Delayed cytopathic onset of VOC Alpha SARS-CoV-2 infection.

Vero E6 cells were infected with B.1, VOC Alpha/v1, and VOC Alpha/v2 (MOI 0.001). Onset of CPE was monitored by live cell imaging until 70 hours postinfection. CPE, cytopathogenic effect; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. (MP4)</p

Primers used for reconstruction of recombinant SARS-CoV-2.

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VOC Alpha and B.1 efficiently dampen induction of innate immunity in hBAECs.

hBAECs were infected with B.1 or VOC Alpha (MOI of 0.5) and cell lysates were generated at the indicated time points followed by total RNA extraction. The experiment was performed with cells derived from 1–5 adult donors and that were infected in duplicates. (A) Cell-associated expression of envelope in hBAECs during the early phase of infection determined by Q-RT-PCR. TBP was used for normalization. (B) Cell-associated expression of sgN in hBAECs during an early phase of infection determined by Q-RT-PCR. (C–I) Expression of the indicated genes was determined by Q-RT-PCR. Shown is the mean fold change +/− SD. (J) Relative change (to preinfection) of cytokines and chemokines concentration in the basal medium of infected hBAECs (MOI 0.5). Concentration of cytokines and chemokines was determined by MagPix Luminex technology. Paired t tests were conducted between B.1 and VOC Alpha-infected groups and scored negative. AEC, airway epithelial cells; hBAEC, human bronchial airway epithelial cell; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein; VOC, variant of concern. See S1 Data.</p

VOC Alpha spike is not superior in mediating entry compared to B.1 spike.

(A) Calu-3 cells were transduced for 72 hours with increasing amounts of lentiviral particles (0.1 μl, 1 μl, and 10 μl) pseudotyped with either B.1 or VOC Alpha spike proteins. Pseudotype entry was analyzed luminometrically in cell lysates. (B) Calu-3 cells were pretreated with 25 μM MDL28170 (Cathepsin L inhibitor), 25 μM pitstop II (clathrin inhibitor), 100 μM Camostat (TMPRSS2 inhibitor), or 15 μM CMK (furin inhibitor), infected and entry efficiency was determined by sgN Q-RT-PCR. (C) Calu-3 cells were infected with Calu-3-derived virus stocks. Entry efficiency was determined by sgN-specific Q-RT-PCR from cell lysates at 4 hours postinfection. Cam, Camostat mesylate; CatL, Cathepsin L; DMSO, Dimethylsulfoxid; PS: PitStop; Q-RT-PCR, quantitative real-time PCR; sgN, subgenomic nucleocapsid; TMPRSS2, transmembrane protease serine subtype 2; VOC, variant of concern. See S1 Data. (TIF)</p

VOC Alpha fails to escape from neutralizing antibodies.

(A) Neutralizing titers against the indicated virus strains were determined in PRNTs. Red line indicates median titers per group. (B) Inhibition of ACE2/RBD interaction was measured using surrogate virus neutralization assays. Sera were tested using RBD proteins of B.1, VOC Alpha-, and Beta-VOC as indicated. Red lines indicate median values. The same set of samples was measured in (A) and (B), vaccinees n = 19, non-VOC convalescent donors n = 50, B1.1.7 patients n = 13. PRNT, plaque reduction neutralization test; VOC, variant of concern. See S1 Data. (TIF)</p

Primers used for quantitative RT-PCR.

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Similar abundance of sgN RNAs, genome replication, and low but similar expression of IFNs, proinflammatory cytokines, and ISGs in B.1 and VOC Alpha-infected H1299 cells.

