Spike mutation D614G alters SARS-CoV-2 fitness

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic^1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.

Extended Data Figure 1.. Experimental design of hamster infection and sample harvest.

(a) Graphical overview of experiment to assess the impact of G614 mutation on replication in the respiratory system of hamsters. (b) Schematic organs harvested from hamsters sacrificed on days 2, 4, and 7 post-infection. Illustration of hamster lung adapted from Reznik, G. et al. Clinical anatomy of the European hamster. Cricetuscricetus, L., For sale by the Supt of Docs, U.S. Govt. Print. Off., 1978.

Extended Data Figure 2.. Validation of competition assay by Sanger sequencing.

(a) The correlation between input PFU ratios and output RT-PCR amplicon ratios determined by Sanger sequencing. D614 and G614 viruses were mixed at PFU ratios of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, or 1:10. Total RNA of the virus mixtures were extracted and amplified by RT-PCR. The D614/G614 ratios were calculated by the peak heights of Sanger sequencing. Data were analyzed by linear regression with correlation coefficients (r) and significance (p). Symbols represent individual replicates and the solid line represents the fitted line. Data is derived from a single experiment conducted in duplicate. (b) Assay range evaluation. The ratio of D614/G614virus mixture calculated from Sanger sequencing was consistent when using a wide range of virus amounts. The D614/G614 viruses were mixed at 1:1PFU ratio. The total titers of the mixed viruses were 10², 10³, 10⁴, 10⁵, and 10⁶ PFU. The total RNA of virus mixture was isolated and amplified by RT-PCR. The D614/G614 ratios were calculated by the peak heights from Sanger sequencing. Symbols represent individual replicates, bar heights represent the mean, and error bars represent the standard deviation. Data is derived from a single experiment conducted in triplicate.

Extended Data Figure 3.. D614G substitution significantly enhances SARS-CoV-2 replication in primary human airway tissues from a different donor.

D614 and G614 viruses were equally mixed and inoculated onto the airway tissue at a total MOI of 5. This airway tissue was produced from a different donor than that used in Figure 3. The tissues were washed by DPBS to collect the secreted viruses every day from days 1 to 5. The total RNAs were isolated and amplified by RT-PCR. The ratio of D614 and G614 viruses after competition were measure by Sanger sequencing. Circles represent individual samples (n=6, two independent experiments conducted in triplicate). The midline represents the sample mean, and the shaded region represents +/− one unit of standard error about the mean. The width of the catseye plot represents the distribution of the model-adjusted means, and the heights extend to span 99.8% of the distribution of the mean. The y-axis is displayed on the log₁₀ scale such that the null value is 1. P values are calculated for the group (strain) coefficient for each linear regression model, and are reported for all instances of p<0.05.

Extended Data Figure 4.. SARS-CoV-2 G614 is more stable than D614.

Equal amounts (10⁵ PFU/ml) of D614 and G614 viruses were incubated in DPBS at 42°C (a), at 37°C (b), or 33°C (c), respectively. At 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h, the viruses were quantified for their infectious levels by plaque assay on Vero E6 cells. The detect limitation of plaque assay is 10 PFU/ml. The percentage of remaining infectious viruses were normalized by the average titers at 0 h. Symbols represent individual replicates, bar heights represent the mean, and error bars represent the standard deviation. P values were determined by two-tailed Mann-Whitney test (n=9, from three independent experiments, each conducted in triplicate), and results of p<0.05 are indicated.

Extended Data Figure 5.. Scheme for preparing the D614 SARS-CoV-2-infected hamster sera for neutralization assay.

Eight sera were collected: Four sera (number 1–4) collected on day 28 post infection and another four sera (number 5–8) collected on day 49 after the second viral infection.

Extended Data Figure 6.. Construction of G614 mNeonGreen SARS-CoV-2.

(a) Diagram of the construction. The D614G mutation was introduced into a mNeonGreen reporter SARS-CoV-2 using the method as described previously. (b) Plaque morphologies of D614 and G614 mNeonGreen SARS-CoV-2.

