key: cord-0807742-m1muu2cq authors: Stravalaci, M.; Pagani, I.; Paraboschi, E. M.; Pedotti, M.; Doni, A.; Scavello, F.; Mapelli, S. N.; Sironi, M.; Varani, L.; Matkovic, M.; Cavalli, A.; Cesana, D.; Gallina, P.; Pedemonte, N.; Capurro, V.; Clementi, N.; Mancini, N.; Invernizzi, P.; Rappuoli, R.; Duga, S.; Bottazzi, B.; Uguccioni, M.; Asselta, R.; Vicenzi, E.; Mantovani, A.; Garlanda, C. title: Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules date: 2021-06-08 journal: nan DOI: 10.1101/2021.06.07.21258350 sha: ffe1b96080b3e07c552a3cd9d01eec9ba8ece774 doc_id: 807742 cord_uid: m1muu2cq The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in COVID-19. The present study was designed to conduct a systematic investigation of the interaction of humoral fluid phase pattern recognition molecules (PRM) with SARS-CoV-2. Out of 10 PRM tested, the long pentraxin PTX3 and Mannose Binding Lectin (MBL) bound the viral Nucleoprotein and Spike, respectively. MBL bound trimeric Spike, including that of variants of concern, in a glycan-dependent way and inhibited SARS-CoV-2 in three in vitro models. Moreover, upon binding to Spike, MBL activated the lectin pathway of complement activation. Genetic polymorphisms at the MBL locus were associated with disease severity. These results suggest that selected humoral fluid phase PRM can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications. hepatitis C virus (HCV) and herpes simplex virus, as well as to the nonenveloped rotavirus 67 (Holmskov et al., 2003) . Interaction may result in opsonization, agglutination, inhibition of 68 viral fusion and entry, or complement activation, generally leading to inhibition of infection 69 (Holmskov et al., 2003) . Among pentraxins, the long pentraxin PTX3 has been show to interact 70 with H3N2-subtype influenza virus type A by interacting with viral envelope hemagglutinin 71 and neuraminidase glycoproteins through a sialic acid residue on its glycosidic moiety 72 (Reading et al., 2008) , with cytomegalovirus (CMV) (Bozza et al., 2006) , and with the 73 protein with other collectins involved in innate immunity, such as Collectin-12 (CLP-1) and 124 the pulmonary surfactant proteins SP-A and SP-D. We also extended the analysis to 125 recombinant Ficolin-1, -2, or -3, a family of proteins known to activate the complement lectin 126 pathway, and structurally-related to MBL. As shown in Figure 2D and 2E, in contrast with 127 MBL, CLP-1, SP-A, SP-D, and ficolins did not bind to SARS-CoV-2 Spike protein, indicating 128 that recognition of Spike is unique to MBL. 129 We further characterized the interaction of SARS-CoV-2 Spike protein with MBL by 130 Surface Plasmon Resonance (SPR). Different concentrations of recombinant, SARS-CoV-2 131 Spike protein or RBD domain were flowed onto MBL immobilized on the biosensor surface. 132 As shown in Figure 2F and Extended Data Figure 2 , trimeric SARS-CoV-2 Spike protein 133 formed a stable calcium-dependent complex with nanomolar affinity (KD=34 nM) whereas 134 MBL did not bind the isolated RBD, confirming the results obtained using the S1 subunit. 135 To mimic the interaction between MBL and SARS-CoV-2 Spike protein in its 136 physiological conformation in the viral envelope, we investigated the binding of viral particles 137 of SARS-CoV-2 Spike protein pseudotyped on a lentivirus vector to MBL-coated plates. The 138 interaction was determined by lysing the bound pseudovirus and measuring the lentiviral vector 139 p24 core protein by ELISA. While control lentiviral particles pseudotyped with the VSV-g 140 glyprotein (VSV-pseudovirus) did not result in any binding, those exposing the SARS-CoV-2 141 Spike protein showed a specific interaction with MBL ( Figure 3A ). These data strongly suggest 142 that MBL can also interact with the SARS-CoV-2 Spike protein exposed on the virus surface. 