The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary human nasal epithelial cells grown at air–liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal [Severe acute respiratory syndrome (SARS)-CoV-2 and Middle East respiratory syndrome-CoV (MERS-CoV)] and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures, though replication is differentially modulated by temperature. Infections conducted at 33 °C vs. 37 °C (reflective of temperatures in the upper and lower airway, respectively) revealed that replication of both seasonal HCoVs (HCoV-NL63 and -229E) is significantly attenuated at 37 °C. In contrast, SARS-CoV-2 and MERS-CoV replicate at both temperatures, though SARS-CoV-2 replication is enhanced at 33 °C late in infection. These HCoVs also diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV receptor availability as well as replication. MERS-CoV receptor DPP4 expression increases with IL-13 treatment, whereas ACE2, the receptor used by SARS-CoV-2 and HCoV-NL63, is down-regulated. IL-13 treatment enhances MERS-CoV and HCoV-229E replication but reduces that of SARS-CoV-2 and HCoV-NL63, reflecting the impact of IL-13 on HCoV receptor availability. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.

The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary nasal epithelial cells grown at air-liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal (SARS-CoV-2 and MERS-CoV) and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures but diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV replication, enhancing MERS-CoV replication but reducing that of SARS-CoV-2 and HCoV-NL63. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.

Background High sensitivity SARS-CoV-2 antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. Methods We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection (LOD) of 0.5 pg/mL (10.6 fmol/L) nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives, and quantified and tracked antigen dynamics in ICU COVID inpatients and immunocompromised COVID patients. Results Compared to qualitative RT-PCR methods, the MSAA demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. Conclusions The MSAA enables sensitive and specific detection of acute infections, quantification and tracking of antigen burden, and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples. Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients. Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections. Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.