Intra-host mutation rate of acute SARS-CoV-2 infection during the initial pandemic wave.

SARS-CoV-2 mutation is minimized through a proofreading function encoded by NSP-14. Most estimates of the SARS-CoV-2 mutation rate are derived from population based sequence data. Our understanding of SARS-CoV-2 evolution might be enhanced through analysis of intra-host viral mutation rates in specific populations. Viral genome analysis was performed between paired samples and mutations quantified at allele frequencies (AF) ≥ 0.25, ≥ 0.5 and ≥ 0.75. Mutation rate was determined employing F81 and JC69 evolution models and compared between isolates with (ΔNSP-14) and without (wtNSP-14) non-synonymous mutations in NSP-14 and by patient comorbidity. Forty paired samples with median interval of 13 days [IQR 8.5-20] were analyzed. The estimated mutation rate by F81 modeling was 93.6 (95%CI 90.8-96.4], 40.7 (95%CI 38.9-42.6) and 34.7 (95%CI 33.0-36.4) substitutions/genome/year at AF ≥ 0.25, ≥ 0.5, ≥ 0.75 respectively. Mutation rate in ΔNSP-14 were significantly elevated at AF ≥ 0.25 vs wtNSP-14. Patients with immune comorbidities had higher mutation rate at all allele frequencies. Intra-host SARS-CoV-2 mutation rates are substantially higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated mutation rate at low AF. Immunosuppressed patients have elevated mutation rate at all AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling.

Alpha to Omicron: Disease Severity and Clinical Outcomes of Major SARS-CoV-2 Variants.

BACKGROUND: Four SARS-CoV-2 variants predominated in the United States since 2021. Understanding disease severity related to different SARS-CoV-2 variants remains limited. METHOD: Viral genome analysis was performed on SARS-CoV-2 clinical isolates circulating March 2021 through March, 2022 in Cleveland, Ohio. Major variants were correlated with disease severity and patient outcomes. RESULTS: 2779 patients identified with either alpha (N = 1153), gamma (N = 122), delta (N = 808) or omicron variants (N = 696) were selected for analysis. No difference in frequency of hospitalization, ICU admission, and death were found among alpha, gamma, and delta variants. However, patients with omicron infection were significantly less likely to be admitted to the hospital, require oxygen, or admission to the ICU (X2 = 12.8 p < 0.001, X2 = 21.6 p < 0.002, X2 = 9.6 p = 0.01, respectively). In patients whose vaccination status was known, a substantial number had breakthrough infections with delta or omicron variants (218/808 [26.9%] and 513/696 [73.7%], respectively). In breakthrough infections, hospitalization rate was similar regardless of variant by multivariate analysis. No difference in disease severity was identified between omicron sub-variants BA.1 and BA.2. CONCLUSIONS: Disease severity associated with alpha, gamma, and delta variants is comparable while omicron infections are significantly less severe. Breakthrough disease is significantly more common in patients with omicron infection.

Antigenic Cross-Reactivity Between SARS-CoV-2 S1-RBD and Its Receptor ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein-protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2.

Genomic Epidemiology of SARS-CoV-2 Infection During the Initial Pandemic Wave and Association With Disease Severity.

Importance: Understanding of SARS-CoV-2 variants that alter disease outcomes are important for clinical risk stratification and may provide important clues to the complex virus-host relationship. Objective: To examine the association of identified SARS-CoV-2 variants, virus clades, and clade groups with disease severity and patient outcomes. Design, Setting, and Participants: In this cross-sectional study, viral genome analysis of clinical specimens obtained from patients at the Cleveland Clinic infected with SARS-CoV-2 during the initial wave of infection (March 11 to April 22, 2020) was performed. Identified variants were matched with clinical outcomes. Data analysis was performed from April to July 2020. Main Outcomes and Measures: Hospitalization, intensive care unit (ICU) admission, mortality, and laboratory outcomes were matched with SARS-CoV-2 variants. Results: Specimens sent for viral genome sequencing originated from 302 patients with SARS-CoV-2 infection (median [interquartile range] age, 52.6 [22.8 to 82.5] years), of whom 126 (41.7%) were male, 195 (64.6%) were White, 91 (30.1%) required hospitalization, 35 (11.6%) needed ICU admission, and 17 (5.6%) died. From these specimens, 2531 variants (484 of which were unique) were identified. Six different SARS-CoV-2 clades initially circulated followed by a rapid reduction in clade diversity. Several variants were associated with lower hospitalization rate, and those containing 23403A>G (D614G Spike) were associated with increased survival when the patient was hospitalized (64 of 74 patients [86.5%] vs 10 of 17 patients [58.8%]; χ21 = 6.907; P = .009). Hospitalization and ICU admission were similar regardless of clade. Infection with Clade V variants demonstrated higher creatinine levels (median [interquartile range], 2.6 [-0.4 to 5.5] mg/dL vs 1.0 [0.2 to 2.2] mg/dL; mean creatinine difference, 2.9 mg/dL [95% CI, 0.8 to 5.0 mg/dL]; Kruskal-Wallis P = .005) and higher overall mortality rates (3 of 14 patients [21.4%] vs 17 of 302 patients [5.6%]; χ21 = 5.640; P = .02) compared with other variants. Infection by strains lacking the 23403A>G variant showed higher mortality in multivariable analysis (odds ratio [OR], 22.4; 95% CI, 0.6 to 5.6; P = .01). Increased variants of open reading frame (ORF) 3a were associated with decreased hospitalization frequency (OR, 0.4; 95% CI, 0.2 to 0.96; P = .04), whereas increased variants of Spike (OR, 0.01; 95% CI, <0.01 to 0.3; P = .01) and ORF8 (OR, 0.03; 95% CI, <0.01 to 0.6; P = .03) were associated with increased survival. Conclusions and Relevance: Within weeks of SARS-CoV-2 circulation, a profound shift toward 23403A>G (D614G) specific genotypes occurred. Replaced clades were associated with worse clinical outcomes, including mortality. These findings help explain persistent hospitalization yet decreasing mortality as the pandemic progresses. SARS-CoV-2 clade assignment is an important factor that may aid in estimating patient outcomes.

Rapid SARS-CoV-2 Spike Protein Detection by Carbon Nanotube-Based Near-Infrared Nanosensors.

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.