BACKGROUND:There is evidence that antiviral defenses are impaired in airway epithelia derived from the lungs of people with cystic fibrosis (CF), however it is unclear whether this phenotype is an intrinsic feature of CF or an acquired trait that develops secondary to chronic airway infection and inflammation. To distinguish between these possibilities, we examined the antiviral responses of newborn CF pigs, prior to the onset of airway inflammation and chronic lung disease. METHODS:We performed an in vivo viral challenge experiment in newborn CF and non-CF pigs, using influenza A (IAV) as a model respiratory virus. To learn more about the contributions of epithelia in this setting, we carried out a parallel in vitro infection experiment using cultured airway epithelia derived from newborn CF and non-CF pig controls. RESULTS:We found that viral growth kinetics and host antiviral responses were relatively similar in the cultured epithelia from CF and non-CF pigs. However, we observed divergent host responses in the animal experiment, with the newborn CF pigs exhibiting comparatively increased innate immune activation and increased viral loads after IAV challenge. CONCLUSIONS:Our findings indicate a reduced effectiveness of antiviral host defense at birth in the CF pig model and suggest that this phenotype is not driven solely by altered airway epithelial cell function. These results highlight a likely contribution of early life viral infections in initiating CF lung disease.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC)2. Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice3,4. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions.

Background: COVID-19 in hospitalized patients may be the result of community acquisition or in-hospital transmission. Molecular epidemiology can help confirm hospital COVID-19 transmission and outbreaks. We describe large COVID-19 clusters identified in our hospital and apply molecular epidemiology to confirm outbreaks. Methods: The University of Iowa Hospitals and Clinics is an 811-bed academic medical center. We identified large clusters involving patients with hospital onset COVID-19 detected during March–October 2020. Large clusters included ≥10 individuals (patients, visitors, or HCWs) with a laboratory confirmed COVID-19 diagnosis (RT-PCR) and an epidemiologic link. Epidemiologic links were defined as hospitalization, work, or visiting in the same unit during the incubation or infectious period for the index case. Hospital onset was defined as a COVID-19 diagnosis ≥14 days from admission date. Admission screening has been conducted since May 2020 and serial testing (every 5 days) since July 2020. Nasopharyngeal swab specimens were retrieved for viral whole-genome sequencing (WGS). Cluster patients with a pairwise difference in ≤5 mutations were considered part of an outbreak. WGS was performed using Oxford Nanopore Technology and protocols from the ARTIC network. Results: We identified 2 large clusters involving patients with hospital-onset COVID-19. Cluster 1: 2 hospital-onset cases were identified in a medical-surgical unit in June 2020. Source and contact tracing revealed 4 additional patients, 1 visitor, and 13 employees with COVID-19. Median age for patients was 62 (range, 38–79), and all were male. In total, 17 samples (6 patients, 1 visitor, and 10 HCWs) were available for WGS. Cluster 2: A hospital-onset case was identified via serial testing in a non–COVID-19 intensive care unit in September 2020. Source investigation, contact tracing, and serial testing revealed 3 additional patients, and 8 HCWs. One HCW also had a community exposure. Patient median age was 60 years (range, 48–68) and all were male. In total, 11 samples (4 patients and 7 HCWs) were sequenced. Using WGS, cluster 1 was confirmed to be an outbreak: WGS showed 0–5 mutations in between samples. Cluster 2 was also an outbreak: WGS showed less diversity (0–3 mutations) and ruled out the HCW with a community exposure (20 mutations of difference). Conclusion: Whole-genome sequencing confirmed the outbreaks identified using classic epidemiologic methods. Serial testing allowed for early outbreak detection. Early outbreak detection and implementation of control measures may decrease outbreak size and genetic diversity.Funding: NoDisclosures: None

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 coronavirus 2 (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 mice susceptible to SARS-CoV-2, indicating its potential to be repositioned for coronavirus disease 2019 (COVID-19) prophylaxis in humans.