NCI-H1299 cells were infected with B.1 or VOC Alpha (MOI of 2), and viral replication, viral transcription, and expression of innate immune genes were determined by Q-RT-PCR from cell lysates at 24 and 48 hours postinfection. (A) Expression of cell-associated envelope. (B) Expression of cell-associated sgN RNA. TBP was used for normalization. (C) Expression of the indicated genes was determined by specific Q-RT-PCR. TBP was used for normalization. Shown is the mean fold change +/− SD of 3 biologically independent experiments that were each conducted in quadruples. RVFV cl.13, which is devoid of its IFN antagonist NSs, was included for the expression of IFNs, ISGs, and pro-inflammatory cytokines. GE, genome equivalents; IFN, interferon; ISG, IFN-stimulated gene; Q-RT-PCR, quantitative real-time PCR; RVFV cl.13, Rift Valley Fever Virus clone 13; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein. See S1 Data. (TIF)</p

Relative sgRNA level normalized to total RNA reads and infection efficiency in B.1- and VOC Alpha-infected Calu-3 cells.

(A) RNA-seq analysis was conducted from total cell lysates that were obtained 24 hours postinfection to quantify sgRNA proportions in SARS-CoV-2-infected cells (MOI of 2). Canonical, as well as ORF9b and N* sgRNAs were quantified from the RNA-seq dataset. Data were normalized to total RNA reads. (B, C) Number of SARS-CoV-2 nucleocapsid (N)-positive Calu-3 cells was determined by flow cytometry. Calu-3 were left either UI or were infected with B.1 and VOC Alpha (MOI of 2) for 24 hours, permeabilized and immunostained with rabbit-anti-SARS-CoV-2 nucleocapsid antibody, followed by goat anti-rabbit Alexa 488 secondary antibody. (B) Percentage of SARS-CoV-2 N-positive cells. (C) Gating strategy of living-, single-, and N-positive cells is depicted for UI, B.1-, and VOC Alpha-infected cells. MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, subgenomic RNA; UI, uninfected; VOC, variant of concern. See S1 Data. (TIF)</p

Competition assay, additional targets.

Calu-3 cells were infected with a mixture of B.1 and VOC Alpha at indicated ratios (B.1:VOC Alpha/v1 ratio of 1:1, 9:1, and 1:9) with a total infectious dose of 10,000 PFU (corresponding to an MOI of 0.04). After serial passaging, viral RNA from the supernatant was isolated, sequenced, and the relative proportion of B.1- and VOC Alpha-corresponding sequences, discriminated by mutations in NSP3 (A), Spike amino acid positions 501 (B) and 681 (C) was plotted. Data show individual values of triplicates of 1 experiment. MOI, multiplicity of infection; PFU, plaque-forming units; p0-p5, passage 0–passage 5; VOC, variant of concern. See S1 Data. (TIF)</p

Absence of detectable fitness advantages of VOC Alpha in primary human respiratory cells, organoids, and hamsters.

(A) Virus growth kinetics were performed in infected hNAECs (MOI 0.1). Samples were collected from the apical and basal side at indicated time points and titrated by plaque assay. n = 3 biological replicates. (B) Virus growth kinetics was conducted in infected bronchial AEC (MOI 0.5). Samples were collected from the apical side and titrated by plaque assay. Data are derived from 1 experiment conducted in triplicates. (C) Intestinal organoids were infected (MOI 0.05) and viral load in supernatant (left) and organoid lysates (right) was quantified at indicated time points by E-gene-specific quantitative RT-PCR. Data are derived from 4 independent experiments. (D) Virus replication was monitored in infected lung organoids (MOI 1). Samples harvested at indicated time points were titrated by plaque assay. Data are derived from 3 independent experiments. (E) Dwarf hamsters were intranasally infected (100,000 PFU) and infectious virus particles from lung homogenates were quantified using plaque assay (left). Donor hamsters were cohoused with naive animals and transmission efficiency was determined from lung homogenates at the indicated time points (right). n = 1–3 animals per experimental condition. Dotted horizontal lines indicate the lower detection limit of the plaque assays. AEC, airway epithelial culture; GE, genome equivalents; hNAEC, human nasal airway epithelial culture; MOI, multiplicity of infection; n.d., not detected; PFU, plaque-forming units; RT-PCR, real-time PCR; VOC, variant of concern. See S1 Data.</p