Article

Spike mutation D614G alters SARS-CoV-2

fitness

https://doi.org/10.1038/s41586-020-2895-3 Jessica A. Plante1,2,3,12, Yang Liu4,12, Jianying Liu2,3,12, Hongjie Xia4, Bryan A. Johnson2,
Kumari G. Lokugamage3, Xianwen Zhang4, Antonio E. Muruato2,3, Jing Zou4,
Received: 1 September 2020
Camila R. Fontes-Garfias4, Divya Mirchandani1,2,3, Dionna Scharton1,2,3, John P. Bilello5,
Accepted: 20 October 2020 Zhiqiang Ku6, Zhiqiang An6, Birte Kalveram7, Alexander N. Freiberg2,7,8,9, Vineet D. Menachery2,3,
Xuping Xie4 ✉, Kenneth S. Plante1,2,3 ✉, Scott C. Weaver1,2,3,8,9,10,11 ✉ & Pei-Yong Shi2,4,8,9,10,11 ✉
Published online: 26 October 2020

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein
substitution D614G became dominant during the coronavirus disease 2019
(COVID-19) pandemic1,2. 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.

Since the identification of SARS-CoV-2 in China in late 20193, For SARS-CoV-2, analyses of more than 28,000 spike gene sequences
COVID-19 has caused more than 47 million confirmed infections and in May 2020 revealed a D614G substitution that was rare before
more than 1.2 million deaths worldwide. Although most infections March 2020, but became more common as the pandemic spread1,
are mild, SARS-CoV-2 can cause severe, life-threatening pneumonia, occurring in over 74% of all published sequences by June 20202. The
particularly in older age groups and those with chronic conditions. D614G substitution was accompanied by three other mutations: a
The exact pathogenesis of severe COVID-19 remains unclear, but it typi- C-to-T mutation in the 5′ untranslated region at position 241, a synony-
cally involves a dysregulated, hyperinflammatory response following mous C-to-T mutation at position 3037, and a nonsynonymous C-to-T
viral infection4. However, in addition to the host response, variations mutation at position 14408 in the RNA-dependent RNA polymerase
in the viral strain could contribute to disease severity and efficiency gene8. The frequency of this set of mutations increased not only glob-
of spread. ally, but also during co-circulation within individual regions during
Coronaviruses have evolved a genetic proofreading mechanism to outbreaks, suggesting that the increase was the result of a fitness advan-
maintain their long RNA genomes5. Despite the low sequence diversity tage rather than founder effects and/or genetic drift. The association of
of SARS-CoV-26, mutations in the spike protein, which interacts with spike amino acid substitutions with coronavirus transmissibility sug-
cellular receptors such as angiotensin-converting enzyme 2 (ACE2) gested that the D614G substitution was critical to this putative selective
to mediate entry into cells, have a strong influence on host range, sweep. The correlation of this mutation with higher nasopharyngeal
tissue tropism and pathogenesis. During the SARS-CoV outbreak in viral RNA loads in patients with COVID-191,9 also supported a putative
2002–2003, one such mutation mediated adaptation for infection advantage of the mutant in transmission. However, direct measure-
of the intermediate civet host as well as interhuman transmission7. ments of fitness are needed to confirm this hypothesis.

1
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA. 2Institute for Human Infections and Immunity, University of Texas
Medical Branch, Galveston, TX, USA. 3Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA. 4Department of Biochemistry and Molecular
Biology, University of Texas Medical Branch, Galveston, TX, USA. 5Gilead Sciences, Foster City, CA, USA. 6Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine,
University of Texas Health Science Center at Houston, Houston, TX, USA. 7Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA. 8Center for Biodefense and
Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA. 9Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA. 10Institute
for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA. 11Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch,
Galveston, TX, USA. 12These Authors contributed equally: Jessica A. Plante, Yang Liu, Jianying Liu. ✉e-mail: [email protected]; [email protected]; [email protected]; [email protected]

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Article
a SARS-CoV-2 b
L ORF 1a ORF 1b S E 6
3 7a 8a N UTR
S1 S2
M
7b 8b 9b

23398 23411

D614 TAT CAG GAT GTT AAC TGC

Tyr612 Gln613 Asp614 Val615 Asn616 Cys617

G614 TAT CAG GGT GTT AAC TGC

Ser612 Gln613 Gly614 Val615 Asn616 Cys617
D614 G614
Mutation
c D614 G614
d e D614 G614
D614 G614

log10[genomes (ml–1)]
8 15 6
log10[PFU (ml–1)]

log10(RNA/PFU)
7 5
P = 0.041
6 10
P = 0.0022 4
5
5
4 3

3 0 2

h
h

12

24

36

12

24

36
12

24

36
f g h
D614 G614 D614 G614 D614 G614

log10[genomes (ml–1)]
6 P = 0.0022 10 6
P = 0.0087
log10[PFU (ml–1)]

log10(RNA/PFU)
P = 0.015 P = 0.0087 P = 0.0022
P = 0.026 5 P = 0.026
5 9
4
4 8
3

3 7 2
h

h
h

24

36

48

24

36

48
24

36

48

i Calu-3 j Vero
Repeat 1 Repeat 2 Repeat 1 Repeat 2
225 225 225 225
FL 150 FL 150
150 150
100 100 100 100
Spike S1/S2 75 Spike S1/S2 75
75 75
S2′ S2′
50 50 50
50
37 37 37
37

50
Nucleocapsid N 50 50 Nucleocapsid N 50
37 37
37 37
14

14

14

14

4
61

61

61

61
D6

D6

D6

D6
G

G
Fig. 1 | Spike D614G substitution increases SARS-CoV-2 replication in Calu-3 were determined by plaque assay and quantitative RT–PCR (RT–qPCR),
cells by increasing virion infectivity. a, Construction of G614 SARS-CoV-2. A respectively. The genomic RNA/PFU ratios (e, h) were calculated to indicate
single-nucleotide A-to-G substitution was introduced to produce the spike virion infectivity. Dots represent individual samples, bar heights represent
D614G substitution in the infectious cDNA clone of SARS-CoV-2. Numbers on the means and error bars show s.d. P values were determined by two-tailed Mann–
main schematic refer to open reading frames (ORFs). E, envelope glycoprotein Whitney test from a sample size of n = 6 (two independent experiments
gene; L, leader sequence; M, membrane glycoprotein gene; N, nucleocapsid performed in triplicate). All P values below 0.05 are shown. i, j, Spike protein
gene; UTR, untranslated region. b, Plaque morphologies of D614 and G614 cleavage of purified virions from Calu-3 cells (i) and Vero E6 cells (j). Purified
viruses, developed on day 2 after infection in Vero E6 cells. c–h, Viral replication D614 and G614 virions were analysed by western blot using polyclonal antibodies
and genomic RNA/PFU ratios of D614 and G614 viruses produced from Vero E6 against spike and nucleocapsid. Full-length spike20, S1/S2-cleavage form and S2′
cells (c–e) and from Calu-3 cells (f–h). Cells were infected at a MOI of 0.01. protein are indicated. Results from two independent experiments are presented.
Infectious viral titres (c, f) and genomic RNA levels (d, g) in the culture medium

