Development and Characterization of a Sublethal-Sequelae Mouse Model of EEEV Infection

This proposal is submitted relevant to the topic Emerging Infectious Diseases, specifically as it relates to the area of encouragement "Research, development, and validation of animal models for the study of emerging viral diseases, including novel infections such as the WHO's [World Health Organization's] Disease X, that demonstrate the pathophysiological mechanism of the disease and provide translational data to advance drug products to human clinical trials." The mosquito-borne encephalitic alphaviruses (eastern, Venezuelan, and western equine encephalitis viruses - EEEV, VEEV, and WEEV) cause disease in both equines and humans, resulting in overt encephalitis in a significant percentage of cases. Infection can result in death or neurological sequelae in surviving patients. The significant mortality and morbidity associated with encephalitic alphavirus infections, especially with EEEV, underscores the need for useful interventions. Well-characterized animal models reflective of human disease are critical to the development of prospective therapeutics and vaccines because drug and vaccine candidates will need to be approved via the Animal Rule. Unfortunately, few studies have described EEEV pathogenesis in lethal mouse models and there is no neurological sequelae model currently available. We propose to address this deficiency by developing and characterizing a novel sublethal-sequelae model of EEEV infection and characterizing the host responses associated with cognitive deficits. Our overarching hypothesis is that characterization of proteomic, neuropathologic, and cognitive changes in mice following EEEV infection will yield a validated sublethal-sequelae mouse model of EEEV infection that can be used for therapeutic approval. The following specific aims and methods will address this hypothesis. Aim 1: Determine the cognitive impact of EEEV infection. We hypothesize that those animals that appear to have recovered from EEEV infection in fact suffer substantial cognitive deficits resulting from neurodegeneration associated with viral infection. To study the effects of sublethal EEEV infection, we will infect mice with a sublethal dose of EEEV and perform a battery of behavioral experiments to investigate potential lasting cognitive impairments in mice that have recovered from EEEV infection. Serum and brains will be collected at the time of sacrifice for analysis focused on correlates of neuropathology (Aim 2) and neuropathological alterations in the hippocampus. Aim 2: Identify circulating correlates of neuropathology. We hypothesize that EEEV infection results in alteration of the circulating proteome, which can be mined for correlates of neuropathology, as well as potentially predictive markers of host outcome. We will analyze circulating proteins and peptides found in the blood using novel bioprospecting microparticles in combination with mass spectrometry analysis. Aim 3: Determine the host responses that contribute to EEEV-induced cognitive defects. We hypothesize that molecular responses in the brain following EEEV infection contribute to neuroinflammation and apoptosis, which promote neurological defects and contribute to cognitive impairment. We will determine alterations in the proteomic landscapes in the brains of EEEV-infected mice through reverse phase protein arrays. Aim 4: Proof-of-concept therapeutic testing in the EEEV neurological sequalae model. We hypothesize that attenuation of the observed alterations in signaling cascades will prevent EEEV-induced pathologies. We will assess the efficacy of small molecule inhibitors at preventing EEEV-induced pathology and cognitive impairment. We aim to determine whether altered signaling pathways contribute to EEEV-induced pathologies and validate our EEEV neurological sequalae model as a viable tool for testing therapeutics. The near-term impacts of our proposed studies include (1) the development of a novel neurological sequelae model of EEEV infection, (2) the identification of correlates of neuropathology induced by EEEV infection, (3) a molecular understanding of host response events contributing to EEEV pathogenesis, and (4) identification of potential therapeutic targets to prevent cognitive deficits. The long-term implications of these studies are (1) correlates of neuropathology as markers to evaluate vaccine or therapeutic efficacy, (2) development of therapeutics that prevent EEEV- induced disease by blocking activation of pathways identified in these studies, and (3) mouse models for use as part of countermeasure approval through the Animal Rule. Less