Mucosal Gene Expression in Response to SARS-CoV-2 Is Associated with Viral Load.

Little is known about the relationships between symptomatic early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and upper airway mucosal gene expression and immune response. To examine the association of symptomatic SARS-CoV-2 early viral load with upper airway mucosal gene expression, we profiled the host mucosal transcriptome from nasopharyngeal swab samples from 68 adults with symptomatic, mild-to-moderate coronavirus disease 19 (COVID-19). We measured SARS-CoV-2 viral load using reverse transcription-quantitative PCR (RT-qPCR). We then examined the association of SARS-CoV-2 viral load with upper airway mucosal immune response. We detected SARS-CoV-2 in all samples and recovered >80% of the genome from 95% of the samples from symptomatic COVID-19 adults. The respiratory virome was dominated by SARS-CoV-2, with limited codetection of other respiratory viruses, with the human Rhinovirus C being identified in 4 (6%) samples. This limited codetection of other respiratory viral pathogens may be due to the implementation of public health measures, like social distancing and masking practices. We observed a significant positive correlation between SARS-CoV-2 viral load and interferon signaling (OAS2, OAS3, IFIT1, UPS18, ISG15, ISG20, IFITM1, and OASL), chemokine signaling (CXCL10 and CXCL11), and adaptive immune system (IFITM1, CD300E, and SIGLEC1) genes in symptomatic, mild-to-moderate COVID-19 adults, when adjusting for age, sex, and race. Interestingly, the expression levels of most of these genes plateaued at a cycle threshold (CT) value of ~25. Overall, our data show that the early nasal mucosal immune response to SARS-CoV-2 infection is viral load dependent, potentially modifying COVID-19 outcomes. IMPORTANCE Several prior studies have shown that SARS-CoV-2 viral load can predict the likelihood of disease spread and severity. A higher detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity. However, the relationship between SARS-CoV-2 viral load, airway mucosal gene expression, and immune response remains elusive. We profiled the nasal mucosal transcriptome from nasal samples collected from adults infected with SARS-CoV-2 during spring 2020 with mild-to-moderate symptoms using a comprehensive metatranscriptomics method. We observed a positive correlation between SARS-CoV-2 viral load, interferon signaling, chemokine signaling, and adaptive immune system in adults with COVID-19. Our data suggest that early nasal mucosal immune response to SARS-CoV-2 infection was viral load dependent and may modify COVID-19 outcomes.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern.

There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.

Topical TMPRSS2 inhibition prevents SARS-CoV-2 infection in differentiated human airway cultures.

BACKGROUND: There are limited effective prophylactic/early treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Viral entry requires spike protein binding to the angiotensin-converting enzyme-2 receptor and cleavage by transmembrane serine protease 2 (TMPRSS2), a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is in clinical trials in early SARS-CoV-2 infection. METHODS: We used differentiated primary human airway epithelial cells at the air-liquid interface to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection. RESULTS: We first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-CoV-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, oral camostat is rapidly metabolised in the circulation, with poor airway bioavailability. We therefore modelled local airway administration by applying camostat to the apical surface of differentiated airway cultures. We demonstrated that a brief exposure to topical camostat effectively restricts SARS-CoV-2 infection. CONCLUSION: These experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.

Comparison of SARS-CoV-2 Evolution in Paediatric Primary Airway Epithelial Cell Cultures Compared with Vero-Derived Cell Lines.

SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality positively associated with increasing host age and presence of co-morbidities. SARS-CoV-2 continues to adapt to the human population, resulting in several variants of concern (VOC) with novel properties, such as Alpha and Delta. However, factors driving SARS-CoV-2 fitness and evolution in paediatric cohorts remain poorly explored. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change in primary airway cell cultures derived from children. Through viral whole-genome sequencing, we explored changes in genetic diversity over time of two pre-VOC clinical isolates of SARS-CoV-2 during passage in paediatric well-differentiated primary nasal epithelial cell (WD-PNEC) cultures and in parallel, in unmodified Vero-derived cell lines. We identified a consistent, rich genetic diversity arising in vitro, variants of which could rapidly rise to near fixation within two passages. Within isolates, SARS-CoV-2 evolution was dependent on host cells, with paediatric WD-PNECs showing a reduced diversity compared to Vero (E6) cells. However, mutations were not shared between strains. Furthermore, comparison of both Vero-grown isolates on WD-PNECs disclosed marked growth attenuation mapping to the loss of the polybasic cleavage site (PBCS) in Spike, while the strain with mutations in Nsp12 (T293I), Spike (P812R) and a truncation of Orf7a remained viable in WD-PNECs. Altogether, our work demonstrates that pre-VOC SARS-CoV-2 efficiently infects paediatric respiratory epithelial cells, and its evolution is restrained compared to Vero (E6) cells, similar to the case of adult cells. We highlight the significant genetic plasticity of SARS-CoV-2 while uncovering an influential role for collaboration between viral and host cell factors in shaping viral evolution and ultimately fitness in human respiratory epithelium.

4'-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.

Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of IFN-mediated innate immune defenses.

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air-liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.

Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression.

In the coronavirus disease 2019 (COVID-19) health crisis, one major challenge is to identify the susceptibility factors of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in order to adapt the recommendations for populations, as well as to reduce the risk of COVID-19 development in the most vulnerable people, especially patients with chronic respiratory diseases such as cystic fibrosis (CF). Airway epithelial cells (AECs) play a critical role in the modulation of both immune responses and COVID-19 severity. SARS-CoV-2 infects the airway through the receptor angiotensin-converting enzyme 2, and a host protease, transmembrane serine protease 2 (TMPRSS2), plays a major role in SARS-CoV-2 infectivity. Here, we show that Pseudomonas aeruginosa increases TMPRSS2 expression, notably in primary AECs with deficiency of the ion channel CF transmembrane conductance regulator (CFTR). Further, we show that the main component of P. aeruginosa flagella, the protein flagellin, increases TMPRSS2 expression in primary AECs and Calu-3 cells, through activation of Toll-like receptor-5 and p38 MAPK. This increase is particularly seen in Calu-3 cells deficient for CFTR and is associated with an intracellular increased level of SARS-CoV-2 infection, however, with no effect on the amount of virus particles released. Considering the urgency of the COVID-19 health crisis, this result may be of clinical significance for CF patients, who are frequently infected with and colonized by P. aeruginosa during the course of CF and might develop COVID-19.

Imiquimod Boosts Interferon Response, and Decreases ACE2 and Pro-Inflammatory Response of Human Bronchial Epithelium in Asthma.

Background: Both anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity. Objective: To investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients. Methods: Effects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses. Results: Imiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1ß, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-ß expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections. Conclusion: Imiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-ß expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.

Prolonged activation of nasal immune cell populations and development of tissue-resident SARS-CoV-2-specific CD8+ T cell responses following COVID-19.

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.

Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb.

Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.

Niclosamide shows strong antiviral activity in a human airway model of SARS-CoV-2 infection and a conserved potency against the Alpha (B.1.1.7), Beta (B.1.351) and Delta variant (B.1.617.2).

SARS-CoV-2 variants are emerging with potential increased transmissibility highlighting the great unmet medical need for new therapies. Niclosamide is a potent anti-SARS-CoV-2 agent that has advanced in clinical development. We validate the potent antiviral efficacy of niclosamide in a SARS-CoV-2 human airway model. Furthermore, niclosamide remains its potency against the D614G, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants. Our data further support the potent anti-SARS-CoV-2 properties of niclosamide and highlights its great potential as a therapeutic agent for COVID-19.

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

Intersubject Variation in ACE2 Protein Expression in Human Airway Epithelia and Its Relationship to Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ) initiates entry into airway epithelia by binding its receptor, angiotensin-converting enzyme 2 (ACE2). METHODS: To explore whether interindividual variation in ACE2 abundance contributes to variability in coronavirus disease 2019 (COVID-19) outcomes, we measured ACE2 protein abundance in primary airway epithelial cultures derived from 58 human donor lungs. RESULTS: We found no evidence for sex- or age-dependent differences in ACE2 protein expression. Furthermore, we found that variations in ACE2 abundance had minimal effects on viral replication and induction of the interferon response in airway epithelia infected with SARS-CoV-2. CONCLUSIONS: Our results highlight the relative importance of additional host factors, beyond viral receptor expression, in determining COVID-19 lung disease outcomes.

