 inactivating mutation results in decreased virulence in weanling mice but not in suckling mice, suggesting that specific host conditions are required for epitope escape mutants to display increased virulence, and the N514S mutation causes increased attenuation in the context of A59 background genes, demonstrating that genes other than that for the spike are also important in determining neurovirulence.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram of recombinant viruses. Recombinant viruses were selected by targeted recombination as described in Materials and Methods. RJHM is wild-type recombinant and RJHMN514S is a recombinant containing the epitope mutation; SJHM/RA59 and SJHMN514S/RA59 encode wild-type JHM spike and JHM spike with the N514S substitution, respectively, both with A59 background genes. RA59 contains all genes derived from A59 (29).
FIG. 2.
FIG. 2.
CD8+ T-cell response following infection with recombinant viruses encoding wild-type JHM spike or mutant spike with the N514S substitution. (A) Mice infected with 10 PFU of either RJHM, RJHMN514S, JHM, or v106 were sacrificed at day 6 postinfection and their spleens were removed. Splenocytes were harvested and incubated with specific peptides, as indicated above each column, followed by surface and intracellular staining for CD8 and IFN-γ (see Materials and Methods). (B) Splenocytes were isolated, at day 7 postinfection, from mice infected with 10 PFU of RA59, SJHM/RA59, or SJHMN514S/RA59 and incubated with specific peptides as indicated. Intracellular IFN-γ secretion in response to peptide stimulation was analyzed by flow cytometry. The percent of IFN-γ- and CD8-positive T cells is represented in the upper right quadrant. A representative animal for each virus is shown from two separate experiments, each including three animals per virus.
FIG. 3.
FIG. 3.
Replication of epitope mutants in B6 mice. B6 mice were infected with 10 PFU of virus and sacrificed days on days 3, 5,7, 12, and 28 p.i.; brains were homogenized and infectious virus was titered on L2 cells. Viral titers are shown for days 3, 5, and 7. (Titers were below the limit of detection on days 12 and 28 and are not shown.) The data were pooled from two experiments that included five mice per virus per time point, except as indicated. (A) Mice were infected with JHM background viruses RJHM (black bar); RJHMN514S (white bar); JHM.IA (gray bar); v106 (striped bar). By 7 days p.i. RJHM and JHM.IA infections resulted in nearly uniform death and the survivors (n = 2) were moribund. Infection with RJHMN514S and v106 resulted in more survivors at day 7 p.i. (n = 4 and n = 5, respectively). There were no significant differences in titer among the viruses at any of the time points. (B) Mice were infected with A59 background viruses SJHMN514S/RA59 (checked bar); SJHM/RA59 (open bar); RA59 (closed bar). At day 5, mice infected with SJHMN514S/RA59 have significantly lower titers than mice infected with SJHM/RA59 or RA59 (Mann-Whitney U test, P < 0.05).
FIG. 4.
FIG. 4.
Viral antigen spread in brains from B6 mice infected with JHM background viruses. Mice were infected with 10 PFU of each virus and sacrificed on day 5; sagittal brain sections, prepared as described in Materials and Methods, were stained with a monoclonal antibody directed against the nucleocapsid protein. Sections represent samples from two separate experiments with at least four animals per group. Represented are two regions that showed consistent antigen staining in all animals examined: the subiculum (A) and the midbrain (B). Magnification, 60×.
FIG. 5.
FIG. 5.
Viral antigen spread in brains from B6 mice infected with the A59 background viruses. Brain sections from mice sacrificed 5 days postinfection with10 PFU of RA59, SJHM/RA59, or SJHMN514S/RA59 were prepared and stained with a monoclonal antibody directed against the nucleocapsid protein as in Fig. 4 for immunohistochemistry for viral antigen. The regions of the brain represented are the subiculum (A) and the midbrain (B). Several sections were stained from at least four different animals from two separate experiments. Magnification, 60×.
FIG. 6.
FIG. 6.
Replication and antigen spread in BALB/c mice infected with A59 background viruses. BALB/c mice infected with 10 PFU of RA59, SJHM/RA59, or SJHMN514S/RA59 were sacrificed at day 5 postinfection. (A) Half of each brain was homogenized and titered for infectious virus as described in Materials and Methods. RA59 (solid bar) and SJHM/RA59 (open bar) demonstrated significantly higher titers than SJHMN514S/RA59 (checked bar) (Mann-Whitney U test, P < 0.01). (B) Half of each brain was sectioned sagitally and stained for viral antigen as described in Materials and Methods. Representative images are shown of the midbrain from a representative animal for each virus. Magnification, 60×.
FIG. 7.
FIG. 7.
Demyelination following infection of B6 mice with A59 background viruses. Demyelination was examined in B6 mice infected with RA59 (solid bar), SJHM/RA59 (open bar), and SJHMN514S/RA59 (checked bar). Animals were infected with a low dose (5 PFU of RA59 or 10 PFU of SJHM/RA59 and SJHMN514S/RA59) and a high dose (2,500 PFU of RA59 and 100 PFU of SJHM/RA59 and SJHMN514S/RA59) of virus and spinal cords were removed day 30 p.i., processed, and stained with luxol fast blue as described in Materials and Methods. Sections were examined for demyelination by counting demyelinated quadrants, at least 40 quadrants per mouse. The scores shown are the averages from 5 to 10 mice per virus per dose. At both doses, RA59 induces a greater percentage of demyelination than the viruses containing the JHM spike. At the high dose this was significantly different (**,P < 0.001, two-tailed t test). SJHM/RA59 and SJHMN514S/RA59 exhibit similar percentages of demyelination at both low and high doses.
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