Extended Data Figure 7.. Neutralization activities of hamster sera against D614 and G614 mNeonGreen SARS-CoV-2.

(a) Neutralizing curves of eight hamster sera against D614 and G614 mNeonGreenSARS-CoV-2. The neutralizing curve for serum 5 is shown in Fig. 4c. Symbols represent individual samples and the solid line represents the fitted curve. Data is derived from a single experiment conducted in duplicate. (b) Calculated NT₅₀ values and ratios of 1/NT₅₀ for all eight hamster sera. The mean ratios were determined by (D614 1/NT₅₀)/(G614 1/NT₅₀).

Extended Data Figure 8.. Neutralization activities of human mAbs against D614 and G614 mNeonGreen SARS-CoV-2 in Experiment I.

(a) Neutralizing curves of eleven mAbs against D614 and G614 reporter SARS-CoV-2. The neutralizing curve for mAb18 is shown in Fig. 4f. Symbols represent individual replicates and the solid line represents the fitted curve. Data is derived from a single experiment conducted in duplicate. (b) Calculated NT₅₀ values for all eleven mAbs.

Extended Data Figure 9.. Neutralization activities of human mAbs against D614 and G614 mNeonGreen SARS-CoV-2 in Experiment II.

(a) Neutralizing curves of eleven mAbs against D614 and G614 reporter SARS-CoV-2. Symbols represent individual replicates and the solid line represents the fitted curve. Data is derived from a single experiment conducted in either duplicate or quadruplicate. (b) Calculated NT₅₀ values for all eleven mAbs. (c) Summary of NT₅₀ ratios from two independent experiments. The ratios were determined by (D614 NT₅₀)/(G614 NT₅₀)

Figure 1.. D614G substitution improves SARS-CoV-2 replication on Calu-3 cells through increased virion infectivity.

(a) Construction of mutant G614 SARS-CoV-2. A single nucleotide A-to-G substitution was introduced to construct the spike D614G mutation in the infectious cDNA clone of SARS-CoV-2. (b) Plaque morphologies of D614 and G614 viruses, developed on day 2 pi in Vero E6 cells. (c-h) Viral replication and genomic RNA/PFU ratios of D614 and G614 viruses produced from Vero E6 cells (c-e) and from Calu-3 cells (f-h). Cells were infected at an MOI of 0.01. Infectious viral titers (c,f) and genomic RNA levels (d,g) in culture medium were determined by plaque assay and real-time RT-qPCR, respectively. The genomic RNA/PFU ratios (e,h) were calculated to indicate virion infectivity. Symbols represent individual samples, bar heights represent means, and error bars represent standard deviations. P values were determined by two-tailed Mann–Whitney test from a sample size of n=6 (two independent experiments, conducted in triplicate). All results of p<0.05 are reported. (i,j) Spike protein cleavage of purified virions. Purified D614 and G614 virions were analyzed by Western blot using polyclonal antibodies against spike and nucleocapsid. Full-length spike , S1/S2 cleavage form, and S2’ protein are annotated. Results from two independent experiments are presented for virions produced from Calu-3 cells (i) and Vero E6 cells (j).

Figure 2.. D614G substitution increases SARS-CoV-2 replication in the upper airway, but not the lungs, of hamsters.

