SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

Abstract

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic ¹ . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

Figure 1.. In vitro characterization of S1 proteins and recombinant SARS-CoV-2^S−614D and SARS-CoV-2^S−614G viruses.

(a) Affinity between S1 and hACE2 determined by Bio-layer interferometry. Biotinylated S1 protein (S1–614D or S1–614G) was loaded onto surface of streptavidin biosensors. Association was conducted using hACE2 protein followed by dissociation. (b) Binding of Fc-tagged or polyhistidine-tagged S1 to BHK-hACE2 cells is shown as peaks of fluorescence detected by flow cytometry. (c) Replication kinetics of recombinant viruses in (left) Vero E6 at 37°C and (right) hNE at 33°C. Supernatant was collected at indicated time points and titrated by plaque assay. Data represent the mean ± s.d. of three replicates (Vero E6) and four replicates (hNE). (d) Replication kinetics of recombinant viruses in NhBE at 33°C (left), 37°C (middle) and 39°C (right). NhBE were infected with 100,000 PFU of each virus. Supernatants were collected daily and titrated by TCID50 assay. Data represent the mean ± s.d. of four replicates. (c-d) Statistical significance was determined by two-sided unpaired Student’s t-test without adjustments for multiple comparisons. (c) P values (left to right): left, NS, P=0.9132; NS P=0.0604; NS P=0.2394; NS P=0.2389; NS P=0.2778; NS P=0.2781; right, NS P=0.1520; NS P=0.3891; NS P=0.9110; NS P=0.8985; NS P=0.1464. (d) P values (left to right): left, NS P=0.7943; NS P=0.5025; NS P=0.6683; NS P=0.8985; *P=0.0220; middle, **P=0.0065; NS P=0.4660; NS P=0.3134; *P=0.0159; right, **P=0.0094; ****P<10⁻⁴; **P=0.0028; ***P=0.0009. (e-f) Competition assay of recombinant viruses in hNE at 33°C and NhBE at 33°C, 37°C and 39°C. The inoculum was prepared by mixing two viruses at 1:1 ratio based on PFU ml⁻¹ and used for infection of hNE and NhBE. Apical wash and supernatant were collected daily, and extracted RNA was used for sequencing. (e-f) Bar graph shows proportion of sequencing reads encoding either S-614D or S-614G, and square dots represent individual data points.

Figure 2.. Replication of SARS-CoV-2^S−614D and SARS-CoV-2^S−614G viruses in hACE2 knock-in mice.

(a) Experimental scheme for infection of hACE2-KI mice intranasally infected recombinant SARS-CoV-2^S−614D and SARS-CoV-2^S−614G viruses. Oropharyngeal swabs were sampled daily and tissue samples were analyzed in sub-groups of 4 mice at 2 and 4 days post infection (dpi) in two independent experiments. (b) Quantitative RT-PCR analysis of oropharyngeal swabs of inoculated hACE2-KI and wild-type mice. (c,d) Pie chart representation of mean frequencies of A or G nucleotide at position 23,403 corresponding to SARS-CoV-2^S−614D and SARS-CoV-2^S−614G, respectively. Each pie chart illustrates the ratio of A/G detected from individual oropharyngeal swab samples (c) and tissues (d) at indicated time post infection. OB, olfactory bulb; ND, not detected.

Figure 3.. Replication and transmission of SARS-CoV-2^S−614D and SARS-CoV-2^S−614G viruses in Syrian hamsters.

Transmission of SARS-CoV-2^S−614D and ^S−614G variant by hamsters in a pairwise one-by-one setup with direct contact of donor and cohoused contact hamsters is illustrated. Samples of nasal washings were taken daily between days 2 to 8 post infection (dpi) and finally at 12 dpi and were analyzed. Pie chart representation of fraction of A or G nucleotide at position 23,403 corresponding to SARS-CoV-2^S−614D and SARS-CoV-2^S−614G, respectively, measured by amplicon sequencing. Genome copies were calculated from RT-qPCR using a standard RNA. Orange coloring of the hamster silhouette refer to detection of G (SARS-CoV-2^S−614G), while blue coloring indicates detection of A (SARS-CoV-2^S−614D) on most time points. Grey coloring signals no infection detected

Figure 4.. Replication and transmission of SARS-CoV-2^S−614D and SARS-CoV-2^S−614G viruses in ferrets.

Schematic illustration of the experimental setup with six pairs of donor ferrets cohoused with naïve contact ferrets. Samples of nasal washings were taken daily between days 2 to 8 post infection (dpi) and finally at 12 dpi and were analyzed. Pie chart representation of fraction of A or G nucleotide at position 23,403 corresponding to SARS-CoV-2^S−614D and SARS-CoV-2^S−614G, respectively. Each pie chart illustrates the ratio of A/G detected from individual nasal washing samples over time. Orange coloring of the ferret silhouette refer to detection of G (SARS-CoV-2^S−614G) on most time points, while blue coloring indicates detection of A (SARS-CoV-2^S−614D). Numbers represent total genome copies ml⁻¹ and grey coloring signals no infection or viral genome number too low for A/G ratio determination in sequencing. ND, not detected.