The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.
We generated a SARS-CoV-2 reverse genetics system; characterized virus RNA transcription profiles; evaluated the effect of ectopically expressed proteases on virus growth; and used reporter viruses to characterize virus tropisms, ex vivo replication, and to develop a high-throughput neutralizing assay. These reagents were utilized to explore aspects of early infectivity and disease pathogenesis relevant to SARS-CoV-2 respiratory infections.
Our single-cell RNA-ISH technology extended the description of ACE2 in respiratory epithelia on the basis of scRNA-seq data. Single-cell RNA-ISH detected ∼20% of upper respiratory cells expressing ACE2 versus ∼4% for scRNA-seq. Most of the RNA-ISH-detected ACE2-expressing cells were ciliated cells, not normal MUC5B+ secretory (club) cells or goblet cells. Notably, the nose contained the highest percentage of ACE2-expressing ciliated cells in the proximal airways. The higher nasal ACE2 expression-level findings were confirmed by qPCR data comparing nasal to bronchial airway epithelia. qPCR data also revealed that ACE2 amounts further waned in the more distal bronchiolar and alveolar regions. Importantly, these ACE2 expression patterns were paralleled by high SARS-CoV-2 infectivity of nasal epithelium with a gradient in infectivity characterized by a marked reduction in the distal lung (bronchioles and alveoli)
Multiple aspects of the variability in SARS-CoV-2 infection of respiratory epithelia were notable in these studies. First, significant donor variations in virus infectivity and replication efficiency were observed. Notably, the variability was less in the nose than lower airways. The reason(s) for the differences in lower airway susceptibility are important but remain unclear. We identified variations in ACE2 receptor expression but not numbers of ciliated cells as potential variables. Second, variation in infectivity of a single cell type, i.e., the ciliated cell, was noted with only a fraction of ciliated cells having access to virus infected at 72 h. Third, the dominant secretory cell, i.e., the MUC5B+ club cell, was not infected in vitro or in vivo, despite detectable ACE2 and TMPRSS2 expression. Collectively, these data suggest that measurements of ACE2 and TMPRSS2 expression do not fully describe cell infectivity and that a description of other variables that mediate susceptibility to infection, including the innate immune system(s), is needed.
The ACE2 receptor gradient in the normal lung raised questions focused on the initial sites of respiratory tract virus infection, the mechanisms that seed infection into the deep lung, and the virus-host interaction networks that attenuate or augment intra-regional virus growth in the lung to produce severe disease, especially in vulnerable patients experiencing chronic lung or inflammatory diseases.
We speculate that nasal surfaces might be the dominant initial site for SARS-CoV-2 respiratory tract infection. First, SARS-CoV-2 RNA has been detected in aerosol particles in the range of aerodynamic sizes exhaled during normal tidal breathing. Aerosol deposition and fomite mechanical delivery deposition modeling suggest that aerosols containing virus inhaled by naive subjects achieve the highest density of deposition, i.e., highest MOI per unit surface area, in the nose. Second, the relatively high ACE2 expression in nasal specimens and the parallel high infectivity of the HNE cultures suggests the nasal cavity is a fertile site for early SARS-CoV-2 infection. Nasal infection likely is dominated by ciliated cells in the superficial epithelium, not nasal submucosal glands. Third, the nose is exposed to high but variable loads of environmental agents, producing a spectrum of innate defense responses. Hence, a portion of the variability of the clinical syndrome of COVID-19 might be affected by environmentally driven variance of nasal infectivity.
Another aspect of the variability of the COVID-19 syndrome is the variable incidence and severity of lower lung disease. It is unlikely SARS-CoV-2 is transmitted to the lung by hematogenous spread, as demonstrated by the absence of infection of MVE cells and by previous reports that indicate airway cultures are difficult to infect from the basolateral surface. Theoretically, infection could be transmitted directly to lower lung surfaces by microaerosol inhalation with deposition on and infection of alveolar surfaces mediated in part by the high ACE2 binding affinity reported for SARS-CoV-2 . However, given the low amounts of ACE2 expression in alveolar cells in health, the correlated poor infectivity in vitro, and the absence of a homogeneous pattern radiographically, the importance of this route remains unclear (In contrast, it is well-known that an oral-lung aspiration axis is a key contributor to many lower airways infectious diseases. Nasal secretions are swept from the nasal surface rostrally by mucociliary clearance and accumulate in the oral cavity at a rate of ∼0.5 mL/h where they are admixed with oropharyngeal or tonsillar fluid. Especially at night, it is predicted that a bolus of relatively high titer virus is aspirated into the deep lung, either via microaspiration or as part of gastro-esophageal reflex-associated aspiration, sufficient to exceed the threshold PFU/unit surface area needed to initiate infection. Note, our data that tracheas exhibited significant viral infection in vivo suggest that small-volume microaspiration could also seed this site. Tracheal-produced virus could then also accumulate in the oropharynx via mucus clearance for subsequent aspiration into the deep lung (Quirouette et al., 2020). Oropharyngeal aspirates also contain enzymes and/or inflammatory mediators that might condition alveolar cells for infection. Aspiration of SARS-CoV-2 into the lung is consistent with the patchy, bibasilar infiltrates observed by chest CT in COVID-19 (Xu et al., 2020). Notably, robust microaspiration and gastro-esophageal aspiration are observed frequently in subjects who are at risk for more severe COVID-19 lower respiratory disease, e.g., older, diabetic, and obese subjects. Finally, our autopsy studies demonstrated patchy, segmental or subsegmental disease, consistent with aspiration of virus into the lung from the oropharynx.
