Emergence and spread of a SARS-CoV-2 variant through Europe in the
summer of 2020
Emma B. Hodcroft,
1, 2, 3
Moira Zuber,
1
Sarah Nadeau,
4, 2
Timothy G. Vaughan,
4, 2
Katharine H. D. Crawford,
5, 6, 7
Christian L. Althaus,
3
Martina L. Reichmuth,
3
John E. Bowen,
8
Alexandra C. Walls,
8
Davide Corti,
9
Jesse
D. Bloom,
5, 6, 10
David Veesler,
8
David Mateo,
11
Alberto Hernando,
11
I˜naki Comas,
12, 13, 14
Fernando Gonz´alez
Candelas,
15, 13, 14
SeqCOVID-SPAIN consortium,
16
Tanja Stadler
∗
,
4, 2
and Richard A. Neher
∗ 1, 2
1
Biozentrum, University of Basel, Basel, Switzerland
2
Swiss Institute of Bioinformatics, Basel, Switzerland
3
Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland
4
Department of Biosystems Science and Engineering, ETH Z¨urich, Basel, Switzerland
5
Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center,
Seattle, WA 98109, USA
6
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
7
Medical Scientist Training Program, University of Washington, Seattle, WA 98195, USA
8
Department of Biochemistry, University of Washington, Seattle, WA, USA
9
Humabs Biomed SA, a subsidiary of Vir Biotechnology, 6500 Bellinzona, Switzerland
10
Howard Hughes Medical Institute, Seattle, WA 98103, USA
11
Kido Dynamics SA, Avenue de Sevelin 46, 1004 Lausanne, Switzerland
12
Tuberculosis Genomics Unit, Biomedicine Institute of Valencia (IBV-CSIC), Valencia, Spain
13
CIBER de Epidemiolog´ıa y Salud P´ublica (CIBERESP), Madrid, Spain
14
on behalf or the SeqCOVID-SPAIN consortium
15
Joint Research Unit ”Infection and Public Health” FISABIO-University of Valencia,
Institute for Integrative Systems Biology (I2SysBio), Valencia, Spain
16
SeqCOVID-SPAIN consortium
Following its emergence in late 2019, severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) has caused a global pandemic resulting in unprecedented efforts to reduce
transmission and develop therapies and vaccines (WHO Emergency Committee, 2020;
Zhu et al., 2020). Rapidly generated viral genome sequences have allowed the spread of
the virus to be tracked via phylogenetic analysis (Worobey et al., 2020; Hadfield et al.,
2018; Pybus et al., 2020). While the virus spread globally in early 2020 before borders
closed, intercontinental travel has since been greatly reduced, allowing continent-specific
variants to emerge. However, within Europe travel resumed in the summer of 2020, and
the impact of this travel on the epidemic is not well understood. Here we report on a
novel SARS-CoV-2 variant, 20E (EU1), that emerged in Spain in early summer, and
subsequently spread to multiple locations in Europe. We find no evidence of increased
transmissibility of this variant, but instead demonstrate how rising incidence in Spain,
resumption of travel across Europe, and lack of effective screening and containment may
explain the variant’s success. Despite travel restrictions and quarantine requirements,
we estimate 20E (EU1) was introduced hundreds of times to countries across Europe
by summertime travellers, likely undermining local efforts to keep SARS-CoV-2 cases
low. Our results demonstrate how a variant can rapidly become dominant even in
absence of a substantial transmission advantage in favorable epidemiological settings.
Genomic surveillance is critical to understanding how travel can impact SARS-CoV-2
transmission, and thus for informing future containment strategies as travel resumes.
Severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) is the first pandemic where the spread of a
viral pathogen has been globally tracked in near real-time
using phylogenetic analysis of viral genome sequences
(Worobey et al., 2020; Hadfield et al., 2018; Pybus et al.,
2020). SARS-CoV-2 genomes continue to be generated
at a rate far greater than for any other pathogen and
more than 500,000 full genomes are available on GISAID
as of February 2020 (Shu and McCauley, 2017).