143 The SARS-CoV-2 Spike protein is highly glycosylated, as recently described 146 (Watanabe et al., 2020) . Out of the 22 N-glycosylation sites, 8 contain oligomannose-types 147 glycans, which could be interaction sites for the MBL carbohydrate recognition domain (CRD). 148 To address this possibility, we performed a solution-based competition assay with D-mannose 149 and N-acetyl-glucosamine, two specific ligands of the lectin. As shown in Figure 3B , D-150 mannose and N-acetyl-glucosamine inhibited MBL binding to the Spike protein, thus 151 confirming the Ca 2+ -dependent interaction between the MBL lectin domain and the glycosidic 152 sites exposed by the Spike protein. D-Glucose, a non-specific ligand of MBL, inhibited the 153 interaction only at higher concentration ( Figure 3B ). Based on the alignment of MBL crystal 154 structure with mannose molecules (Fig. 3C) , we identified 14 putative binding sites on the 155 Spike protein (Fig. 3D ). Next, we considered sites having a high (>80%) oligomannosylation 156 occupancy (Watanabe et al., 2020 ). This analysis provided two possible MBL binding sites, 157 namely N603, N801 and N1074 all on the same Spike chain, or N603, N1074 and N709 with 158 N709 on a neighboring chain ( Figure 3E ). Interestingly, in both cases, the hypothesized MBL 159 binding sites spans across the S1 and S2 region ( Figure 3F ) of the Spike protein providing hints 160 to a possible inhibition mechanism. These data indicate that the glycosylation state of the 161 SARS-CoV-2 Spike protein is important for its interaction with MBL. 162 163 We then tested whether MBL recognized Spike proteins from VoC. First, we analyzed 165 whether the known 22 glycosylation sites of each protomer are affected by the reported 166 mutations. Figure 3G We asked whether the interaction of MBL with Spike could activate the complement 177 lectin pathway. We incubated SARS-CoV-2 Spike protein-coated plates with human serum, or 178 C1q-or C4-or C3-depleted serum, and we assessed the deposition of C5b-9. As shown in 179 incubation with a serum depleted of C4 strongly reduced C5b-9 deposition, with levels 182 comparable to those observed with heat-inactivated serum or C3-depleted serum. 183 Reconstitution of C4-depleted serum with purified C4 restored C5b-9 deposition levels similar 184 to those observed with normal human serum. To further address the role of MBL in SARS-185 CoV-2 Spike protein-mediated complement activation, we assessed C5b-9 deposition by 186 incubating normal human serum or MBL-immunodepleted serum over captured SARS-CoV-2 187 Spike protein, either as active, or non-covalent trimer ( Figure 3I , right panel). In agreement 188 with binding data, no complement deposition was observed with the non-covalent trimeric 189 Spike protein. Notably, immunodepletion of MBL from human serum resulted in a significant 190 reduction in C5b-9 deposition, which could be fully reverted by addition of rhMBL ( Figure 3I , 191 right panel). These data clearly indicate that SARS-CoV-2 Spike, by interacting with MBL, 192 activates the complement lectin pathway. 193 To validate the relevance of the interaction between MBL and SARS-CoV-2 Spike 196 protein, we investigate whether MBL inhibited SARS-CoV-2 entry in susceptible cells. We 197 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Among the soluble PRMs tested, MBL was found to be the only molecule with anti-SARS-201 CoV-2 activity. Spike-mediated viral entry was inhibited by 90% at the highest concentration 202 of 10 µg/ml (34 nM) with an EC50 value of approximately 0.5 µg/ml (1.7 nM) ( Figure 4A ). 203 As control, entry of lentiviral particles pseudotyped with the VSV-g glycoprotein was not 204 inhibited by MBL ( Figure 4A ). Figure 3B ). The calculated EC50 was 0.08 µg/mL (0.27 nM) at 72h. Notably, MBL showed 222 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. a concentration-dependent inhibition of infection of Calu-3 cells also by SARS-CoV-2 variant 223 20I/501Y.V1 (B.1.1.7) at MOI 1 ( Figure 4C ) and MOI 0.1 (Extended Data Figure 3C ), as well 224 as by 20H/501Y.V2 (B.1.351) at MOI 1 ( Figure 4D) . 