Project Summary/Abstract Infection of the olfactory epithelium in COVID-19 patients is thought to underlie loss of smell (anosmia), a pathognomonic symptom that can be long-term in some patients, significantly affecting quality of life. The olfactory epithelium is also believed to be a major entry point for systemic SARS-CoV-2 infection, which can result in neurological as well as respiratory symptoms. Wild-type SARS-CoV-2 cannot bind the mouse ACE2 receptor, and existing human ACE2 (hACE2)-expressing mouse models either do not permit conditional analysis or do not confer severe illness after infection. Due to the lack of genetically manipulable models that display severe disease, the infected cell types responsible for acute and long-term anosmia, and the route(s) by which the virus penetrates the brain, have not been definitively identified. To address these gaps in knowledge and test cell type-specific requirements for COVID-19-related pathologies we generated conditional hACE2fl knockin mice that express hACE2 in similar cell types to humans. hACE2fl mice nasally inoculated with a high dose of wild-type SARS-CoV-2 display initial infection of olfactory epithelium and rapidly develop anosmia. This is followed by infection of neurons in the olfactory bulb and brain, which is associated with lethality and requires neuronal hACE2 expression. Importantly, specific destruction of olfactory epithelium via methimazole treatment prevents olfactory bulb and brain infection and lethality, identifying the olfactory epithelium as an essential gateway to CNS infection. hACE2fl mice inoculated with a low dose of wild-type SARS-CoV-2 show reversible disease and survive, but a subset displays a long-term decrease in odor sensitivity (hyposmia) like that observed in humans. We propose to use hACE2fl mice to provide definitive genetic evidence for cellular mechanisms of short- and long-term loss of smell and identify the pathways for brain infection during COVID-19. These studies are expected to complement existing descriptive human studies to identify causal pathogenic mechanisms and preventative and therapeutic targets. Three specific aims will be pursued: (i) define the cellular requirements for acute loss of smell; (ii) determine the mechanisms of long-term hyposmia; and (iii) uncover the cellular mechanisms of olfactory bulb and brain infection.

Project Narrative Loss of smell and other neurological symptoms are prevalent in COVID-19 disease and can be long-lasting, significantly impairing quality of life; however, the underlying cellular mechanisms are poorly defined. We will use a new, genetically manipulatable mouse model that is susceptible to SARS-CoV-2 infection to delineate the mechanisms that underly acute and long-term loss of smell and identify the routes by which the virus can invade the brain.