Initial phenotypic characterizations of the spike D614G substitu-

tion were performed using pseudotyped viruses, in which replica- D614G effect on viral replication and infectivity
tion kinetics were studied in vesicular stomatitis virus and lentiviral We first examined the effect of the spike D614G substitution on
particles incorporating the SARS-CoV-2 spike protein. The D614G viral replication in cell cultures. Site-directed mutagenesis was
substitution resulted in significantly higher pseudovirus titres in performed on an infectious cDNA clone of SARS-CoV-2 to prepare a
multiple cell types, suggesting that spike G614 might be associated pair of recombinant isogenic viruses with spike D614 or G614 (Fig. 1a).
with enhanced entry into cells and enhanced replication in airways1,2. Similar amounts of infectious D614 and G614 viruses were recovered
However, these results need to be confirmed in studies with authentic from Vero E6 cells (monkey kidney epithelial cells). The two viruses
G614 SARS-CoV-2, as well as using in vivo studies with a suitable animal formed similar plaque morphologies (Fig. 1b). In Vero E6 cells, the
model. Therefore, using an infectious cDNA clone for SARS-CoV-210, G614 virus replicated with a higher infectious titre than D614 at 12 h
we generated the D614G substitution in the USA-WA1/2020 strain11 post-infection (hpi), after which the two viruses replicated at compa-
and performed experiments in cell culture, primary human airway rable levels (Fig. 1c). A similar trend was observed for extracellular viral
tissue and a hamster infection model12. We also developed D614 and RNA production from the infected Vero E6 cells (Fig. 1d). Sequencing of
G614 mNeonGreenSARS-CoV-2 viruses for rapid testing of neutraliza- the spike gene of viruses purified from Vero E6 cells did not reveal any
tion by serum specimens and monoclonal antibodies. Our study has further mutations. To compare infectivity, we calculated the genomic
major implications for understanding the evolution and transmission RNA/plaque-forming units (PFU) ratio; we found no significant differ-
of SARS-CoV-2 as well as the development of COVID-19 vaccines and ences between the two viruses (Fig. 1e), indicating that the D614G muta-
therapeutic antibodies. tion does not affect viral replication or virion infectivity in Vero E6 cells.

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 a 15 Mock D614 G614 b Fig. 2 | Spike D614G substitution increases SARS-CoV-2
8
replication in the upper airway, but not the lungs, of
Weight change (%)
10 2 dpi
5 7 hamsters. a–i,Three- to four-week-old male Syrian

log10PFU
0 golden hamsters were infected intranasally with 2 × 104
6
–5
PFU of D614 or G614 SARS-CoV-2 or PBS (mock).
5 All data are from a single experiment. a, Weight loss
–10
4
was monitored for seven days after infection. Data are
–15
0 1 2 3 4 5 6 7 Nasal Trachea Lung Lung Lung Lung mean ± s.d.; n = 18 (D614 and G614) and n = 14 (mock) at
Time after infection (d) wash cranial middle caudal accessory days 0–2; n = 12 (D614 and G614) and n = 10 (mock) at
c d
7 14
days 3 and 4; n = 6 (all cohorts) at days 5–7. Weight loss
4 dpi
was analysed by two-factor analysis of variance (ANOVA)

log10(genomes)
P = 0.0352
6 12
with Tukey’s post hoc test. P > 0.05 for D614 vs G614 at all
log10PFU

P = 0.0026
5 10
time points. b–d, Infectious titres (b, c) and amount of
4 8
viral genomes (d) were measured in the nasal wash,
3 6 trachea and lung on days 2 (b, d), 4 (c, d) and 7 (d) after
dpi: 2 4 7 2 4 7 2 4 7 2 4 7 2 4 7 2 4 7
2 infection. e, f, Genome/PFU ratios on days 2 (e) and 4 (f)
Nasal Trachea Lung Lung Lung Lung Nasal Trachea Lung Lung Lung Lung
wash cranial middle caudal accessory wash cranial middle caudal accessory after infection were calculated as a measure of
infectivity. In b–f, symbols represent individual
e f 8
P = 0.0003
hamsters (n = 6). Centre lines (b, c, e, f) and bar heights
7.0 2 dpi 4 dpi
P = 0.0149
6.5 (d) represent means, and error bars represent s.d.
log10(RNA/PFU)
log10(RNA/PFU)

7
6.0 Two-factor ANOVA with Sidak’s post hoc test. g–i,
5.5 6 Hamsters were inoculated with 1:1 mixtures of D614 and
5.0 G614 viruses (104 PFU each). Nasal wash, trachea and
5
4.5 lungs were collected on days 2 (g), 4 (h) and 7 (i) after
4.0 4
Nasal Trachea Lung Lung Lung Lung Nasal Trachea Lung Lung Lung Lung
infection. Relative amounts of D614 and G614 RNAs were
wash cranial middle caudal accessory wash cranial middle caudal accessory assessed by RT–PCR and Sanger sequencing. In g–i, dots
represent individual hamsters (n = 6), the centre line
g D614:G614 h D614:G614 i D614:G614
P = 0.0004

1:1 1:1 1:1 represents the mean, shaded regions represent s.e.m.,
10 10 10
P = 0.0008

P = 0.0007

the width represents the distribution of the

Relative replicative fitness

Relative replicative fitness

Relative replicative fitness

P = 0.017

P = 0.0001

P = 0.004
P < 0.0001

P < 0.0001
P = 0.0004

P = 0.0044

P = 0.0047
P < 0.0001

model-adjusted means, and heights extend to 99.8% of

the distribution; y-axes use a log10 scale. P values are
calculated for the group (strain) coefficient for each
1 1
1 linear regression model.