In addition to tracking the viral spread, these genome
sequences have been used to monitor mutations which
might change the transmission, pathogenesis, or anti-
genic properties of the virus. One mutation in partic-
ular, D614G in the spike protein, has received much at-
tention. This variant (Nextstrain clade 20A) seeded large
outbreaks in Europe in early 2020 and subsequently dom-
inated the outbreaks in the Americas, thereby largely re-
placing previously circulating lineages. This rapid rise
led to the suggestion that this variant is more transmis-
sible, which has since been corroborated by phylogenetic
(Korber et al., 2020; Volz et al., 2020) and experimental
evidence (Plante et al., 2020; Yurkovetskiy et al., 2020).
Following the global dissemination of SARS-CoV-2
in early 2020 (Worobey et al., 2020), intercontinental
travel dropped dramatically. Within Europe, however,
travel and in particular holiday travel resumed in sum-
mer (though at lower levels than in previous years)
with largely uncharacterized effects on the pandemic.
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2
Here we report on a novel SARS-CoV-2 variant 20E
(EU1) (S:A222V) that emerged in early summer 2020,
presumably in Spain, and subsequently spread to mul-
tiple locations in Europe. Over the summer, it rose in
frequency in parallel in multiple countries. As we re-
port here, this variant, 20E (EU1), and a second variant
20A.EU2 with mutation S477N in the spike protein ac-
counted for the majority of sequences in Europe in the
autumn of 2020.
Multiple variants emerged in Summer 2020 in Europe
Figure 1 shows a time scaled phylogeny of sequences
sampled in Europe through the end of November and
their global context, highlighting the variants in this
manuscript. Clade 20A and its daughter clades 20B and
20C have variant S:D614G and are colored in yellow. A
cluster of sequences in clade 20A has an additional muta-
tion S:A222V colored in orange. We designate this cluster
as 20E (EU1) (this cluster consists of lineage B.1.177 and
its sublineages (Rambaut et al., 2020)).
In addition to the 20E (EU1) cluster we describe here,
an additional variant (20A.EU2; blue in Fig. 1) with
several amino acid substitutions, including S:S477N and
mutations in the nucleocapsid protein, has become com-
mon in some European countries, particularly France
(Fig. S6). The S:S477N substitution has arisen mul-
tiple times independently, for example in a variant in
clade 20B that has dominated the outbreak in Oceania
during the southern-hemisphere winter (now identified as
20F). The position 477 is close to the receptor binding
site (Fig. S1). Residue S477 is part of the epitope rec-
ognized by the S2E12 and C102 neutralizing antibodies
(Barnes et al., 2020; Tortorici et al., 2020) and the detec-
tion of multiple variants at this position, such as S477N,
might have resulted from the selective pressure exerted
by the host immune response.
Several other smaller clusters defined by the spike mu-
tations D80Y, S98F, N439K are also seen in multiple coun-
tries (see Table I and Fig. S6). While none of these have
reached the prevalence of 20E (EU1) or 20A.EU2, some
have attracted attention in their own right: S:N439K has
appeared twice in the pandemic (Thomson et al., 2020) as
well as numerous times independently. It is found across
Europe (particularly Ireland, the UK, and Czech Repub-
lic), is located in the receptor binding domain (RBD),
and reduces neutralization by antibody C135 (Barnes
et al., 2020; Weisblum et al., 2020). Focal phylogenies
for these, and other variants mentioned in this paper,
as well as updated phylogenies of SARS-CoV-2 in Eu-
rope and individual European countries can be found at
nextstrain.org/groups/neherlab. Further detailed analy-
ses of the individual clusters discussed here are available
at CoVariants.org.
Antigenic and functional characterization of S:A222V
Our analysis here focuses on the variant 20E
(EU1) with substitution S:A222V. S:A222V is in the spike
protein’s domain A (Figure S1) also referred to as the N-
terminal domain (NTD) (Tortorici et al., 2020; McCal-
lum et al., 2020; Walls et al., 2020), which is not known
to play a direct role in receptor binding or membrane
fusion for SARS-CoV-2. However, mutations can some-
times mediate long-range effects on protein conformation
or stability.
