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

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

Evaluation of titration method on Vero E6 and Calu-3 cells for the calibration of B.1 and VOC Alpha seeding doses and comparison of E gene copy numbers versus infectious units (determined by Q-RT-PCR and plaque assay on Vero E6 cells) in SARS-CoV-2 stocks.

(A) No significant differences in the titers were observed when titers of B.1 and VOC Alpha SARS-CoV-2 stocks were compared on Calu-3 vs. Vero E6 using TCID50 titration method. Although plaque morphology of B.1- and VOC Alpha-infected Vero E6 cells differ, Vero E6 cells are suitable to determine titers by plaque titration assay. N = 3 biologically independent experiments each conducted in triplicates. (B) Overview on virus infectivity and viral RNA concentrations, determined by plaque assay (log10 PFU/ml) and E gene assay (log10 GE/ml), of all virus stocks is shown. Direct comparison of all B.1 and VOC Alpha stocks. Statistical analysis was conducted between both viruses for genomic E gene and infectious titers, respectively. Stocks applied in gene expression analysis (triangle) and growth kinetics (square) are highlighted by symbols. GE, genome equivalents; n.s., not significant; PFU, plaque-forming units; Q-RT-PCR, quantitative real-time PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. See S1 Data. (TIF)</p

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 0.01). 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.

(XLSX)</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

Inoculum data from all experiments conducted in this study.

(XLSX)</p

Enhanced cell–cell fusion and reduced virus particle entry by VOC Alpha SARS-CoV-2 spike.

(A) For Tat-mediated cell–cell fusion assay, CHO cells were cotransfected with plasmids expressing indicated spike-HA and HIV-1 Tat. LTR-luciferase-expressing target TZM-bl cells were transfected with plasmids encoding human ACE2 and TMPRSS2. Transfected cells were cocultured for 8 hours and luciferase expression resulting from intercellular Tat transfer was quantified luminometrically. All values were normalized to B.1 spike (indicated by a dotted line). Shown are results from 3–6 biological replicates, each performed in triplicates. (B) Calu-3 cells were transduced with lentiviral pseudoparticles expressing luciferase and decorated with indicated spike-HA. Transduction efficiency was quantified luminometrically. Dotted line indicates background levels of luciferase nontransduced cultures. Shown are results from 6 independent biological replicates (using independent lentivirus particle preparations), each performed in triplicates, indicated by symbols. (C) Indicated A549 cells were transduced with increasing quantities (0.5 μl, 5 μl, and 50 μl) of lentiviral, luciferase-expressing particles pseudotyped with B.1- or VOC Alpha-spike. Transduction efficiency was determined luminometrically. Dotted line indicates luciferase background level of luciferase detected in nontransduced cells. Symbols represent individual values of 3 biological replicates, each performed in triplicates. (D, E) Indicated A549 (D) and Calu-3 (E) cells were infected at 4°C with B.1 or VOC Alpha isolates (MOI 1) to allow synchronized infection. Total cellular RNA was isolated at the indicated time points and nucleocapsid-encoding sgRNA was quantified by Q-RT-PCR. N = mean of 3–4 biological replicates, indicated by symbols. (F) Synchronized infection of Calu-3 cells was performed with B.1 and VOC Alpha virions that were pretreated with trypsin for 1 hour at 37°C. Data were normalized to the respective log10 relative N sgRNA level of the untreated (0 μg/ml) 4 hours postinfection sample (dotted line). Means +/− SD of 6 independently performed experiments are shown. (G) Synchronized infection of Calu-3 cells was performed with B.1 and VOC Alpha virions that were pretreated with saliva (pooled saliva from 3 healthy donors) for 1 hour at 37°C. Data were normalized to the respective log10 relative sgRNA N level of the untreated (-) 4 hours postinfection sample (dotted line). Results show means of 4 independently performed experiments, which were each performed in triplicates. Del, deletion; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; RLU, relative light units; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA N, subgenomic nucleocapsid RNA; TMPRSS2, transmembrane protease serine subtype 2; VOC, variant of concern. See S1 Data.</p

Primers used for quantitative RT-PCR.

(XLSX)</p

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