Nasal Trachea Lung Lung Lung Lung Nasal Trachea Lung Lung Lung Lung Nasal Trachea Lung Lung Lung Lung
wash cranial middle caudal accessory wash cranial middle caudal accessory wash cranial middle caudal accessory

Next, we compared the replication kinetics of D614 and G614 viruses hamsters were infected intranasally with 2 × 104 PFU of D614 or G614
in the human lung epithelial cell line Calu-3. With a multiplicity of virus. Infected hamsters from both groups exhibited similar weight
infection (MOI) of 0.01, the G614 virus produced modest 1.2-, 2.4- and loss (Fig. 2a). On day 2 after infection, infectious viral titres from nasal
1.9-fold more infectious virus than the D614 virus at 24, 36 and 48 hpi, washes, trachea and various lobes of the lung were consistently higher
respectively (Fig. 1f), indicating that D614G enhances viral replication. in the hamsters infected with G614 virus compared with those infected
By contrast, the cells infected with G614 virus produced less (at 24 and with D614 virus, particularly in the upper airway, although the differ-
36 hpi) or equivalent (at 48 hpi) extracellular viral RNA compared with ences did not reach statistical significance (Fig. 2b, Extended Data
cells infected with D614 virus (Fig. 1g). The genomic RNA/PFU ratios of Fig. 1b). By day 4 after infection the differences in infectious viral titres
D614 virus were therefore 1.9- to 3.0-fold higher than those of G614 virus became more pronounced in the upper airway, with 1.3 log10 PFU ml−1
(Fig. 1h), indicating that the D614G mutation increases the infectivity and 0.9 log10 PFU g−1 higher titres of G614 virus than D614 virus in the
of SARS-CoV-2 produced from a human lung cell line. nasal washes and tracheae, respectively (Fig. 2c), whereas the viral
To explore the mechanism of increased infectivity of G614 virus pro- loads in the lungs were essentially identical. No infectious virus was
duced from Calu-3 cells, we compared the processing of the spike pro- detected on day 7 after infection (data not shown).
tein from D614 and G614 viruses. We purified virions from the culture We compared the infectivity of the D614 and G614 viruses produced
medium of infected Calu-3 cells using ultracentrifugation and a sucrose in hamsters by determining their viral RNA levels and viral RNA/PFU
cushion. The pelleted viruses were analysed for spike protein process- ratios. The two viruses produced nearly identical levels of viral RNA
ing by western blot, using nucleocapsid protein as a loading control. across all organs and time points (Fig. 2d). The RNA/PFU ratios of G614
In both viruses, full-length spike was almost completely processed to virus were 0.3 log10 to 0.7 log10 lower than those of D614 virus across
the S1/S2 cleaved form and S2′, with similar cleavage efficiencies of 93% airway tissues on day 2 after infection (Fig. 2e). On day 4 after infec-
for D614 and 95% for G614 (Fig. 1i). When virions produced from Vero tion, the RNA/PFU ratio of G614 was 1.1 log10 lower than that of D614
E6 cells were analysed, less full-length spike protein was processed to in nasal wash, whereas the differences in the trachea and lungs were
the S1/S2 form, with cleavage efficiencies of 73% for D614 and 67% for negligible (Fig. 2f). On day 7 after infection, despite there being no
G614 (Fig. 1j; uncropped images in Supplementary Fig. 1). These results detectable infectious virus (detection limit 40 PFU ml−1), more than
suggest that more spike protein is cleaved to the S1/S2 form in virions 108 viral genomes per ml were detected in the nasal washes (Fig. 2d),
produced from Calu-3 cells compared with Vero E6 cells, and that the demonstrating high levels of viral RNA persistence after the clearance
D614G substitution does not significantly affect the spike cleavage ratio. of infectious virus. This result recapitulates findings in human patients
with COVID-19, who frequently test positive by PCR with reverse tran-
scription (RT–PCR) for several weeks but have low or undetectable
Increased fitness in the hamster upper airway infectious virus. One caveat of the above calculations is that the total
The in vivo relevance of the D614G mutation was evaluated in the Syrian RNA could include viral RNAs from both virions and intracellular sub-
golden hamster model (Extended Data Fig. 1a). Four- to five-week-old genomic RNAs during sample processing.

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Article
a Fig. 3 | Spike D614G substitution enhances SARS-CoV-2
replication in primary human airway tissues. a,
Virus 2 h infection at 37 °C Days 1 to 5 after infection
DPBS Experimental scheme. D614 and G614 viruses were
Basal medium inoculated onto primary human airway tissues at a MOI
SARS-Cov-2 infection on Culture cells without Incubate the cells with DPBS to of 5. After incubation for 2 h, the culture was washed
human primary airway tissue medium in apical well collect secreted viruses daily with Dulbecco’s PBS (DPBS), then maintained for 5 days.
For collection, DPBS was added at 37 °C for 30 min to
b D614 G614 c D614 G614
7 P = 0.0022 P = 0.0022 10 elute the virus. b–d, Viral replication and genomic RNA/

log10[genomes (ml–1)]
P = 0.026 PFU ratios. The amounts of infectious virus (b) and
log10(PFU ml–1)

6 9
P = 0.0022
5 P = 0.0022 genomic RNA (c) were quantified by plaque assay and
8
4 RT–qPCR, respectively. d, The genomic RNA/PFU ratio
3
7 was calculated as an indication of virion infectivity.
2 6 In b–d, symbols represent individual samples, bar
dpi 1 2 3 4 5 dpi 1 2 3 4 5
heights represent means, and error bars represent
D614:G614 standard deviations. P values were determined by

P = 0.0041
d e 1:1
D614 G614 two-tailed Mann–Whitney test (n = 6, two independent
6

P = 0.0029
P = 0.0018
P = 0.0022 P = 0.0087 experiments conducted in triplicate). e–g, Competition
Relative replicative fitness
log10(RNA/PFU)

5 P = 0.041 P = 0.0087
assay. A mixture of D614 and G614 viruses with initial

P = 0.013
100
4 ratios of 1:1 (e), 3:1 (f) or 9:1 (g) were inoculated onto
3 human airway tissue cultures at a total MOI of 5. Virus
10 ratios after competition were measured by Sanger
2
dpi 1 2 3 4 5 sequencing. Dots represent individual samples (n = 6,
1 two independent experiments conducted in triplicate);
the centre line represents the sample mean; the shaded
dpi 1 2 3 4 5 region represents s.e.m.; the width represents the
D614:G614
f 3:1 g D614:G614 distribution of the model-adjusted means; and the
P = 0.0001

P = 0.0001

100 9:1 heights extend to 99.8% of the distribution of the mean;

P = 0.0002

100
the y-axes use a log10 scale such that the null value is 1.
Relative replicative fitness

P = 0.0001
Relative replicative fitness

P = 0.0001

P = 0.0001
P = 0.0001

P values are calculated for the group (strain) coefficient

P = 0.0006

for each linear regression model.