Upregulation of Human Endogenous Retroviruses in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.

Drug repurposing for COVID-19 based on an integrative meta-analysis of SARS-CoV-2 induced gene signature in human airway epithelium.

Drug repurposing has the potential to bring existing de-risked drugs for effective intervention in an ongoing pandemic-COVID-19 that has infected over 131 million, with 2.8 million people succumbing to the illness globally (as of April 04, 2021). We have used a novel `gene signature'-based drug repositioning strategy by applying widely accepted gene ranking algorithms to prioritize the FDA approved or under trial drugs. We mined publically available RNA sequencing (RNA-Seq) data using CLC Genomics Workbench 20 (QIAGEN) and identified 283 differentially expressed genes (FDR<0.05, log2FC>1) after a meta-analysis of three independent studies which were based on severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection in primary human airway epithelial cells. Ingenuity Pathway Analysis (IPA) revealed that SARS-CoV-2 activated key canonical pathways and gene networks that intricately regulate general anti-viral as well as specific inflammatory pathways. Drug database, extracted from the Metacore and IPA, identified 15 drug targets (with information on COVID-19 pathogenesis) with 46 existing drugs as potential-novel candidates for repurposing for COVID-19 treatment. We found 35 novel drugs that inhibit targets (ALPL, CXCL8, and IL6) already in clinical trials for COVID-19. Also, we found 6 existing drugs against 4 potential anti-COVID-19 targets (CCL20, CSF3, CXCL1, CXCL10) that might have novel anti-COVID-19 indications. Finally, these drug targets were computationally prioritized based on gene ranking algorithms, which revealed CXCL10 as the common and strongest candidate with 2 existing drugs. Furthermore, the list of 283 SARS-CoV-2-associated proteins could be valuable not only as anti-COVID-19 targets but also useful for COVID-19 biomarker development.

Genetic Dissection of the Regulatory Mechanisms of Ace2 in the Infected Mouse Lung.

Acute lung injury (ALI) is an important cause of morbidity and mortality after viral infections, including influenza A virus H1N1, SARS-CoV, MERS-CoV, and SARS-CoV-2. The angiotensin I converting enzyme 2 (ACE2) is a key host membrane-bound protein that modulates ALI induced by viral infection, pulmonary acid aspiration, and sepsis. However, the contributions of ACE2 sequence variants to individual differences in disease risk and severity after viral infection are not understood. In this study, we quantified H1N1 influenza-infected lung transcriptomes across a family of 41 BXD recombinant inbred strains of mice and both parents-C57BL/6J and DBA/2J. In response to infection Ace2 mRNA levels decreased significantly for both parental strains and the expression levels was associated with disease severity (body weight loss) and viral load (expression levels of viral NA segment) across the BXD family members. Pulmonary RNA-seq for 43 lines was analyzed using weighted gene co-expression network analysis (WGCNA) and Bayesian network approaches. Ace2 not only participated in virus-induced ALI by interacting with TNF, MAPK, and NOTCH signaling pathways, but was also linked with high confidence to gene products that have important functions in the pulmonary epithelium, including Rnf128, Muc5b, and Tmprss2. Comparable sets of transcripts were also highlighted in parallel studies of human SARS-CoV-infected primary human airway epithelial cells. Using conventional mapping methods, we determined that weight loss at two and three days after viral infection maps to chromosome X-the location of Ace2. This finding motivated the hierarchical Bayesian network analysis, which defined molecular endophenotypes of lung infection linked to Ace2 expression and to a key disease outcome. Core members of this Bayesian network include Ace2, Atf4, Csf2, Cxcl2, Lif, Maml3, Muc5b, Reg3g, Ripk3, and Traf3. Collectively, these findings define a causally-rooted Ace2 modulatory network relevant to host response to viral infection and identify potential therapeutic targets for virus-induced respiratory diseases, including those caused by influenza and coronaviruses.