Age-dependent resistance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central nervous system and identification of a novel interferon-inducible protective gene, mouse ISG12

Abstract

Several different mammalian neurotropic viruses produce an age-dependent encephalitis characterized by more severe disease in younger hosts. To elucidate potential factors that contribute to age-dependent resistance to lethal viral encephalitis, we compared central nervous system (CNS) gene expression in neonatal and weanling mice that were either mock infected or infected intracerebrally with a recombinant strain, dsTE12Q, of the prototype alphavirus Sindbis virus. In 1-day-old mice, infection with dsTE12Q resulted in rapidly fatal disease associated with high CNS viral titers and extensive CNS apoptosis, whereas in 4-week-old mice, dsTE12Q infection resulted in asymptomatic infection with lower CNS virus titers and undetectable CNS apoptosis. GeneChip expression comparisons of mock-infected neonatal and weanling mouse brains revealed developmental regulation of the mRNA expression of numerous genes, including some apoptosis regulatory genes, such as the proapoptotic molecules caspase-3 and TRAF4, which are downregulated during development, and the neuroprotective chemokine, fractalkine, which is upregulated during postnatal development. In parallel with increased neurovirulence and increased viral replication, Sindbis virus infection in 1-day-old mice resulted in both a greater number of host inflammatory genes with altered expression and greater changes in levels of host inflammatory gene expression than infection in 4-week-old mice. Only one inflammatory response gene, an expressed sequence tag similar to human ISG12, increased by a greater magnitude in infected 4-week-old mouse brains than in infected 1-day-old mouse brains. Furthermore, we found that enforced neuronal ISG12 expression results in a significant delay in Sindbis virus-induced death in neonatal mice. Together, our data identify genes that are developmentally regulated in the CNS and genes that are differentially regulated in the brains of different aged mice in response to Sindbis virus infection.

FIG. 1.

dsTE12Q infection produces more severe CNS disease in 1-day-old CD1 mice than in 4-week-old CD1 mice. (A) Survival curves of 1-day-old and 4-week-old CD1 mice infected intracerebrally with 1,000 PFU Sindbis virus, strain dsTE12Q. The data represent combined survival probabilities for five independent litters with 10 to 12 mice per litter. (B) Viral growth of dsTE12Q in brains of CD1 mice infected at 1 day and 4 weeks of age. Each datum point represents the geometric mean viral titer and standard error of the mean for three mouse brains. (C) Immunoperoxidase labeling to detect Sindbis virus antigen and TUNEL labeling to detect apoptotic nuclei in the brains of mice infected at 1 day or four weeks of age. Representative regions are shown from the colliculus 60 h after infection. Magnification, ×194.

FIG. 2.

mRNA expression patterns of apoptosis-related genes in the brains of mock-infected 4-week-old mice compared with mock-infected 1-day-old mice.

FIG. 3.

Caspase-3, TRAF4 and fractalkine expression differs in the brains of 1-day-old versus 4-week-old CD1 mice. (A) RiboQuant Apo-1 Multi-Probe RNase protection assay measuring caspase mRNA expression in the brains of dsTE12Q- or mock-infected mice of different ages at serial time points after infection. RNA was pooled from three mice per time point per treatment group. (B) RT-PCR analysis of RNA samples from triplicate mouse brains harvested at serial time points after infection with dsTE12Q or mock infection to detect GAPDH, Sindbis virus E2 envelope glycoprotein, fractalkine, and TRAF4. (C) Immunoperoxidase staining to detect fractalkine expression in mock-infected 1-day-old and 4-week-old brain tissue. Representative sections from the hippocampus by using a ×4 (upper panels) or ×10 (lower panels) objective lens. C-, caspase; p.i., postinfection; fract, fractalkine.

FIG. 4.

Age-related difference in upregulation of mouse ISG12 expression after dsTE12Q infection. (A) Northern blot analysis of ISG12 and GAPDH mRNA expression in mouse brains harvested at 60 h after intracerebral mock or virus infection of 1-day-old or 4-week-old mice. (B) RT-PCR analysis of ISG12, GAPDH, and Sindbis virus E2 envelope glycoprotein gene in RNA harvested from triplicate mouse brains at serial time points after mock or virus infection. SIN, Sindbis virus, strain dsTE12Q; d/o, day-old; p.i., postinfection.

FIG. 5.

Deduced amino acid sequence of mouse ISG12 (GenBank accession no. AY090098) (A), Kaplan-Meier survival curves of 1-day-old mice infected intracerebrally with 1,000 PFU of dsTE12Q/ISG12 or dsTE12Q/ISG12control (B), and viral growth curves in mouse brains infected with dsTE12Q/ISG12 or dsTE12Q/ISGcontrol. The results in panel B represent combined survival curves for eight independent litters with 10 to 12 mice per litter. Similar differences in survival curves were observed in each independent litter. Significance of survival difference between dsTE12Q/ISG12- and dsTE12Q/ISG12 control-infected mice is P = 0.0001 (log-rank sum test). The results in panel C represent the geometric mean viral titers of five to six mice per time point per virus group.