10 10
P = 0.008
P = 0.0001

1 1

dpi 1 2 3 4 5 dpi 1 2 3 4 5

The above results prompted us to directly compare the finesses of titres from day 1 to 5, up to 7.8 × 105 PFU ml−1 (Fig. 3b), demonstrating
D614 and G614 viruses in a competition experiment. This approach that the airway tissue supports SARS-CoV-2 replication. The infectious
has major advantages over infecting replicate hosts with individual viral titres of G614 virus were significantly higher (2.1- to 8.6-fold)
strains in that each competition is internally controlled, eliminating than those of D614 virus (Fig. 3b). By contrast, there were no differences
host-to-host variation that can reduce the power of experiments. in viral RNA yields between the variants (Fig. 3c). The genomic RNA/PFU
Also, the ratios of infective strains can be assayed with more precision ratios of D614 virus were 1.4- to 5.3-fold higher than those of G614 virus
than individual virus titres. Thus, competition assays have been used (Fig. 3d). Sequencing of the viruses collected on day 5 after infection
for many studies of microbial fitness, including viruses13–16. We infected did not show any acquired mutations. Collectively, these results demon-
hamsters intranasally with equal amounts of the two viruses (104 PFU strate that the spike D614G mutation enhances viral replication through
per virus), collected nasal wash and respiratory organs on days 2, 4 and increased virion infectivity in primary human upper airway tissues.
7 after infection, and quantified the relative amounts of D614 and G614 Next, we performed competition experiments in the human airway
RNAs by RT–PCR and Sanger sequencing. This method was validated tissue culture model. After infecting the cells with a 1:1 infectious ratio
to reliably quantify the relative amounts of D614 and G614 viruses in of D614 and G614 viruses, the G614/D614 ratio increased from 1.2 at 1 day
a mixed specimen (Extended Data Fig. 2). A G614/D614 ratio greater post-infection (dpi) to 13.9 at 5 dpi (Fig. 3e). In addition, after infecting
than one indicates a replication advantage for G614. All respiratory the airway culture with 3:1 ratio of D614 and G614 viruses, the G614 virus
tissues showed G614/D614 ratios of 1.2 to 2.6 on days 2, 4 and 7 after rapidly overcame its initial deficit to reach a slight advantage with a G614/
infection, indicating that G614 virus has a consistent advantage over D614 ratio of 1.2 by 1 dpi, with that advantage increasing to 9.1 by 5 dpi
D614 virus (Fig. 2g–i). (Fig. 3f). Furthermore, when infecting the airway tissue with a 9:1 ratio
of D614 to G614, the G614/D614 ratios increased from1.4 to 5.2 from 1 dpi
to 5 dpi (Fig. 3g). Similar results were obtained when the experiment
Enhanced replication in a human airway model was repeated using a different donor-derived human airway culture
To further define the effect of the spike D614G mutation in the human (Extended Data Fig. 3). These results confirm that the G614 virus can
respiratory tract, we characterized the replication of D614 and G614 rapidly outcompete the D614 virus when infecting human airway tissues,
viruses in a primary human airway tissue culture model (Fig. 3a). This even when it is initially present as a minor variant in a mixed population.
model contains human tracheal and bronchial epithelial cells in layers
resembling the epithelial tissue of the respiratory tract. The primary
tissue is cultured at an air–liquid interface to recapitulate the barrier, Stability of D614 and G614 viruses
microciliary response and infection of human airway tissues in vivo17,18. To examine the effect of D614G on virus stability, we measured the decay
After infecting the airway tissue at a MOI of 5 (as determined in Vero of infectivity of D614 and G614 viruses over time at 33 °C, 37 °C and 42 °C
E6 cells), both D614 and G614 viruses produced increasing infectious (Extended Data Fig. 4). The infectivity of both viruses decreased at a

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 a b 2.5 Serum 5
c 120
0.0024 Serum 5 D614

1/NT50 ratio (D614/G614)

Relative viral activity

100 G614
0.0020

(% of control)
2.0 80
0.0016

1/NT50
0.0012 60 50%
Mean = 1.7
0.0008 1.5 40

0.0004 20

0 1.0 0
D614 G614 1 2 3 4 5
Serum 5 (log dilution)
d e 2.5 f 120
140 mAb18

NT50 ratio (D614/G614)

120 100

Relative viral activity

2.0
NT50(μg ml–1)

100

(% of control)
80
80 1.5
8 60 50%
6 1.0 Mean = 1.0
mAb18 40
4 D614
0.5 20 G614
2
0 0.0 0
D614 G614 –2 –1 0 1 2
log10[mAb18 (μg ml–1)]

Fig. 4 | The spike D614G substitution affects the susceptibility of fitted curve, and the dotted line indicates 50% viral inhibition. d, Neutralizing
SARS-CoV-2 to neutralization by antibodies. a, Neutralizing activities of activities of 11 human monoclonal antibodies against D614 and G614
hamster sera against D614 and G614 mNeonGreen reporter SARS-CoV-2. Sera mNeonGreen SARS-CoV-2. Symbols represent individual monoclonal
from hamsters infected with D614 virus (n = 8) were tested for neutralizing antibodies. Data represent one of two independent experiments. e, Ratio of
titres against D614- and G614-reporter SARS-CoV-2, and the 1/NT50 values are NT50 between D614 and G614 viruses. The averages of the NT50 ratios from two
plotted. Symbols represent sera from individual hamsters. b, Ratio of 1/NT50 independent experiments performed in duplicate are shown. Dots represent
between D614 and G614 viruses. Symbols represent sera from individual individual monoclonal antibodies, the centre line represents the mean, and the
hamsters (n = 8), the centre line represents the mean, and error bars represent error bars represent the s.d. f, Representative neutralizing curve of mAb18.
s.d. c, Representative neutralizing curve of serum from an individual hamster Symbols represent individual replicates, the solid line represents the fitted
(serum 5). Symbols represent individual replicates, the solid line represents the curve, and the dotted line indicates 50% viral inhibition.

