Characterization of the CDV-H protein from CDV strains and their interactions with carnivore SLAM receptors.

<p>(a) Alignment of partial SLAM binding region of CDV-H protein sequences of the strains used in this study. Amino acids 528–550 are shown, and the amino acid at position 549 for each strain is indicated. (b) Variation in the extent of syncytia formation (mean number of nuclei per syncytium) induced by proteins from two domestic dog strains (<i>Dog94SE</i> and <i>A75/17</i>), two non-dog strains (<i>5804P</i> and <i>Lion94SNP</i>) and one CDV-H protein mutated at position 549 (<i>A75/17-549H</i>) in domestic dog (black bars), African lion (grey bars) or domestic cat SLAM-expressing cells (white bars). Error bars indicate standard deviations of six independent experiments. (c) Appearance of syncytia upon expression of CDV-H and CDV-F proteins from two dog strains (<i>Dog94SE</i> and <i>A75/17</i>), two non-dog strains (<i>5804P</i> & <i>Lion94SNP</i>) and one mutated CDV-H protein coding 549H (<i>A75/17-549H</i>) in domestic dog SLAM, African lion SLAM and domestic cat SLAM cells. Photos were taken 12 hours post transfection at 100× magnification.</p

Analysis of SLAM (CD150) receptors from different carnivore species.

<p>(a) Phylogenetic analysis of amino acid sequences of carnivore SLAM (CD150) protein (for details see supplementary information-methods), (b) Flow cytometry analysis of parental Vero cells and derived cell lines stably expressing domestic dog, lion, and cat SLAM (CD150) receptors.</p

Predicted expression of specialist and generalist traits by canine distemper virus strains in relation to different host species receptors (SLAM CD150).

<p>Predictions designed for CDV-H proteins were tested in an <i>in vitro</i> fusion assay (FA) and for CDV strains tested in an <i>in vitro</i> virus titration assay (TA). Strong co-evolution of CDV in a homogeneous environment (domestic dog) should produce strains that express specialist traits. Weak co-evolution of CDV in heterogeneous environments (non-dog carnivores) should produce strains expressing generalist traits. The homogeneous experimental environment is Vero cells expressing domestic dog SLAM. The heterogeneous experimental environment is Vero cells expressing African lion SLAM. The last two columns summarize the experimental results from this study.</p

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

(XLSX)</p

VOC Alpha and Delta display a postentry, spike-dependent replication advantage in NCI-H1299 cells, a human bronchial cell line with undetectable ACE2 protein level.

(A) Virus growth of B.1, VOC Alpha, and Delta was assessed on NCI-H1299 cells. Cells were infected (MOI 0.01) and supernatants of indicated time points were titrated on Vero E6 cells (left panel). Plaque morphology of NCI-H1299-derived B.1, VOC Alpha, and Delta on Vero E6 cells is shown (right panel). Results from 1 representative experiment out of 3 is shown. (B) NCI-H1299 cells were infected with rB.1, rB.1/VOC Alpha spike, or rVOC Alpha/B.1 spike viruses (MOI 0.01) and supernatant was titrated on Vero E6 cells. The growth experiment was performed once in triplicates. (C) ACE2 expression levels were analyzed by immunoblotting. Beta-actin was used as a loading control. (D) Expression of ACE2, TMPRSS2, and FURIN was quantified by quantitative RT-PCR in indicated cells. (E) Virus growth of B.1 and VOC Alpha was investigated on parental-NCI-H1299 (left) and respective ACE2-KO (right) cells. Cells were infected (MOI 0.01) and supernatants of indicated time points were titrated on Vero E6 cells. Three independent experiments, each in triplicates, were performed and are indicated by symbols. (F) NCI-H1299 cells were treated with increasing amounts of camostat mesylate (0–100 μM) for 2 hours at 37°C prior to infection with B.1 and VOC Alpha (MOI 0.01). After infection for 1 hour at 37°C, camostat mesylate was replenished to the infection medium and cells were incubated for 48 hours. Virus replication was determined from the supernatant by E gene assay. All values were normalized to the respective B.1 or VOC Alpha-infected, untreated control cells (dotted line at 100%). The experiment was performed once in duplicates. (G) NCI-H1299 cells were pretreated for 2 hours at 37°C prior to infection with B.1 and VOC Alpha (MOI 2) with MDL28170 (Cathepsin L inhibitor, 12.5 and 25 mM), pitstop II (clathrin inhibitor, 12.5 and 25 μM), camostat mesylate (TMPRSS2 inhibitor, 50 and 100 μM), or CMK (furin inhibitor, 2.5 and 5 μM). Entry efficiency was determined at 8 hours postinfection from cell lysates by sgN quantitative RT-PCR. The experiment was performed once in triplicates. (H, I) NCI-H1299, ACE2-KO-H1299, and ACE2+-H1299 cells were infected in triplicates at 4°C with (H) B.1 and VOC Alpha isolates or (I) rB.1, rB.1/VOC Alpha spike, and rVOC Alpha/B.1 spike viruses (MOI 2) to allow synchronized entry. Relative quantities of cell-associated nucleocapsid-specific subgenomic RNA were determined by Q-RT-PCR. Three independent experiments were performed, each conducted in 4 replicates. Symbols represent the arithmetic means of each experiment. (J) Synchronized infection of NCI-H1299 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 sgRNA N transcription of the untreated (UT) 8 hours postinfection sample (dotted line). Results of 3 independently conducted experiments, which were each performed in triplicates, are shown. (K) NCI-H1299 and Calu-3 cells were infected with B.1 and VOC Alpha isolates (MOI 0.01) for 48 hours and supernatant was titrated by plaque assay on Vero E6 cells to determine PFU/ml. Genome equivalents (GE/ml) were determined by E gene assay. Two independent experiments, each in triplicates, were performed. Gray numbers indicate mean relative virus infectivity of VOC Alpha to B.1 of the independent experiments. (L) NCI-H1299 and Calu-3 cells were infected with rB.1, rB.1/VOC Alpha spike, and rVOC Alpha/B.1 spike viruses (MOI 0.01) for 72 hours and supernatant was titrated by plaque assay on Vero E6 cells to determine PFU/ml. Genome equivalents (GE/ml) were determined by E gene assay. Two independent experiments, each in triplicates, were performed. Gray numbers indicate mean relative virus infectivity to rB.1 of the independent experiments. Bars represent arithmetic means of independent experiments. KO, knock-out; MOI, multiplicity of infection; PFU, plaque-forming units; RT-PCR, real-time PCR; 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

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

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

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

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