Background: Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 RNA can be detected by real-time reverse-transcription polymerase chain reaction (RT-PCR) for several weeks after infection. Discerning persistent RT-PCR positivity versus reinfection is challenging and the frequency of COVID-19 reinfections is unknown. We aimed to determine the frequency of clinically suspected reinfection in our center and confirm reinfection using viral whole-genome sequencing (WGS). Methods: The University of Iowa Hospitals and Clinics (UIHC) is an 811-bed academic medical center. Patients with respiratory complaints undergo COVID-19 RT-PCR using nasopharyngeal swabs. The RT-PCR (TaqPath COVID-19 Combo kit) uses 3 targets (ORF1ab, S gene, and N gene). We identified patients with previous laboratory-confirmed COVID-19 who sought care for new respiratory complaints and underwent a repeated SARS-CoV-2 test at least 45 days from their first positive test. We then identified patients with median RT-PCR cycle threshold (Ct) values. Results: During the study period, 13,603 patients had a SARS-CoV-2– positive RT-PCR. Of these, 296 (2.2%) had a clinical visit for new onset of symptoms and a repeated RT-PCR assay >45 days from the first test. Moreover, 29 patients (9.8%) had a positive RT-PCR assay in the repeated testing. Ct values were available for samples from 25 patients; 7 (28%) had Ct values. Conclusions: In patients with a recent history of COVID-19 infection, repeated testing for respiratory symptoms was infrequent. Some had a SARS-CoV-2–positive RT-PCR assay on repeated testing, but only 1 in 4 had Ct values suggestive of a reinfection. We confirmed 1 case of reinfection using WGS.Funding: NoDisclosures: None

Molecular epidemiology of large coronavirus disease 2019 (COVID-19) clusters before and after the implementation of routine serial testing at an academic medical center in Iowa, 2020 - Volume 42 Issue 12

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 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 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.

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.

The association between atrial fibrillation (AF) and thromboembolic (TE) complications in left ventricular assist device (LVAD) recipients is controversial, and there is paucity of large-scale data evaluating the impact of AF on early outcomes after device implantation. Using the National Inpatient Sample, we identified hospitalizations where patients underwent LVAD implantation from 2010 to 2015. Multivariate logistic regression was used to evaluate the association of AF on in-hospital outcomes. A total of 18,378 patients (41.7% with AF) underwent LVAD implantation. Patients with AF were older (59.9 vs. 54.0 years, p < 0.001), more commonly male (79.9 vs. 74.1%, p < 0.001), and had a greater burden of comorbidities as measured by the Elixhauser comorbidity index (7.2 vs. 6.3, p < 0.001). Patients with AF had less incidence of ischemic stroke (3.1 vs. 4.7%, p = 0.04, OR 0.68), hemorrhagic stroke (1.0 vs. 2.4%, p = 0.006, OR 0.43), and other systemic embolism (1.8 vs. 3.7%, p = 0.01, OR 0.55). There was no significant difference in the incidence of bleeding requiring transfusion between AF and no AF cohorts (29.3 vs. 24.2%, p = 0.09, OR 1.15). LOS was shorter in patients with AF (32.9 vs. 36.7 mean days, p < 0.001). Patients with AF had lower in-hospital mortality (8.9 vs. 14.9%, p < 0.001, OR 0.48). In a large real-world US cohort of patients undergoing LVAD implantation, a diagnosis of AF was common among device recipients. After adjustment for demographics and comorbidities, AF was associated with reduced TE events and in-hospital mortality.

BACKGROUND:Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors.MATERIALS AND METHODS:We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19.FINDINGS:Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19.INTERPRETATION:These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.

Based on the largest publicly available all-payer inpatient database in the United States, this study sought to evaluate real world outcomes after bariatric surgery among patients with heart failure.

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.

Supplemental Digital Content is available in the text.

After heart disease, cancer is the second leading cause of death in the United States. Evolving cancer treatments have improved overall survival and many survivors now die of non-oncologic causes. Advanced heart failure (HF) therapies, including ventricular assist devices (VAD), traditionally have

Background: Morbidly obese patients with end-stage heart failure (HF) are routinely ineligible for heart transplantation. Left ventricular assist devices (LVAD) are commonly used in an attempt to a...