faster rate at 42 °C than at 37 °C or 33 °C. However, the G614 virus retained infectivity. Compared with the original D614 virus, the emergent G614
higher infectivity than the D614 virus at all temperatures, suggesting that virus exhibited increased viral replication in the human lung cell line
the D614G mutation may increase the stability of SARS-CoV-2. Calu-3 and in primary human upper airway tissues. The differences
in replication were higher in the primary human airway culture, with
an advantage of up to 13.9-fold in a competition assay. The increased
Susceptibility to neutralization replication fitness was accompanied by increases in specific infectivity
All of the COVID-19 vaccines currently in clinical trials are based on the and stability of the G614 virions. Previous studies with pseudotyped
original D614 spike sequence19,20. We therefore investigated whether virus have shown that the efficiency of spike protein cleavage into S1
the D614G substitution could reduce the efficacy of these vaccines, and S2 fragments modulates SARS-CoV-2 infection22,23; we therefore
assuming that the G614 virus continues to circulate. To address this compared the spike cleavage with D614 and G614 virions. Although
question, we measured the neutralization titres of a panel of sera virions produced from Calu-3 cells showed more complete S1/S2
collected from hamsters that were previously infected with D614 cleavage than those produced form Vero E6 cells, no substantial dif-
SARS-CoV-2 (Extended Data Fig. 5). Each serum was analysed using ferences in spike cleavage were detectable between the D614 and G614
mNeonGreen reporter D614 or G614 SARS-CoV-2 viruses21 (Extended virions, suggesting that the enhanced virion infectivity is unlikely to
Data Fig. 6). The mNeonGreen gene was engineered in open reading be caused by differences in spike cleavage. In contrast with previous
frame 7 of the SARS-CoV-2 genome10. All sera exhibited 1.4- to 2.3-fold studies reporting that the D614G substitution changes the cleavage and
higher neutralization titres (mean 1.7-fold) against heterologous G614 shedding of spike protein when expressed alone on pseudotyped viri-
virus compared with homologous D614 virus (Figs. 4a–c, Extended ons24,25, we found no such difference in experiments with SARS-CoV-2.
Data Fig. 7), suggesting that the D614G mutation may confer higher Mechanistically, recent studies have shown that the D614G substitution
susceptibility to serum neutralization. abolishes a hydrogen-bond interaction with T859 from a neighbouring
Next, we evaluated the activity of a panel of 11 monoclonal antibodies protomer of the spike trimer1, which allosterically shifts the RBD to an
to the SARS-CoV-2 receptor-binding domain (RBD) against the D614 and ‘up’ conformation, promoting binding with the ACE2 receptor2, leading
G614 mNeonGreen SARS-CoV-2 viruses. The details of these antibodies to enhanced virion infectivity.
will be reported elsewhere (Z. An et al., submitted). One of the antibod- The higher viral loads of G614 in the upper airway of patients with
ies (mAb18) showed a 2.1-fold higher neutralizing activity against G614 COVID-1926 and infected hamsters suggest a role of the D614G substitu-
than against D614 virus, whereas the others showed similar neutraliza- tion in viral transmissibility. The robust replication of SARS-CoV-2 in the
tion activities against both viruses (Fig. 4d–f, Extended Data Figs. 8, 9). human upper airway may be partially conferred by higher expression
The results suggest that the D614G mutation may modulate spike pro- of the ACE2 receptor in the nasal cavity compared with the lower res-
tein conformation, thereby affecting neutralization by monoclonal piratory tract8,27. Compared with the D614 virus, the G614 virus showed
antibodies in an epitope-specific manner. increased replication in the upper airway—but not in the lungs—of
hamsters, suggesting that the D614G substitution may select against
replication in the lung. Patients infected with G614 virus developed
Discussion higher levels of viral RNA in nasopharyngeal swabs than those with
We have shown that the spike D614G substitution enhances SARS-CoV-2 D614 virus, but did not develop more severe disease1,2,9. Our hamster
replication in the upper respiratory tract through increased virion model of infection recapitulated these clinical findings: the G614 virus