225 Furthermore, a model of 3D-human bronchial epithelial cells (HBEC) was used to 226 test whether MBL inhibited SARS-CoV-2 replication. SARS-CoV-2 production at the 227 epithelial apical surface increased sharply at 48 h PI (not shown), reaching 48x10 6 ± 6x10 6 228 (mean ± SEM) PFU/ml 72 h PI. Treatment of HBEC with MBL decreased viral production 229 to 4x10 6 ± 0.8x10 6 PFU/ml 72 h PI at the highest concentration of 50 µg/ml (170 nM) ( Figure 230 5A). In contrast PTX3 treatment was ineffective at inhibiting virus production (Extended 231 Data Figure 3D ). We then assessed whether in these experimental conditions, MBL affected 232 inflammatory responses in HBEC upon SARS-CoV-2 infection. As shown in Extended Data 233 Figure 3E , MBL treatment inhibited the production of IL-8 and CXCL5, chemokines 234 involved in myeloid cell recruitment and activation. 235 We finally evaluated occurrence of MBL-Spike protein interaction in SARS-CoV-2 236 infected HBEC by confocal microscopy. As shown in Figure 5B and C, MBL colocalized with 237 SARS-CoV-2 Spike protein in infected cells. In 3D rendered images of the HBEC cell cultures 238 ( Figure 5D and Movie S1), colocalization was preferentially associated to the apical side of 239 cytokeratin 14 positive cells. Evidence of the interaction between MBL and SARS-CoV-2 240 Spike protein in infected HBEC at molecular scale (<100nm XY spatial resolution) were also 241 obtained in STED-based super-resolution microscopy ( Figure 5E ). 242 243 MBL2 haplotypes are associated with severe COVID-19 244 MBL2 genetic variants have been shown to correlate with increased susceptibility to 245 selected infections, including SARS (Ip et al., 2005) . To explore the significance of our in vitro 246 results in the frame of COVID-19 pandemic, we investigated the possible association of MBL2 247 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 6 ), whereas haplotype-based analysis 266 disclosed 7 haplotypes of different lengths, from 2 to 24 SNPs, strongly associated with severe 267 COVID-19 (all surviving the correction for multiple tests; Figure 6 ; Table 1D ). Among them, 268 the one composed of polymorphisms rs10824844-rs10824845 incorporates one of the two top-269 markers evidenced by the single-SNP association analysis and is present in 12.2% of cases and 270 6.9% of controls (TA haplotype, OR=1.88, 95%CI=1.44-2.45, P=1.04*10 -5 ; Table 1D ). Hence, 271 we performed a meta-analysis based on the rs10824845 polymorphism by including the GHS 272 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Table 1E) attached to the residues N603, N801 and N1074 on the same chain or N603, N709 and N1074 295 with N709 on a different chain. In both cases the hypothesized MBL binding site spans across 296 the S1 and S2 region of SARS-CoV-2 Spike, suggesting a possible neutralization mechanism. 297 The binding of MBL could prevent the detachment of the S1 region and the release of the 298 fusion peptide at position 815, thus inhibiting virus entry into host cells. However, the 299 mechanisms responsible for the antiviral activity of MBL remain to be fully defined. It is 300 noteworthy that C-type lectins have been reported to act as entry receptors (or coreceptors) 301 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.07.21258350 doi: medRxiv preprint (https://www.encodeproject.org/) for H3K27Ac, H3K4Me1, H3K4Me3 histone modifications 506 marks, all derived from 7 cell lines; viii) the GeneHancer regulatory elements track. 507 (B) The Manhattan plot of the single-SNP association analysis is reported. The horizontal line 508 represents the suggestive P=5*10 −5 significance level. SNPs showing lowest P value signals 509 are indicated by an arrow. Bonferroni threshold for significance corresponds to P < 1.5*10 -5 . 