To evaluate if the A222V mutation affects the confor-
mation of the SARS-CoV-2 spike glycoprotein, we probed
binding of the benchmark COVID-19 convalescent pa-
tient plasma from the National Institute for Biologicals
Standards and Control, two neutralizing monoclonal an-
tibodies recognizing the RBD, namely S2E12 and S309
(Tortorici et al., 2020; Pinto et al., 2020; Walls et al.,
2020) and a NTD-specific neutralizing monoclonal anti-
body (4A8) (Chi et al., 2020). The dose-response curves
were indistinguishable for the SARS-CoV-2 2P S and the
SARS-CoV-2 2P A222V D614G S ectodomain trimers,
as observed by ELISA, Fig.S3a-d S3. These results are
in agreement with a recent study showing that binding of
several NTD-specific neutralizing antibodies were unaf-
fected by the A222V mutation (McCallum et al., 2021).
Collectively, these data indicate that the A222V substi-
tution does not affect the SARS-CoV-2 S antigenicity
appreciably.
To test whether the S:A222V mutation had an obvious
functional effect on spike’s ability to mediate viral entry,
we produced lentiviral particles pseudotyped with spike
either containing or lacking the A222V mutation in the
background of the D614G mutation and deletion of the
end of spike’s cytoplasmic tail. Lentiviral particles with
the A222V mutant spike had slightly higher titers than
those without (mean 1.3-fold higher), although the differ-
ence was not statistically significant after normalization
by p24 concentration (Fig. S2). Therefore, A222V does
not lead to the same large increases in the titers of spike-
pseudotyped lentivirus that has been observed for the
D614G mutation (Korber et al., 2020; Yurkovetskiy et al.,
2020), which is a mutation that is now generally consid-
ered to have increased the fitness of SARS-CoV-2 (Volz
et al., 2020; Plante et al., 2020). However, we note that
this small effect must be interpreted in equivocal terms,
as the effects of mutations on actual viral transmission in
humans are not always paralleled by measurements made
in highly simplified experimental systems such as the one
used here.
In addition to S:A222V, 20E (EU1) has the amino acid
mutations ORF10:V30L, N:A220V and ORF14:L67F. How-
ever, there is little evidence of the functional relevance
of ORF10 and ORF14 (Pancer et al., 2020; Finkel et al.,
2020). Different mutations at positions 180 and 220 in N
are observed in almost every major lineage and we are not
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3
Variant Lineage Representative Mutations Spike Substitution
20E (EU1) B.1.177 C22227T, C28932T, G29645T A222V
20A.EU2 B.1.160 C4543T, G5629T, G22992A S477N
S:S98F B.1.221 C21855T, A25505G, G25996T S98F
S:D80Y B.1.367 C3099T, G21800T, G27632T D80Y
S:N439K B.1.258 T7767C, C8047T, C22879A N439K
TABLE I Representative mutations of 20E (EU1) (the focus of this study) and other notable variants. When a lineage definition
matches the variant definition, it is given in column 2 (Rambaut et al., 2020).
20E (EU1)
(S:A222V)
20A.EU2
(S:S477N)
S:S98F
S:D80Y
S:N439K
FIG. 1 Phylogenetic overview of SARS-CoV-2 in Europe through the end of November. The tree shows a representative sample
of isolates from Europe colored by clade and by the variants highlighted in this paper. A novel variant (orange; 20E (EU1)) with
mutation S:A222V on a S:D614G background emerged in early summer and is common in most countries with recent sequences.
A separate variant (20A.EU2, blue) with mutation S:S477N is prevalent in France. On the right, the proportion of sequences
belonging to each variant (through the duration of the pandemic) is shown per country. Tree and visualization were generated
using the Nextstrain platform (Hadfield et al., 2018) as described in methods.
aware of any evidence suggesting that these mutations
have important phenotypic consequence. Therefore, we
examined epidemiological and evolutionary evidence to
assess if the variant showed evidence of enhanced trans-
missibilty in humans.
Early observations of 20E (EU1)
The earliest sequences identified date from the 20th of
June, when 7 Spanish sequences and 1 Dutch sequence
were sampled. The next non-Spanish sequence was from
the UK (Wales) on the 7th July, with a Belgian sequence
sampled on the 17th and a Swiss sampled on the 22nd.
By the end of July, samples from Spain, the Netherlands,
the UK (England, Northern Ireland, Wales), Switzerland,
Ireland, Belgium, and Norway were identified as being
part of the cluster. By the 22nd August, the cluster
also included sequences from France, Denmark, more of
the UK (Scotland), Germany, Latvia, Sweden, and Italy.