Nature | www.nature.com | 5
Article
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6 | Nature | www.nature.com
Methods LightCycler 480 system following the manufacturers ’protocols. Prim-
ers are listed in Extended Data Table 1. The absolute quantification of
No statistical methods were used to predetermine sample size. viral RNA was determined by a standard curve method using an RNA
The experiments were not randomized. The investigators were not standard (in vitro transcribed 3,839-bp RNA containing genomic nucle-
blinded to allocation during experiments and outcome assessment. otide positions 26044 to 29883 of SARS-CoV-2 genome).
To quantify D614:G614 ratios for competition assays, a 663-bp RT–
Ethics statement PCR product was amplified from extracted RNA using a SuperScript III
Hamster studies were performed in accordance with the guidance One-Step RT–PCR kit (Invitrogen). A 20-μl reaction was assembled in
for the Care and Use of Laboratory Animals of the University of Texas PCR 8-tube strips through the addition of 10 μl 2 × reaction mix, 0.4 μl
Medical Branch (UTMB). The protocol was approved by the Institutional SuperScript III RT/Platinum Taq Mix, 0.8 μl Forward Primer (10 μM)
Animal Care and Use Committee (IACUC) at UTMB. All the hamster (Extended Data Table 1), 0.8 μl reverse primer (10 μM) (Extended Data
operations were performed under anaesthesia by isoflurane to mini- Table 1), 4 μl RNA, and 6 μlRnase-free water. Reverse transcription and
mize animal suffering. amplification was completed using the following protocol: (i) 55 °C, 30
min; 94 °C, 2 min; (ii) 94 °C, 15 s; 60 °C, 30 s; 68 °C, 1 min; 40 cycles; (iii)
Animals and Cells 68 °C, 5 min; (iv) indefinite hold at 4 °C. The presence and size of the
The Syrian hamsters (HsdHan:AURA strain) were purchased from desired amplicon was verified with 2 μl of PCR product on an agarose
Envigo. African green monkey kidney epithelial Vero E6 cells (pro- gel. The remaining 18 μl were purified by a QIAquick PCR Purification
vided by R. Baric) were grown in Dulbecco’s modified Eagle’s medium kit (Qiagen) according to the manufacturer’s protocol.
(DMEM) with 10% fetal bovine serum (FBS; HyClone Laboratories) and Sequences of the purified RT–PCR products were generated using
1% antibiotic/streptomycin (Gibco). Human lung adenocarcinoma a BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems).
epithelial Calu-3 cells (ATCC) were maintained in a high-glucose DMEM The sequencing reactions were purified using a 96-well plate format
supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C (EdgeBio) and analysed on a 3500 Genetic Analyzer (Applied Biosys-
with 5% CO2. The EpiAirway system is a primary human airway 3D tissue tems). The peak electropherogram height representing each mutation
model purchased from MatTek Life Science. This EpiAirway system site and the proportion of each competitor was analysed using the
was maintained with the provided culture medium at 37 °C with 5% QSVanalyser program31.
CO2following the manufacturer’s instruction. All other culture media
and supplements were purchased from ThermoFisher Scientific. All Plaque assay
cell lines were verified and tested negative for mycoplasma. No further Approximately 1.2 × 106 Vero E6 cells were seeded to each well of 6-well
authentication of cell lines was performed. plates and cultured at 37 °C, 5% CO2 for 16 h. Virus was serially diluted
in either DMEM with 2% FBS (for viral stocks and in vitro-generated
Generation of SARS-CoV-2 spike D614G mutant viruses samples) or DPBS (for hamster tissues) and 200 μl was transferred to
One single-nucleotide substitution was introduced into a subclone the monolayers. The viruses were incubated with the cells at 37 °C with
puc57-CoV-2-F5-7 containing the spike gene of the SARS-CoV-2 wild-type 5% CO2 for 1 h. After the incubation, overlay medium was added to the
infectious clone10 to convert amino acid 614 from aspartic acid to gly- infected cells per well. The overlay medium contained either DMEM
cine by overlap fusion PCR. The full-length infectious cDNA clone of with 2% FBS and 1% sea-plaque agarose (Lonza) in the case of in vitro
SARS-CoV-2 D614G was assembled by in vitro ligation of seven contigu- samples or Opti-MEM with 2% FBS, 1% penicillin–streptomycin and
ous cDNA fragments following the previously described protocol10. For 0.8% agarose in the case of in vivo samples. After a 2-day incubation,
construction of D614G mNeonGreen SARS-CoV-2, seven SARS-CoV-2 plates were stained with neutral red (Sigma-Aldrich) and plaques were
genome fragments (F1 to F5, F6 containing D614G mutation, and counted on a light box.
F7-mNG containing the mNeonGreen reporter gene) were prepared
and ligated in vitro as previously described10. In vitro transcription Viral infection on cells
was then preformed to synthesize full-length genomic RNA. To recover Approximately 3 × 105 Vero E6 or Calu-3 cells were seeded onto each well
the mutant viruses, the RNA transcripts were electroporated into Vero of 12-well plates and cultured at 37 °C, 5% CO2 for 16 h. Either SARS-CoV-2
E6 cells. The viruses from electroporated cells were collected at 40 h D614 or G614 virus was inoculated into the cells at a MOI of 0.01. The
after electroporation and served as seed stocks for subsequent experi- virus was incubated with the cells at 37 °C for 2 h. After the infection,
ments. The D614G mutation from the recovered viruses was confirmed the cells were washed with DPBS 3 times to remove the unattached
by sequence analysis. Viral titres were determined by plaque assay on virus. One millilitre of culture medium was added into each well to
Vero E6 cells. All virus preparation and experiments were performed in maintain the cells. At each time point, 100 μl of culture supernatants
a biosafety level 3 (BSL-3) facilities. Viruses and plasmids are available were collected for the RT–qPCR detection and plaque assay. Meanwhile,
from the World Reference Center for Emerging Viruses and Arboviruses 100 μl fresh medium was added into each well to replenish the culture
at the University of Texas Medical Branch. volume. The cells were infected in triplicate for each virus. All samples
were stored at −80 °C until plaque or RT–qPCR analysis.
RNA extraction, RT–PCR, and Sanger sequencing
Cell culture supernatants or clarified tissue homogenates were mixed Virion purification and spike protein cleavage analysis
with a fivefold excess of TRIzol LS Reagent (Thermo Fisher Scientific). Vero E6 or Calu-3 2B4 cells were infected with D614 or G614 viruses at
Viral RNAs were extracted according to the manufacturer’s instruc- a MOI of 0.01. At 24 (for Vero) or 48 (Calu-3) hours after infection, the
tions. The extracted RNAs were dissolved in 20 μl nuclease-free water. culture media were collected and clarified by low speed spin. Virions in
Two microlitres of RNA samples were used for reverse transcription by the medium were pelleted by ultracentrifugation through a 20% sucrose
using the SuperScript IV First-Strand Synthesis System (ThermoFisher cushion at 26,000 rpm for 3 h at 4 °C in a BeckmanSW28 rotor. The
Scientific) with random hexamer primers. Nine DNA fragments flank- purified virions were analysed by western blot using polyclonal antibod-
ing the entire viral genome were amplified by PCR. The resulting DNAs ies against spike protein and nucleocapsid as previously described32.
were cleaned up by the QIAquick PCR Purification Kit, and the genome
sequences were determined by Sanger sequencing at Genewiz. Viral infection in a primary human airway tissue model
The quantify viral RNA samples, quantitative RT–qPCR assays were The EpiAirway system is a primary human airway 3D mucociliary tis-
performed using the iTaq SYBR Green One-Step Kit (Bio-Rad) on the sue model consisting of normal, human-derived tracheal/bronchial
Article
epithelial cells. For viral replication kinetics, either D614 or G614 virus
was inoculated onto the culture at a MOI of 5 in DPBS. After 2 h infec- Neutralization assay
tion at 37 °C with 5% CO2, the inoculum was removed, and the culture Neutralization assays were preformed using D614 and G614 mNeon-
was washed three times with DPBS. The infected epithelial cells were Green SARS-CoV-2 as previously described21. In brief, Vero (CCL-81) cells
maintained without any medium in the apical well, and medium was were plated in black CLEAR flat-bottom 96-well plate (Greiner Bio-One).
provided to the culture through the basal well. The infected cells were On the following day, sera or monoclonal antibodies were serially
incubated at 37 °C, 5% CO2. From day 1 to day 5, 300 μl DPBS was added diluted from 1/20 starting dilution and nine twofold dilutions to the
onto the apical side of the airway culture and incubated at 37 °C for final dilution of 1/5,120 and incubated with D614 or G614 mNeonGreen
30 min to elute the released viruses. All virus samples in DPBS were SARS-CoV-2 at 37 °C for 1 h. The virus–serum mixture was transferred to
stored at −80 °C. the Vero cell plate with the final MOI of 2.0. After 20 h, Hoechst 33342
solution (400-fold diluted in Hank’s Balanced Salt Solution; Gibco) was
Hamster infection added to stain cell nucleus, sealed with Breath-Easy sealing membrane
Four- to five-week-old male Syrian golden hamsters, strain (Diversified Biotech), incubated at 37 °C for 20 min, and quantified for
