Limited brain metabolism changes differentiate between the progression and clearance of rabies virus

Abstract

Central nervous system (CNS) metabolic profiles were examined from rabies virus (RABV)-infected mice that were either mock-treated or received post-exposure treatment (PET) with a single dose of the live recombinant RABV vaccine TriGAS. CNS tissue harvested from mock-treated mice at middle and late stage infection revealed numerous changes in energy metabolites, neurotransmitters and stress hormones that correlated with replication levels of viral RNA. Although the large majority of these metabolic changes were completely absent in the brains of TriGAS-treated mice most likely due to the strong reduction in virus spread, TriGAS treatment resulted in the up-regulation of the expression of carnitine and several acylcarnitines, suggesting that these compounds are neuroprotective. The most striking change seen in mock-treated RABV-infected mice was a dramatic increase in brain and serum corticosterone levels, with the later becoming elevated before clinical signs or loss of body weight occurred. We speculate that the rise in corticosterone is part of a strategy of RABV to block the induction of immune responses that would otherwise interfere with its spread. In support of this concept, we show that pharmacological intervention to inhibit corticosterone biosynthesis, in the absence of vaccine treatment, significantly reduces the pathogenicity of RABV. Our results suggest that widespread metabolic changes, including hypothalamic-pituitary-adrenal axis activation, contribute to the pathogenesis of RABV and that preventing these alterations early in infection with PET or pharmacological blockade helps protect brain homeostasis, thereby reducing disease mortality.

Figure 1. Post-exposure treatment of DOG4 RABV-infected mice with TriGAS dramatically reduces the accumulation of RABV N protein mRNA in the brain.

Mice were infected i.n. with 10⁵ FFU of DOG4 RV and 4 hr later either mock treated with PBS or treated with 10⁷ FFU of TriGAS. Ten to 15 mice per group were euthanized for each tissue at each indicated time point, and the number of DOG4 RABV N mRNA copies in A) whole brain, B) olfactory bulb and C) hypothalamus was quantified by qRT-PCR as described in Materials and Methods. The results are presented as the mean RABV N mRNA copy numbers (+SE) per 1,000 copies of L13 mRNA. The bars indicate significant differences in copy numbers between mock-treated and PET-treated animals. *p<.05, **p<.01 and ***p<.001, respectively.

Figure 2. PET with TriGAS prevents the RABV infection-induced decline in acetylcholine.

Mice were infected i.n. with 10⁵ FFU of DOG4 and 4 hr later either mock treated i.m. with PBS or treated with 10⁷ FFU of TriGAS. An additional group of uninfected, untreated mice served as a reference. Six mice per group were euthanized for each time point, and the levels of acetylcholine were determined via LC-GC/MS-MS2 as described in Materials and Methods. The results are presented as comparative levels (+/− SE). Statistical differences between groups were analyzed using Welch's two sample t test and are identified by *p<0.05. The horizontal lines represent significant differences between control and DOG4-infected/mock-treated mice while the asterisks over bars represent differences between mock- and TriGAS –treated, infected mice.

Figure 3. Energy metabolites 3-hydroxybutyrate (BHBA) and glucose increase in the CNS during the course of WT RABV infection.

Mice were infected i.n. with 10⁵ FFU of DOG4 and 4 hr later either mock treated i.m. with PBS or treated with 10⁷ FFU of TriGAS. An additional group of uninfected, untreated mice served as a reference. Six mice per group were euthanized for each time point, and the levels of BHBA and glucose were determined via LC-GC/MS-MS2 as described in Materials and Methods. The results are presented as comparative levels (+/− SE). *p<.05, **p<.01.

Figure 4. Levels of carnitine and several acylcarnitines increase in the brains of TriGAS treated mice late in RABV infection.

Mice were infected i.n. with 10⁵ FFU of DOG4 and 4 hr later either mock treated i.m. with PBS or treated with 10⁷ FFU of TriGAS. An additional group of uninfected, untreated mice served as a reference. Six mice per group were euthanized for each time point, and the levels of carnitine (A) and acylcarnitines: hexanoylcarnitine (B), octanoylcarnitine (C), laurylcarnitine (D), palmitoylcarnitine (E), stearoylcarnitine (F), oleoylcarnitine (G), and butyrylcarnitine (H) were determined via LC-GC/MS-MS2 as described in Materials and Methods. The results are presented as comparative levels (+/− SE). *p<.05, **p<.01, ***p<.001.

Figure 5. Postexposure treatment of DOG4 RV-infected mice with TriGAS prevents the elevation of corticosterone associated with pathogenic RABV in brain and serum.

A) Mice were infected i.n. with 10⁵ FFU of DOG4 and 4 hr later either mock treated with PBS or treated with 10⁷ FFU of TriGAS. An additional group of uninfected, untreated mice served as a reference. Six mice per group were euthanized for each time point, and the levels of brain corticosterone were determined via LC-GC/MS-MS2 as described in Materials and Methods. B) Groups were identical to those in A), with one additional group of animals receiving only TriGAS. Ten mice per group were bled at each time point and serum corticosterone was measured by EIA as described in Materials and Methods. The results represent comparative levels of corticosterone (+/− SE) *p<.05, **p<.01, and ***p<.001, respectively.

Figure 6. Post-exposure treatment of DOG4 RV-infected mice with metyrapone blocks the rise in corticosterone associated with pathogenic RABV.

Mice were infected with 10⁴ FFU of DOG4 RABV i.n. and either mock treated with PBS or treated with metyrapone (100 mg/kg) i.p. each day for 21 days. Ten mice per group were bled 1 hr after injection for each time point. The results represent comparative levels of corticosterone (+/− SE). *p<.05, **p<.01, and ***p<.001, respectively.

Figure 7. PET with metyrapone, a corticosterone synthesis inhibitor, reduces the pathogenicity of WT RABV.

Groups of 20 mice were infected with 10⁴ FFU of DOG4 RABV i.n. and either mock-treated with PBS or treated with metyrapone (100 mg/kg) i.p. each day for 21 days. The mice were observed for 4 weeks, and body weight (A), clinical signs (B) and survival (C) are presented (+/− SE). For (A), two-way anova was used to determine significance effects in treatment per day. (B) Two-way anova and Bonferroni post hoc tests were used to indicate daily differences in clinical severity (*p<.05, **p<.01). (C) The Mantel-Cox log-rank test was used to determine differences in survivorship (*p<.05).