ABSTRACT Severe acute respiratory syndrome coronavirus SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19) has led to a pandemic with a mortality of approximately 3.5% and a wide range of morbidity outcomes negatively impacted by pre-existing conditions. Given the prevalence of pre-existing comorbid conditions in Veterans, it is imperative to understand the mechanisms of how SARS-CoV-2 invades and replicates within the barrier defense cells of the nose, which is the primary portal for viral entry. Furthermore, current data suggests that the nasal carriage functions as a potential reservoir for viral persistence and transmission (i.e., shedding) at times that are both prior to and during the manifestation of severe respiratory symptoms. This project utilizes a unique biobank of cryopreserved nasal cells collected from over 1000 individuals over 15 years to understand the critical issues surrounding SARS-CoV-2 interaction with the human nasal epithelia. Paradoxically, while SARS-CoV-2 can be detected in nasal swabs prior to its detection in sputum, there is a paucity of rhinologic symptoms (<5% with nasal congestion) associated with COVID-19, with the exception of reversible anosmia in 30-70% of patients. This is particularly problematic because up to 25% of infected individuals remain asymptomatic, but can continue to spread SARS-CoV-2 through airborne droplets. This work seeks to elucidate both the mechanisms controlling which epithelial cell lineages become infected with virus and the type of immune response generated within infected or neighboring epithelia. Through this approach, we will shed light on the issue of why certain individuals never develop symptoms while others progress to severe respiratory failure and death. We will focus on the SARS-CoV-2 receptor Angiotensin Converting Enzyme 2 (ACE2), which is essential and sufficient for the virus to enter cells. Our preliminary data generated from single cell RNA analysis of primary human sinonasal tissue demonstrates that ACE2 is expressed in discrete clusters of nasal epithelia. ACE2- specific immunostaining of human nasal epithelial cells ex vivo and primary ciliated air liquid interface (ALI) cultures corroborates the sc-RNAseq data. Furthermore, our data show that inoculation of primary ALI cultures with SARS-CoV-2 results in approximately 1%-25% of cells becoming infected, suggesting a selective process. These data indicate that we are uniquely poised to test the hypothesis that ACE2 expressing cells constitute a unique reservoir of viral replication and are likely to mount an inflammatory cytokine response that is distinct from non-infected epithelia. Using our established team of experts in nasal epithelial cell biology, viral pathogenesis, inflammatory cytokine biology and genetics we will determine the following: A) which types of epithelia are virally infected, B) what are the local inflammatory cascades in infected vs. non-infected cells, and C) will pharmacologic manipulation of the epithelial innate defense pathways significantly alter SARS-CoV-2 ability to infect, replicate and be released from human nasal epithelia. Successful completion of this work is likely to have a major impact on development of novel strategies to combat COVID19 disease progression within the general population and especially in the U.S. Veteran population.

NARRATIVE Severe acute respiratory syndrome coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19) has led to a pandemic with a mortality of approximately 3-4% and a wide range of morbidity outcomes negatively impacted by pre-existing conditions. Given the prevalence of pre-existing conditions in Veterans, it is imperative to understand how SARS-CoV-2 infects cells within the nose which serves as a primary portal for viral entry, and acts as a reservoir for viral persistence and transmission prior to manifestation of symptoms. Utilizing primary human nasal epithelial cultures from a multitude of donors we will (1) identify which cells in the nose are targeted by SARS-CoV-2 for infection and replication, (2) investigate the inflammatory pathways triggered in the upper airway by SARS-CoV-2, and (3) determine whether pharmacologic stimulation of nasal innate defenses can decrease the viral infection.