Immune Clearance of Attenuated Rabies Virus Results in Neuronal Survival with Altered Gene Expression

Abstract

Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent “mark” on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these “cured” neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that can persist or progress in the absence of sustained viral antigen.

Abortively Infected Astrocytes Appear To Represent the Main Source of Interferon Beta in the Virus-Infected Brain

“…Slow recombination of a green fluorescent protein (GFP) reporter gene was also observed in neurons infected with rabies virus expressing Cre recombinase. In this case, GFP became visible only at day 15 postinfection, although high levels of viral RNA were already observed at day 7 (31). It is conceivable that various cellular processes, including nuclear import of host cell proteins, get altered in rabies virus-infected cells, which might slow down Cre-mediated recombination.…”

“…It is conceivable that various cellular processes, including nuclear import of host cell proteins, get altered in rabies virus-infected cells, which might slow down Cre-mediated recombination. Indeed, strongly dysregulated gene expression was observed in neurons derived from rabies virus-infected mouse brains (31).…”

Rabies Virus Is Recognized by the NLRP3 Inflammasome and Activates Interleukin-1β Release in Murine Dendritic Cells

“…Vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) was obtained from John Hiscott (McGill University) (17). For pathogenicity studies, recombinant BNSP-Cre was used (28).…”

“…Ϫ/Ϫ mice were infected intranasally with 1 ϫ 10 5 focus-forming units (FFU) of BNSP-Cre (28). The mice were monitored daily for weight loss and signs of RABV pathology.…”

“…The above-described experiments suggested that IL-1 receptor signaling would be important in the mediation of the innate immune response to RABV in vivo. To study the importance of this pathway in vivo, we infected wild-type and IL-1R-deficient mice with a strain of RABV that is moderately pathogenic after intranasal inoculation (28). As shown in Fig.…”