These speculations describing the early pathogenesis of SARS-CoV-2 upper and lower respiratory tract disease are consistent with recent clinical observations. The data from in COVID-19-positive subjects support the concept of early infection in the upper respiratory tract (0–5 d) followed by subsequent aspiration and infection of the lower lung. These authors focused on the oropharynx as a potential site of the early virus propagation. As noted above, however, a nasal-oropharyngeal axis also exists, which has two implications. First, the nasal surfaces could seed the oropharynx for infection. Second, it is likely that oropharyngeal secretions reflect a mixture of local secretions admixed with a robust contribution of nasal mucus and virus.
Animal model data are also compatible with the scenario of aspiration-induced focal SARS-CoV-2 lung disease. The data of noted focal lung disease after combined intranasal versus intratracheal dosing with SARS-CoV-2 in cynomolgus monkeys. Notably, other findings in this model phenocopied our observations of human disease, e.g., early nasal shedding of virus, infection of nasal ciliated cells, and infection of AT2 and likely AT1 cells. Perhaps more definitive data describing nasal cavity seeding of the lower lung by microaspiration emanate from the studies. These investigators demonstrated in ferret models that genetically marked virus delivered to the nasal cavity more efficiently transmitted infection to the lower lungs than a virus with a distinct genetic marker delivered directly into the lungs.
In addition to identifying possible microaspiration risk factors associated with COVID-19 disease severity in the elderly, diabetic, and obese, our studies provide insights into variables that control disease severity in subjects at risk because of pre-existing pulmonary disease. For example, ACE2 expression was increased in the lungs of CF patients excised at transplantation. A major cytokine that produces the muco-inflammatory CF airways environment, IL-1β, was associated in vitro with increased ACE2 expression . The clinical outcome of increased ACE2 expression in CF is not yet known. The simple prediction is that increased ACE2 expression might be associated with more frequent or severe SARS-CoV-2 disease in CF populations. However, increased ACE2 expression is reported to be associated with improved lung function by negatively regulating ACE and the angiotensin II and the angiotensin II type 1a receptor (AT1a) in models of alveolar damage (pulmonary edema) and bacterial infection . Consequently, CF subjects might exhibit reduced severity of disease once acquired. Data describing outcomes of COVID-19 in the CF populations should emerge soon.
Our autopsy studies also provide early insights into the variable nature of the severity and pathogenesis related to post-COVID-19 lung health or function . Our study has identified another feature of COVID-19, i.e., the accumulation of apparently aberrantly secreted MUC5B in the alveolar region. Accumulation of MUC5B in the peripheral (alveolar) lung is characteristic of subjects who develop IPF, and polymorphisms in the MUC5B promoter associated with IPF have been reported . Future studies of the long-term natural history of SARS-CoV-2 survivors, in combination with studies delineating the cell types responsible for MUC5B secretion (AT2 versus airway cells) and genetics, e.g., MUC5B polymorphisms, might aid in understanding the long-term favorable versus fibrotic outcomes of COVID-19 disease.
Our study also provides a SARS-CoV-2 infectious full-length cDNA clone for the field. Several strategies have been developed to construct stable coronavirus molecular clones, including the bacterial artificial chromosome (BAC) and vaccinia viral vector systems . In contrast, our in vitro ligation method solves the stability issue by splitting unstable regions and cloning the fragmented genome into separate vectors, obviating the presence of a full-length genome. Our in vitro ligation strategy has generated reverse genetic systems for at least 13 human and animal coronaviruses and produced hundreds of mutant recombinant viruses. In contrast to other reports , reporter recombinant SARS-CoV-2 viruses generated herein replicated to normal WT amounts in continuous cell lines, allowing for robust ex vivo studies in primary cultures.
Using this infectious clone, we generated a high-throughput luciferase reporter SARS-CoV-2 assay for evaluation of viral nAbs. In line with previous reports, our data show that several SARS-CoV RBD-binding nAbs fail to neutralize SARS-CoV-2, suggesting distant antigenicity within the RBD domains between the two viruses. Although more samples are needed, early convalescent sera demonstrated ∼1.5 log variation in neutralizing titers at ∼day 30 after infection, demonstrating a need to fully understand the kinetics, magnitude, and durability of the neutralizing antibody response after a primary SARS-CoV-2 infection. The detection of low-level SARS-CoV-2 cross-neutralizing antibodies in 2003 SARS-CoV serum samples is consistent with recent studies suggesting that existence of common neutralizing epitopes between the two CoVs. Interestingly, convalescent COVID-19 sera failed to cross-neutralize SARS-CoV in vitro, suggesting cross-neutralizing antibodies might be rare after SARS-CoV-2 infection. The location of these epitopes is unknown. The nLuc recombinant viruses described herein will be powerful reagents for defining the antigenic relationships between the Sarbocoviruses, the kinetics and durability of neutralizing antibodies after natural infection, and the breadth of therapeutic neutralizing antibodies and vaccine countermeasures.
In summary, our studies have quantitated differences in ACE2 receptor expression and SARS-CoV-2 infectivity in the nose (high) versus the peripheral lung (low). These studies should provide valuable reference data for future animal model development and expand the pool of tissues, e.g., nasal, for future study of disease pathogenesis and therapy. Although speculative, if the nasal cavity is the initial site mediating seeding of the lung via aspiration, these studies argue for the widespread use of masks to prevent aerosol, large droplet, and/or mechanical exposure to the nasal passages. Complementary therapeutic strategies that reduce viral titer in the nose early in the disease, e.g., nasal lavages, topical antivirals, or immune modulation, might be beneficial. Finally, our studies provide key reagents and strategies to identify type-specific and highly conserved neutralizing antibodies that can be assessed most easily in the nasal cavity as well as in the blood and lower airway secretions.
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