Mechanical circulatory support has become an essential component in the therapeutic arsenal for heart failure. The use of short-term devices as a bridge to a durable one and combinations of short-term devices used in parallel or when escalating support, is increasing. There is a paucity of data

BackgroundDurable left-ventricular assist devices (LVAD) have become an increasingly frequent treatment strategy for end-stage heart failure (HF) but their use remains limited by complications including bleeding, thrombosis, and stroke. The impact of LVAD therapy on outcomes in HF patients with postulated higher risk comorbid conditions, such as diffuse connective tissue diseases (DCTD), has not been well studied to date. Given that these patients have an inherently higher risk of hematological complications than the general population, we aimed to investigate demographic characteristics and outcomes after LVAD implantation in this unique patient cohort.MethodsUsing the 2010-2014 Nationwide Inpatient Sample (NIS) database, we identified the overall demographic characteristics and the incidence of common LVAD complications in patients with DCTD. When possible, univariate logistic regression and t-test were used to compare baseline characteristics, specific outcomes and in-hospital mortality after LVAD implantation in patients with and without DCTD.ResultsA total of 50 patients (weighted) with DCTD were admitted for LVAD implantation from 2010-2014. Patients with DCTD were mostly female (70%), white (80%) and admitted electively for LVAD surgery (70%) (Table 1). The most common disorder was systemic lupus erythematosus (60%), followed by polymyositis (20%), systemic sclerosis (20%), and combined syndromes (10%). During the index admission, there were no in-hospital deaths or stroke in DCTD patients undergoing LVAD. The risk of transfusion (50% vs 39.8%; p 0.514; OR 1.51), hemorrhage or hematoma complicating the procedure (30% vs 20.9%; p 0.486; OR 1.61), gastrointestinal bleeding (10% vs 8.5%; p 0.86; OR 1.2), or device-related complications such as pump thrombosis (10% vs 8.6%; p 0.873; OR 1.18), was not higher when compared to patients without DCTD (Table 1).ConclusionTo our knowledge, there is no published data with advanced HF therapies in patients with DCTD. The present analysis found that patients with DCTDs undergoing LVAD therapy do not appear to have a higher risk of bleeding, thrombotic events, stroke or in-hospital mortality after device implantation, but the majority of the DCTD patients in this cohort underwent elective LVAD surgery. As LVADs become increasingly utilized in the advanced HF population, including those with systemic conditions that disqualify them for transplantation, further research is needed to identify additional adverse factors and comorbidities that could significantly impact post-LVAD outcomes. Durable left-ventricular assist devices (LVAD) have become an increasingly frequent treatment strategy for end-stage heart failure (HF) but their use remains limited by complications including bleeding, thrombosis, and stroke. The impact of LVAD therapy on outcomes in HF patients with postulated higher risk comorbid conditions, such as diffuse connective tissue diseases (DCTD), has not been well studied to date. Given that these patients have an inherently higher risk of hematological complications than the general population, we aimed to investigate demographic characteristics and outcomes after LVAD implantation in this unique patient cohort. Using the 2010-2014 Nationwide Inpatient Sample (NIS) database, we identified the overall demographic characteristics and the incidence of common LVAD complications in patients with DCTD. When possible, univariate logistic regression and t-test were used to compare baseline characteristics, specific outcomes and in-hospital mortality after LVAD implantation in patients with and without DCTD. A total of 50 patients (weighted) with DCTD were admitted for LVAD implantation from 2010-2014. Patients with DCTD were mostly female (70%), white (80%) and admitted electively for LVAD surgery (70%) (Table 1). The most common disorder was systemic lupus erythematosus (60%), followed by polymyositis (20%), systemic sclerosis (20%), and combined syndromes (10%). During the index admission, there were no in-hospital deaths or stroke in DCTD patients undergoing LVAD. The risk of transfusion (50% vs 39.8%; p 0.514; OR 1.51), hemorrhage or hematoma complicating the procedure (30% vs 20.9%; p 0.486; OR 1.61), gastrointestinal bleeding (10% vs 8.5%; p 0.86; OR 1.2), or device-related complications such as pump thrombosis (10% vs 8.6%; p 0.873; OR 1.18), was not higher when compared to patients without DCTD (Table 1). To our knowledge, there is no published data with advanced HF therapies in patients with DCTD. The present analysis found that patients with DCTDs undergoing LVAD therapy do not appear to have a higher risk of bleeding, thrombotic events, stroke or in-hospital mortality after device implantation, but the majority of the DCTD patients in this cohort underwent elective LVAD surgery. As LVADs become increasingly utilized in the advanced HF population, including those with systemic conditions that disqualify them for transplantation, further research is needed to identify additional adverse factors and comorbidities that could significantly impact post-LVAD outcomes.