Highlights d A SARS-CoV-2 infectious cDNA clone and reporter viruses are generated d SARS-CoV-2 and SARS-CoV neutralization assays show limited cross neutralization d SARS-CoV-2 shows a gradient infectivity from the proximal to distal respiratory tract d Ciliated airway cells and AT-2 cells are primary targets for SARS-CoV-2 infection

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective epithelial cAMP-dependent Cl(-) secretion and increased airway Na(+) absorption. The mechanistic links between these altered ion transport processes and the pathogenesis of cystic fibrosis lung disease, however, are unclear. To test the hypothesis that accelerated Na(+) transport alone can produce cystic fibrosis-like lung disease, we generated mice with airway-specific overexpression of epithelial Na(+) channels (ENaC). Here we show that increased airway Na(+) absorption in vivo caused airway surface liquid (ASL) volume depletion, increased mucus concentration, delayed mucus transport and mucus adhesion to airway surfaces. Defective mucus transport caused a severe spontaneous lung disease sharing features with cystic fibrosis, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation and poor bacterial clearance. We conclude that increasing airway Na(+) absorption initiates cystic fibrosis-like lung disease and produces a model for the study of the pathogenesis and therapy of this disease.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them via mucociliary clearance (MCC)1,2. However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases1. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus1,3. Genetic variants are linked to diverse lung diseases4-6, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in the lungs. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally7. Apoptotic macrophages accumulated, phagocytosis was impaired, and IL-23 production was reduced inMuc5b−/− mice. By contrast, in Muc5b transgenic (Tg) mice, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum1,8. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%9-11. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Rationale: Chronic obstructive pulmonary disease is a leading cause of death worldwide, but its pathogenesis is not well understood. Previous studies have shown that airway surface dehydration in b-epithelial Na 1 channel (bENaC)-overexpressing mice caused a chronic lung disease with high neonatal pulmonary mortality and chronic bronchitis in adult survivors. Objectives: The aim of this study was to identify the initiating lesions and investigate the natural progression of lung disease caused by airway surface dehydration. Methods: Lung morphology, gene expression, bronchoalveolar lavage, and lung mechanics were studied at different ages in bENaCoverexpressing mice. Measurements and Main Results: Mucus obstruction in bENaCoverexpressing mice originated in the trachea in the first days of life and was associated with hypoxia, airway epithelial necrosis, and death. In surviving bENaC-overexpressing mice, mucus obstruction extended into the lungs and was accompanied by goblet cell metaplasia, increased mucin expression, and airway inflammation with transient perinatal increases in tumor necrosis factor-a and macrophages, IL-13 and eosinophils, and persistent increases in keratinocytederived cytokine (KC), neutrophils, and chitinases in the lung. bENaCoverexpressing mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance.Conclusions: Our studies demonstrate that airway surface dehydration is sufficient to initiate persistent neutrophilic airway inflammation with chronic airways mucus obstruction and to cause transient eosinophilic airway inflammation and emphysema. These results suggest that deficient airway surface hydration may play a critical role in the pathogenesis of chronic obstructive pulmonary diseases of different etiologies and serve as a target for novel therapies.

YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yap tm1Smil allele (Yap ؊/؊ ) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostaining, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in Yap ؊/؊ embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper. Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap ؊/؊ embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithelium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H ؉ ,K ؉ -ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent HCO 3 ؊ secretory pathway that was defective in CF: (i) ASL [HCO 3 ؊ ] was higher in NL than CF ASL; (ii) activating CFTR with forskolin͞3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pH ASL is abnormally regulated under basal conditions because of absent CFTRdependent HCO 3 ؊ secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

The identification of small molecules that target specific CFTR variants has ushered in a new era of treatment for cystic fibrosis (CF), yet optimal, individualized treatment of CF will require identification and targeting of disease modifiers. Here we use genome-wide association analysis to identify genetic modifiers of CF lung disease, the primary cause of mortality. Meta-analysis of 6,365 CF patients identifies five loci that display significant association with variation in lung disease. Regions on chr3q29 (MUC4/MUC20; P=3.3 × 10−11), chr5p15.3 (SLC9A3; P=6.8 × 10−12), chr6p21.3 (HLA Class II; P=1.2 × 10−8) and chrXq22-q23 (AGTR2/SLC6A14; P=1.8 × 10−9) contain genes of high biological relevance to CF pathophysiology. The fifth locus, on chr11p12-p13 (EHF/APIP; P=1.9 × 10−10), was previously shown to be associated with lung disease. These results provide new insights into potential targets for modulating lung disease severity in CF.

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human ␣1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3-5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helperdependent vectors with sequential delivery of vectors of different serotypes.