(a-i) 3–4-Week-old male golden Syrian hamsters were infected intranasally with 2×10⁴ PFU of D614 or G614 SARS-CoV-2, or PBS. All data are from a single experiment. (a) Weight loss was monitored for seven days pi. Symbols represent means, error bars represent standard deviation. Sample sizes were n=18 for infected cohorts and n=14 for the mock cohort at days 0–2, n=12 for infected cohorts and n=10 for the mock cohort at days 3–4, and n=6 for all cohorts at days 5–7. Weight loss was analyzed by two-factor ANOVA with Tukey’s post-hoc test, with p>0.05 at all timepoints. (b,c) Infectious titers and (d) viral genomes were measured in the nasal wash, trachea, and lung on days 2 (b,d), 4 (c,d), and 7 (d) pi. (e,f) Genome/PFU ratios on days 2 (e) and 4 (f) pi were calculated as a measure of infectivity. (b-f) Symbols represent individual animals (n=6). Midlines (b,c,e,f) and bar heights (d) represent means. (b-f) Error bars represent standard deviation. Two-factor ANOVA with Sidak’s post-hoc test are reported for p<0.05. (g-i) Hamsters were inoculated with 1:1 mixtures of D614 and G614 viruses (10⁴ PFU each). Nasal wash, trachea, and lung were collected on days 2 (g), 4 (h), and 7 (i) pi. Relative fitness was assessed by Sanger sequencing. (g-i) Circles represent individual animals (n=6). Midline represents the mean. Shaded regions represent one unit of standard error about the mean. The width of the catseye plot represents the distribution of the model-adjusted means, and heights extend to 99.8% of the distribution. The y-axis is log₁₀ scaled. P values are calculated for the group (strain) coefficient for each linear regression model, and are reported for p<0.05.

Figure 3.. D614G substitution significantly enhances SARS-CoV-2 replication in primary human airway tissues.

(a) Experimental scheme. D614 and G614 viruses were inoculated onto the primary human airway tissues at an MOI of 5. After incubation for 2 h, the culture was washed with DPBS, then maintained at for 5 days. To harvest, DPBS was added and allowed to incubate at 37°C for 30 min to elute the virus. (b-d) Viral replication and genomic RNA/PFU ratios. The amounts of infectious virus (b) and genomic RNA (c) were quantified by plaque assay and real-time RT-qPCR, respectively. The genomic RNA/PFU ratio (d) was calculated to indicate virion infectivity. Symbols represent individual samples, bar heights represent means, and error bars represent standard deviations. P values were determined by two-tailed Mann–Whitney test (n=6, two independent experiments conducted in triplicate). The results of the Mann-Whitney test are reported for all instances of p<0.05. (e,f) Competition assay. A mixture of D614 and G614 viruses with initial ratios of 1:1 (e), 3:1(f), or 9:1(g) were inoculated onto the human airway tissues at a total MOI of 5. Ratios after competition were measure by Sanger sequencing. Circles represent individual samples (n=6, two independent experiments conducted in triplicate). The midline represents the sample mean, and the shaded region represents +/− one unit of standard error about the mean. The width of the catseye plot represents the distribution of the model-adjusted means, and the heights extend to span 99.8% of the distribution of the mean. The y-axis is displayed on the log₁₀ scale such that the null value is 1. P values are calculated for the group (strain) coefficient for each linear regression model, and are reported for all instances of p<0.05.

Figure 4.. D614G substitution affects the neutralization susceptibility of SARS-CoV-2.

(a) Neutralizing activities of hamster sera against D614 and G614 mNeonGreen reporter SARS-CoV-2. Sera from D614 virus-infected hamsters (n=8) were tested for neutralizing titers against D614 and G614 reporter SARS-CoV-2, and the 1/NT₅₀ values are plotted. Symbols represent sera from individual animals. (b) Ratio of 1/NT₅₀ between D614 and G614 viruses. Symbols represent sera from individual animals (n=8), the midline represents the mean, and error bars represent standard deviation. (c) Representative neutralizing curve of hamster serum 5. Symbols represent individual replicates, the solid line represents the fitted curve, and the dotted line indicates 50% viral inhibition. (d) Neutralizing activities of eleven human mAbs against D614 and G614 mNeonGreen SARS-CoV-2. Symbols represent individual mAbs. The data represents one of the two independent experiments. (e) Ratio of NT₅₀ between D614 and G614 viruses. The averages of the NT₅₀ ratios from two independent experiments performed in duplicates are shown. Symbols represent individual mAbs, the midline represents the mean, and the error bars represent the standard deviation. (f) Representative neutralizing curve of mAb18. Symbols represent individual replicates, the solid line represents the fitted curve, and the dotted line indicates 50% viral inhibition.