HsdHan:AURA (Envigo), were inoculated intranasally with 2 × 104 PFU mNeonGreen fluorescence using Cytation 7 (BioTek). The raw images
SARS-CoV-2 in a 100 μl volume. Eighteen hamsters received wild-type (2 × 2 montage) were acquired using a 4× objective, processed and
D614 virus, 18 received mutant G614 virus, and 18 received a mixture stitched using the default setting. The total cells (indicated by nucleus
containing 104 PFU of D614virus and 104 PFU of G614 virus. The infected staining) and mNeonGreen-positive cells were quantified for each well.
hamsters were weighed and monitored for signs of illness daily. On Infection rates were determined by dividing the mNeonGreen-positive
days 2, 4 and 7 after infection, cohorts of 6 infected hamsters and 4 cell number to total cell number. Relative infection rates were obtained
(days 2 and 4) or 6 (day 7) mock-infected hamsters were anaesthetized by normalizing the infection rates of serum-treated groups to those of
with isoflurane and nasal washes were collected in 400 μl sterile DPBS. non-serum-treated controls. The curves of the relative infection rates
Hamsters were humanely euthanized immediately following the nasal versus the serum dilutions (log10 values) were plotted using Prism 8
wash. The trachea and the four lobes of the right lung were collected in (GraphPad). A nonlinear regression method was used to determine
maintenance medium (DMEM supplemented with 2% FBS and 1% peni- the dilution fold that neutralized 50% of mNeonGreen fluorescence
cillin–streptomycin) and stored at −80 °C. Samples were subsequently (NT50). Each serum was tested in duplicates.
thawed, tissues consisting of individual, intact lung lobes or the trachea
were homogenized for 1 min at 26 strokes per s, and debris was pelleted Statistics
by centrifugation for 5 min at 16,100g. Infectious titres were determined Male hamsters were randomly allocated into different groups. The
by plaque assay. Genomic RNAs were quantified by quantitative RT–PCR investigators were not blinded to allocation during the experiments or
(Extended Data Table 1). Ratios of D614/G614 RNA were determined via to the outcome assessment. A priori power analysis for two independ-
RT–PCR with quantification of Sanger peak heights. ent groups having a 1.0 log10 titre difference with a standard deviation of
0.5 log10, assuming an alpha of 0.05 and a power of 0.8 with a two-tailed
Competition assay t-test, dictated that animal experiments include n = 6 subjects per
For the competition in primary human airway 3D tissue model, the D614 cohort. Descriptive statistics have been provided in the figure legends.
and G614 mutant viruses were mixed and inoculated onto the cells at For in vitro replication kinetics, Kruskal–Wallis analysis of variance
a final MOI of 5. The initial ratio of D614 and G614 viruses was 1:1, 3:1 was conducted to detect any significant variation among replicates.
or 9:1 based on PFU titres determined on Vero E6 cells. The DPBS with If no significant variation was detected, the results were pooled for
viruses was collected every day from day 1 to 5 following the protocol further comparison. Differences between continuous variables were
described above. For the competition in hamsters, 100-μl mixtures of assessed with a non-parametric Mann–Whitney test. Hamster weights
D614 and G614 viruses (total 2 × 104 PFU per hamster) were inoculated were analysed by two-factor ANOVA, with the per cent weight change
intranasally into 4–5-week-old Syrian hamsters. On days 2, 4, and 7 as the dependent variable and the strain and time as fixed factors.
pi, 6 infected hamsters were sampled for competition detection. An Tukey’s post hoc test was used to compare all cohort pairs on days 1–7
aliquot of the inoculum for both hamster and human airway infections after inoculation. log10-tranformed titres were analysed by two-factor
was backtitred to estimate the initial ratio of viruses. All samples were repeated measures ANOVA with the organ and strain as fixed factors.
stored at −80 °C before analysis. Sidak’s post hoc test was used to compare strains within each organ.
Genomic RNA/PFU ratios were calculated from non-transformed val-
Validation of competition assay by Sanger sequencing ues, and the resulting ratios were log10-transformed before two-factor
To validate the consistency and accuracy of competition assay repeated measures ANOVA with the organ and strain as fixed factors
by Sanger sequencing, the D614 and G614 viruses were mixed at and Sidak’s post hoc test to compare strains within a given organ. When
ratios of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5 and 1:10 based on their PFU titres a sample was below the limit of detection, it was treated as half of the
(total 106 PFU viruses) or mixed with 106, 105, 104, 103 and 102 PFU of limit of detection value for statistical and graphing purposes. Analysis
the two viruses at a ratio of 1:1. The total RNA of these mixed viruses was was performed in Prism v.7.03 (GraphPad).
isolated and amplified by RT–PCR. The ratios of D614/G614 were For virus competition experiments, relative replicative fitness values
calculated by the peak heights of Sanger sequencing. Data were ana- for G614 strain over D614 strain were analysed according to w = (f0/i0), in
lysed by linear regression with correlation coefficients (r) and sig- which i0 is the initial D614/G614 ratio and f0 is the final D614/G614 ratio
nificance (P). after competition. Sanger sequencing (initial time point T0) counts for
each virus strain being compared were based on average counts over
Stability assay of SARS-CoV-2 three replicate samples of inocula per experiment, and post-infection
D614 and G614 viruses were diluted to a final titre of 105 PFU ml−1 in (time point T1) counts were taken from samples of individual subjects.
DPBS. The diluted viruses were incubated at 42 °C, 37 °C or 33 °C. At For the primary human airway samples, multiple experiments were
indicated time points, 200 μl DPBS containing the viruses were taken performed, so that f0/i0 was clustered by experiment. To model f0/i0,
out and stored at −80 °C. All the samples were then analysed by plaque the ratio T0/T1 was found separately for each subject in each strain
assay on Vero E6 cells. The percentages of remaining infectious viruses group, log10-transformed to an improved approximation of normality,
were calculated by dividing the viral titres at each time point by the and modelled by analysis of variance with relation to group, adjust-
mean viral titre at 0 h. ing by experiment when appropriate to control for clustering within
experiment. Specifically, the model was of the form Log10_Count- 34. Andersen, C. Catseyes: create catseye plots illustrating the normal distribution of the
means. R package version 0.2.3 (2019).
T1overCountT0 ~ Experiment + Group. Fitness ratios between the two 35. Reznik, G. et al. Clinical Anatomy of the European Hamster Cricetus cricetus, L. (Supt of
groups (the model’s estimate of w = (f0/i0)) were assessed per the coef- Docs, US Govt. Print. Off., 1978).
ficient of the model’s Group term, which was transformed to the original
scale as 10 to the power of the coefficient. This modelling approach Acknowledgements This research was supported by grants from NIA and NIAID of the NIH
compensates for any correlation due to clustering within experiment (AI153602 and AG049042 to V.D.M.; R24AI120942 (WRCEVA) to S.C.W.) and by STARs Award
provided by the University of Texas System to V.D.M. X.X. was partially supported by
similarly to that of corresponding mixed-effect models, and is effective
NIH5UC7AI094660. P.-Y.S. was supported by NIH grants AI142759, AI134907, AI145617 and
since the number of experiments was small. Statistical analyses were UL1TR001439, and awards from the Sealy & Smith Foundation, the Kleberg Foundation, the
performed using R statistical software (R Core Team, 2019, v.3.6.1). John S. Dunn Foundation, the Amon G. Carter Foundation, the Gilson Longenbaugh Foundation,
and the Summerfield Robert Foundation. A.E.M. is supported by a Clinical and Translational
In all statistical tests, two-sided alpha = 0.05. Cat’s-eye plots33, which Science Award NRSA (TL1) Training Core (TL1TR001440) from NIH. J.L. and C.R.F.-G. were
illustrate the normal distribution of the model-adjusted means, were supported by the McLaughlin Fellowship at the University of Texas Medical Branch.
produced using the catseyes package34.
Author contributions Conceptualization: Y.L., V.D.M., X.X., K.S.P., S.C.W. and P.-Y.S.;
methodology: J.A.P., Y.L., J.L., H.X., B.A.J., K.G.L., X.Z., A.E.M., J.Z., C.R.F.-G., A.N.F., V.D.M., X.X.,
Reporting summary K.S.P., S.C.W. and P.-Y.S.; investigation: J.A.P., Y.L., J.L., H.X., B.A.J., K.G.L., X.Z., A.E.M., J.Z.,
Further information on research design is available in the Nature C.R.F.-G., D.M., D.S., J.P.B., B.K., A.N.F., V.D.M., X.X., K.S.P., S.C.W. and P.-Y.S.; resources: Z.K. and
Z.A.; data curation: J.A.P., Y.L., J.L., H.X., B.A.J., K.G.L., X.Z., A.E.M., J.Z., A.N.F., V.D.M., X.X.,
Research Reporting Summary linked to this paper. K.S.P., S.C.W. and P.-Y.S.; writing, original draft: J.A.P., Y.L., J.L., X.X., K.S.P., S.C.W. and P.-Y.S;
writing, review and editing: J.A.P., Y.L., J.L., H.X., B.A.J., K.G.L., X.Z., A.E.M., J.Z., A.N.F., V.D.M.,
X.X., K.S.P., S.C.W. and P.-Y.S.; supervision: A.N.F., V.D.M., X.X., K.S.P., S.C.W. and P.-Y.S.; and
Data availability funding acquisition: A.N.F., V.D.M., S.C.W. and P.-Y.S.

