The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs 1 . Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. While COVID-19 severity ranges from asymptomatic to lethal 2 , most experimental infections provide insights into mild disease 3 . Here, using K18-hACE2 mice that we originally developed for SARS studies 4 , we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient protected against lethal disease. Mice developed anosmia at early times after infection. Notably, while pretreatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.

The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs. Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. COVID-19 severity ranges from asymptomatic to lethal. Most experimental infections provide insights into mild disease. Here, using K18-hACE2 mice that we originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient provided protection against lethal disease. Mice developed anosmia at early times after infection. Notably, while treatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays.Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.

15Running title: Expression of ACE2 in the human respiratory tract 16 17 Impact: The mapping of ACE2, the receptor for SARS-CoV-2, to specific anatomical regions 18 and to particular cell types in the human respiratory tract will help guide future studies and 19 provide molecular targets for antiviral therapies. We saw no increase of receptor expression in 20 the presence of known risk factors for severe coronavirus disease 2019. 21 22 Abstract: 35 Rationale: Zoonotically transmitted coronaviruses are responsible for three disease 36 outbreaks since 2002, including the current coronavirus disease 2019 pandemic, caused by 37 SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding 38 the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the cellular 39 receptor for SARS-CoV-2. Receptor expression on the cell surface facilitates viral binding and 40 entry. However, reports of the abundance and distribution of ACE2 expression in the respiratory 41 tract are limited and conflicting. Objectives: To determine ACE2 expression in the human 42 respiratory tract and its association with demographic and clinical characteristics. Methods: 43Here, we systematically examined human upper and lower respiratory tract cells using single-cell 44 RNA sequencing and immunohistochemistry to determine where the receptor is expressed. 45Measurements and main results: Our results reveal that ACE2 expression is highest within the 46 sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe 47 disease development, respectively. In the lung parenchyma where severe disease occurs, ACE2 48 was found on the apical surface of a small subset of alveolar type II cells. We saw no increase of 49 receptor expression in the presence of known risk factors for severe coronavirus disease 2019. 50Conclusions: The mapping of ACE2 to specific anatomical regions and to particular cell types in 51 the respiratory tract will help guide future studies and provide molecular targets for antiviral 52 therapies. 53 54 Word count: 223 55