510 511 512 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Anti-C1q polyclonal antibody was purchased from Dako. Anti-CRP and anti-SAP antibodies 558 were from Merck. 559 Recombinant His-Tag SARS-CoV-2 proteins were immobilized at different 561 concentrations (ranging from 6.25 to 50 pmol/mL) on 96-well Nickel coated plates (Thermo 562 Fisher Scientific) for 1 h at room temperature. Plates were then blocked for 2 h at 37°C with 563 200 µL of 2% BSA diluted in 10 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 2 564 mM CaCl2 and 0.1% Tween-20 (TBST-Ca 2+ ). Following blocking, plates were washed three 565 times with TBST-Ca 2+ and incubated for 1 h at 37°C with 100 µL PTX3 (4 µg/mL -12 nM in 566 TBST-Ca 2+ ), MBL (1-2 µg/mL -3.4-6.7 nM in TBST-Ca 2+ ), C1q (4 µg/mL -10 nM in TBST-567 Ca 2+ ), CRP (3 µg/mL -25 nM in TBST-Ca 2+ ) and SAP (4 µg/mL -32 nM in TBST-Ca 2+ ). 568 After washes, plates were incubated for 1 h at 37°C with specific primary antibodies, followed 569 by the corresponding HRP-conjugated secondary antibodies. Both primary and secondary 570 antibodies were diluted in TBST-Ca 2+ buffer. After development with the chromogenic 571 substrate 3,3',5,5'-tetramethylbenzidine (TMB, Thermo Fisher Scientific, USA), binding was 572 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In another set of experiments, 100 µL of 2 µg/mL rhMBL (6.7 nM), CLP-1 (6.7 nM), site of mannose molecules was determined aligning the MBL2 structure to the crystal structure 621 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. After washing, bound pseudotyped virus particles were lysed with 0.5% Triton X-100 and HIV 645 p24 core protein was detected by ELISA (Perkin Elmer; USA). 646 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.07.21258350 doi: medRxiv preprint Complement deposition assay 647 100 µL of biotinylated SARS-CoV-2 Spike protein (either active trimer or non-covalent 648 trimer, 1µg/mL in PBS) were captured on 96 well plates for 1 h at 37°C. After washing, wells 649 were incubated for 1 h at 37°C with either 10% normal human serum (NHS, ComplemenTech 650 Inc, USA), 10% C1q-depleted serum (C1qDHS), 10% C4-depleted serum (C4DHS) 651 reconstituted or not with 25 µg/mL purified C4 (Calbiochem, USA). 10% heat-inactivated 652 human serum (30' at 56°C, HI-NHS) and 10% C3-depleted serum (C3DHS) were used as 653 negative control. All the sera were diluted in 10 mM Tris-buffered saline containing 0.5 mM 654 MgCl2, 2 mM CaCl2 and 0.05% Tween-20, also used as washing buffer. For MBL 655 immunodepletion, 10% NHS was incubated overnight with 0.6 µg/mL rabbit anti-MBL 656 antibody. Bound MBL-antibody complexes were separated by Dynabeads Protein G (Thermo 657 Fisher Scientific), and the supernatant (termed MBL-ID) was used in the assay (final 658 concentration, 10%). After washing, C5b-9 deposition was assayed by incubation for 1 h at 659 37°C with rabbit anti-sC5b-9 antibody (ComplemenTech Inc., Usa) diluted 1:2000 in washing 660 buffer as described before (Stravalaci et al., 2020) , followed by specific HRP-conjugated 661 secondary antibody incubation and TMB development. 662 The Vero and Vero E6 cell line was obtained from the Istituto Zooprofilattico of 664 Brescia, Italy, and ATCC, respectively. Cells were maintained in Eagle's minimum essential 665 medium (EMEM; Lonza) supplemented with 10% fetal bovine serum (FBS; Euroclone) and 666 penicillin-streptomycin (complete medium). 667 Human embryonic kidney 293T cells, a continuous human embryonic kidney cell line 668 containing the mutant gene of SV40 Large T Antigen (ATCC code CRL-3216), were cultured 669 as described (Follenzi et al., 2000) . 