Data associated with all figures may be accessed via Figshare at https:// Competing interests X.X., V.D.M. and P.-Y.S. have filed a patent on the reverse genetic system
doi.org/10.6084/m9.figshare.13030430. Source data are provided and reporter SARS-CoV-2. The other authors declare no competing interests.
with this paper.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
31. Carr, I. M. et al. Inferring relative proportions of DNA variants from sequencing 2895-3.
electropherograms. Bioinformatics 25, 3244–3250 (2009). Correspondence and requests for materials should be addressed to X.X., K.S.P., S.C.W. or P.-Y.S.
32. Johnson, B. A. et al. Furin cleavage site is key to SARS-CoV-2 pathogenesis. Preprint at Peer review information Nature thanks Stanley Perlman, K. Y. Yuen and the other, anonymous,
https://doi.org/10.1101/2020.08.26.268854 (2020). reviewers for their contribution to the peer review of this work.
33. Cumming, G. The new statistics: why and how. Psychol. Sci. 25, 7–29 (2014). Reprints and permissions information is available at http://www.nature.com/reprints.
Article

Extended Data Fig. 1 | Experimental design of hamster infection and sample organs collected from hamsters killed on days 2, 4, and 7 post-infection.
collection. a, Graphical overview of experiment to assess the impact of G614 Illustration of hamster lung adapted from ref. 35.
mutation on replication in the respiratory system of hamsters. b, Schematic
Extended Data Fig. 2 | Validation of competition assay by Sanger evaluation. The ratio of D614/G614virus mixture calculated from Sanger
sequencing. a, The correlation between input PFU ratios and output RT–PCR sequencing was consistent when using a wide range of virus amounts.
amplicon ratios determined by Sanger sequencing. D614 and G614 viruses were The D614/G614 viruses were mixed at 1:1PFU ratio. The total titres of the mixed
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 viruses were 102, 103, 104, 105, and 106 PFU. The total RNA of virus mixture was
mixtures were extracted and amplified by RT–PCR. The D614/G614 ratios were isolated and amplified by RT–PCR. The D614/G614 ratios were calculated by the
calculated by the peak heights of Sanger sequencing. Data were analysed by peak heights from Sanger sequencing. Symbols represent individual
linear regression with correlation coefficients (r) and significance (p). Symbols replicates, bar heights represent the mean, and error bars represent the
represent individual replicates and the solid line represents the fitted line. Data standard deviation. Data are derived from a single experiment conducted in
are derived from a single experiment conducted in duplicate. b, Assay range triplicate.
Article

Extended Data Fig. 3 | D614G substitution significantly enhances SARS-CoV-2 experiments conducted in triplicate). The midline represents the sample mean,
replication in primary human airway tissues from a different donor. D614 and and the shaded region represents s.e.m. The width of the catseye plot represents
G614 viruses were equally mixed and inoculated onto the airway tissue at a total the distribution of the model-adjusted means, and the heights extend to span
MOI of 5. This airway tissue was produced from a different donor than that used 99.8% of the distribution of the mean. The y-axis is displayed on the log10 scale
in Fig. 3. The tissues were washed by DPBS to collect the secreted viruses every such that the null value is 1. P values are calculated for the group (strain)
day from days 1 to 5. The total RNAs were isolated and amplified by RT–PCR. The coefficient for each linear regression model, and are reported for all instances of
ratio of D614 and G614 viruses after competition were measure by Sanger P < 0.05.
sequencing. Circles represent individual samples (n = 6, two independent
Extended Data Fig. 4 | SARS-CoV-2 G614 is more stable than D614. a–c, Equal remaining infectious viruses were normalized by the average titres at 0 h.
amounts (105 PFU/ml) of D614 and G614 viruses were incubated in DPBS at 42 °C Symbols represent individual replicates, bar heights represent the mean, and
(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 error bars represent the standard deviation. P values were determined by
viruses were quantified for their infectious levels by plaque assay on Vero E6 two-tailed Mann–Whitney test (n = 9, from three independent experiments,
cells. The detect limitation of plaque assay is 10 PFU/ml. The percentage of each conducted in triplicate), and results of P < 0.05 are indicated.
Article

Extended Data Fig. 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 Fig. 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 previously described10. b, Plaque morphologies of D614 and G614 mNeonGreen SARS-CoV-2.
Article

Extended Data Fig. 7 | Neutralization activities of hamster sera against D614 line represents the fitted curve. Data are derived from a single experiment
and G614 mNeonGreen SARS-CoV-2. a, Neutralizing curves of eight hamster conducted in duplicate. b, Calculated NT50 values and ratios of 1/NT50 for all eight
sera against D614 and G614 mNeonGreenSARS-CoV-2. The neutralizing curve for hamster sera. The mean ratios were determined by (D614 1/NT50)/(G614 1/NT50).
serum 5 is shown in Fig. 4c. Symbols represent individual samples and the solid
Extended Data Fig. 8 | Neutralization activities of human mAbs against replicates and the solid line represents the fitted curve. Data are derived from a
D614 and G614 mNeonGreen SARS-CoV-2 in experiment I. a, Neutralizing single experiment conducted in duplicate. b, Calculated NT50 values for all
curves of eleven mAbs against D614 and G614 reporter SARS-CoV-2. The eleven mAbs.
neutralizing curve for mAb18 is shown in Fig. 4f. Symbols represent individual
Article

Extended Data Fig. 9 | Neutralization activities of human mAbs against Data are derived from a single experiment conducted in either duplicate or
D614 and G614 mNeonGreen SARS-CoV-2 in experiment II. a, Neutralizing quadruplicate. b, Calculated NT50 values for all eleven mAbs. (c) Summary of
curves of eleven mAbs against D614 and G614 reporter SARS-CoV-2. Symbols NT50 ratios from two independent experiments. The ratios were determined by
represent individual replicates and the solid line represents the fitted curve. (D614 NT50)/(G614 NT50).
Extended Data Table 1 | Primers for gene cloning and qPCR

Primer sequences for the generation of the D614G mutation, as well as for the detection, sequencing, and quantification of SARS-CoV-2 are reported.
 nature research | reporting summary
Xuping Xie, Kenneth S. Plante, Scott C.
Weaver, and Pei-Yong Shi*
Corresponding author(s): * = Lead Contact

Last updated by author(s): Oct 8, 2020

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Raw data used to generate all graphs have been deposited in figshare and may be accessed at: https://figshare.com/articles/dataset/
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Replication Replication kinetics in Vero and Calu3 cells were performed in two independent experiments, each in triplicate. Replication kinetics in primary
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Antibodies used Western blots used rabbit polyclonal antibodies against SARS-CoV spike (Novus Biologicals, NB100-56578) and SARS-CoV
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October 2018