Four sequences from Hong Kong, 17 from Australia, 27
from New Zealand, and 8 sequences from Singapore, pre-
sumably exports from Europe, were first detected in mid-
August (Hong Kong, Australia), mid-September (New
Zealand), and mid-October (Singapore).
The proportion of sequences from several countries
which fall into 20E (EU1), by ISO week, is plotted in
Fig. 2. 20E (EU1) first rose in frequency in Spain, jump-
ing to around 50% prevalence within a month of the first
sequence being detected before rising to 80%. In many
countries, including the United Kingdom, France, Ire-
land, Denmark, and Switzerland we observe a gradual
rise starting in mid-July before settling at a level between
15 and 80% in September or October. In contrast, Nor-
way observed a sharp peak in early August, but seemed
to bring 20E (EU1)cases down quickly, though they be-
gan growing again in September. The date ranges and
number of sequences observed in this cluster are summa-
rized in Table SI.
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4
Norway to Spain
UK to Spain
Denmark to Spain Advised to quarantine, not required
Switzerland to Spain
Netherlands to Spain
Spain to Europe
France to Spain
Quarantine-free
Travel to/from Spain
(on return)
2020-05
2020-06
2020-07
2020-08
2020-09
2020-10
2020-11
2020-12
0.0
0.2
0.4
0.6
0.8
1.0
frequency
n<=10
n>10
n>50
n>100
n>200
Spain opens borders
France
Norway
United Kingdom
Netherlands
Spain
Belgium
Switzerland
Ireland
Italy
Denmark
FIG. 2 Frequency of submitted samples are 20E (EU1) in selected countries, with quarantine-free travel dates
shown above. We include the eight countries which have at least 200 sequences from 20E (EU1), as well as Norway and
France, to illustrate points in the text. The symbol size indicates the number of available sequence by country and time point
in a non-linear manner. Travel restrictions from selected countries are shown to/from Spain, as this is the probable origin of
the cluster. Most European countries allowed quarantine-free travel to other (non-Spanish) countries in Europe for a longer
period. When the last data point included only very few sequences, it has been dropped for clarity. Frequencies are smoothing
using a Gaussian with σ = 1w.
Initial expansion and spread across Europe
Fig. 3 shows a phylogeny based on data from samples
collected before 2020-09-30 and available on GISAID in
Jan 2021, as described in Methods. The phylogeny is col-
lapsed to group diversity possibly stemming from within-
country transmission into sectors of the pie-charts (see
Fig. S12) for selected countries. The tree indicates that
20E (EU1) harbors substantial diversity and most major
genotypes within the 20E (EU1) cluster have been ob-
served in many European countries. Since it is unlikely
that phylogenetic patterns that are sampled in multiple
countries arose independently, it is reasonable to assume
that the majority of mutations observed in the tree arose
once and were carried (possibly multiple times) between
countries. We use this rationale to provide lower bounds
on the number of introductions to different countries.
Throughout July and August 2020, Spain had a higher
per capita incidence than most other European countries
(see Fig S4) and 20E (EU1) was much more prevalent
in Spain then elsewhere, suggesting Spain as likely origin
of most 20E (EU1) introductions to other countries. The
first sequences in 20E (EU1) were sampled on the 20th of
June in Spain and the Netherlands. This Dutch sequence
nests within the diversity of early sequences from Spain,
suggesting this sequence is the result of the earliest sam-
pled export of the variant outside of Spain, consistent
with and early uptick of travel from Spain to the Nether-
lands (Fig. 4 A). Other sequences in 20E (EU1) that are
dated prior to June 2020 have implausible phylogenetic
positions and are likely mis-dated (these are excluded,
see Methods).
Epidemiological data from Spain indicates the earliest
sequences in the cluster are associated with two known
outbreaks in the north-east of the country. The variant
seems to have initially spread among agricultural work-
ers in Aragon and Catalonia, then moved into the local
population, where it was able to travel to the Valencia
Region and on to the rest of the country (though se-
quence availability varies between regions). This initial
expansion may have been critical in increasing the clus-
ter’s prevalence in Spain just before borders re-opened.