670 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. University of Trento). Lentiviral vector stock expressing ACE2 was produced as described 695 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.07.21258350 doi: medRxiv preprint above. The entry assay was then optimized in 96-well plate by seeding 5x10 4 ACE2 696 overexpressing 293T cells/well. Twenty-four h later, cells and SARS-CoV-2 Spike-697 pseudotyped lentivirus stock (1:500) were incubated with serial dilutions of soluble innate 698 immunity molecules for 30 min. The SARS-CoV-2 Spike-pseudotyped was added to the cells 699 and forty-eight h later, cells were treated with accutase, in order to detach them from the wells, 700 fixed and analyzed for GFP expression by cytofluorimetry. 701 The (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Half of the ALI medium (1 ml) was collected from each well of the lower chamber 744 every 24 h PI and replaced with fresh ALI medium. The harvested medium was stored at -70 745 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. respectively. Confocal images were processed, 3D rendered and analyzed as colocalization rate 793 between Spike and MBL with Leica Application Suite X software (LASX; version 794 3.5.5.19976) and presented as medium intensity projection (MIP). STED images were de-795 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.07.21258350 doi: medRxiv preprint the server options to filter by an imputation of R²>0.1. In the post-imputation steps, we only 821 retained those SNPs with R²≥0.6 and minor allele frequency (MAF) ≥1%. Next, we accurately 822 checked cases and controls for solving within-Italian relationships and for testing the possible 823 existence of population stratification within and across batches: to this aim, we performed 824 principal component analysis (PCA), using a LD-pruned subset of SNPs across chromosome 825 10 and the Plink v.1.9 package (Chang et al., 2015) . The final set of analyzed variants 826 comprised 3,425 SNPs, distributed in the MBL2 region (the gene +/-500 kb). 827 Prism GraphPad software v. 8.0 (www.graphpad.com) was used for the statistical 829 analyses. Comparison among groups were performed using one or two-way analysis of 830 variance (ANOVA) and the Bonferroni's correction. Non-linear fit of transformed data was 831 determined by using the log (agonist) vs. response (three or four parameters). 832 For genetic studies, case-control allele-dose association tests were performed using the 833 PLINK v.1.9 logistic-regression framework for dosage data. Age, sex, age*age, sex*age, and 834 the first 10 principal components from PCA were introduced in the model as covariates. 835 Analyses were conducted always referring to the minor allele. All P values are presented as not 836 corrected and accompanied by odds ratio (OR) and 95% confidence interval (CI); however, in 837 the relevant (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.07.21258350 doi: medRxiv preprint In the meta-analysis, we took advantage of association data deposited in the Regeneron 846 -Genetic Center database (https://rgc-covid19.regeneron.com/home) for the GHS study 847 (Geisinger Health System; data available for 869 cases and 112,862 controls of European 848 ancestry). Pooled Ors and Cis were calculated using the Mantel-Haenszel model (Mantel and 849 Haenszel, 1959) 850 851 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. Movie S1. SARS-CoV-2 S protein and MBL colocalized on infected HBEC cells. 3D 886 rendering showing a blended reconstruction of the colocalization between SARS-CoV-2 S 887 protein and MBL in HBEC cultures, preferentially associated to the apical side. 888 889 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.07.21258350 doi: medRxiv preprint with Huygens Professional software (Scientific Volume Imaging B. 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These included: 799 i) 332 patients with severe COVID-19, which was defined as hospitalization with respiratory 800 failure and a confirmed SARS-CoV-2 viral RNA PCR test from nasopharyngeal swabs.