Most basal genotypes have been observed both in
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5
Spain and a remarkably large number of other countries,
suggesting repeated exports. However, the 801 sequences
from Spain contributing to Fig. 3 likely do not repre-
sent the full diversity. Variants found only outside of
Spain may reflect diversity that arose in secondary coun-
tries, or may represent diversity not sampled in Spain.
In particular, as the UK sequences much more than any
other country in Europe, it is not unlikely they may have
sampled diversity that exists in Spain or elsewhere but
has only been sampled in the UK. Despite limitations
in sampling, Fig. 3 clearly shows that most major geno-
types in this cluster were distributed to multiple coun-
tries, suggesting that many countries have experienced
multiple introductions of identical genotypes that can-
not be fully identified from the phylogeny. This is con-
sistent with the large number of introductions estimated
from travel data, discussed below. While initial intro-
ductions of the variant likely originated from Spain, 20E
(EU1) cases outside of Spain surpassed those in Spain
in late September and later cross-border transmissions
likely originated in other European countries (see Fig
S4B and 20E (EU1) Nextstrain build online). In the
supplementary text we provide a brief discussion of travel
restrictions and measures imposed by selected European
countries and the associated patterns of 20E (EU1) in-
troductions.
Fig. S5 shows the distribution of sequence clusters
compatible with onward transmission within countries
outside of Spain, highlighting two different patterns.
Norway and Iceland, for example, seem to have experi-
enced only a small number of introductions over the sum-
mer that led to substantial further spread. In Fig. 3, the
majority of sequences from these countries fall into one
sector, the remainder are singletons or very small clus-
ters that have not spread. Unlike the initial introduction,
later sequences in Norway or Iceland often cluster more
closely with diversity in European countries other than
Spain, which may suggest further introductions came
from third countries (see 20E (EU1) Nextstrain build on-
line).
In contrast, countries like Switzerland, the Nether-
lands, or the United Kingdom have sampled sequences
that correspond to a large number of independent in-
troductions that include most major and many minor
genotypes observed in Spain. Many sequences sampled
in Ireland are closely associated with sequences sampled
in the UK, which might indicated exchange with the UK
or shared holiday destinations. As described in the sup-
plementary text, Ireland never allowed quarantine-free
travel to Spain, but travel figures (Fig 4 A) suggest it
was a popular destination nonetheless.
No evidence for transmission advantage of 20E (EU1)
During a dynamic outbreak, it is particularly difficult
to unambiguously tell whether a particular variant is in-
creasing in frequency because it has an intrinsic advan-
tage, or because of epidemiological factors (Grubaugh
et al., 2020). In fact, it is a tautology that every novel
big cluster must have grown recently and multiple lines
of independent evidence are required in support of an
intrinsically elevated transmission potential.
The cluster we describe here – 20E (EU1) (S:A222V)
– was dispersed across Europe initially mainly by trav-
elers to and from Spain. Many EU and Schengen-area
countries, including Switzerland, the Netherlands, and
France, opened their borders to other countries in the
bloc on 15th June. Travel resumed quickly and peaked
during July and August, see Fig. 4. The number of con-
firmed SARS-CoV-2 cases in Spain rose from around 10
cases per 100k inhabitants per week in early July to 100
in late August, while case number remained low in most
of Europe during this time. To explore whether repeated
imports are sufficient to explain the rapid rise in fre-
quency and the displacement of other variants, we first
estimated the number of expected introductions of 20E
(EU1) based on the number of visitors from a particular
country to different provinces of Spain and the SARS-
CoV-2 incidence in the provinces. Taking reported inci-
dence in the provinces at face value and assuming that
returning tourists have a similar incidence, we expect 380
introductions of 20E (EU1) into the UK over the sum-
mer (6 July-27 Sept, see Table SII and Fig. 4 for tourism
summaries (Instituto Nacional de Estadistica, 2020) and
departure statistics (Aena.es, 2020)). Similarly, for Ger-
many and Switzerland we would expect around 320 and
90 introductions of 20E (EU1), respectively. We then
create a simple model that also incorporates the inci-
dence in the country where travellers are returning to
and onward spread of imported 20E (EU1) cases to es-
timate the frequency of 20E (EU1) in countries across
Europe over time (see Fig. 4). This model assumes that
20E (EU1) spread at the same rate as other variants in
the resident countries and predicts that the frequencies
of 20E (EU1) would start rising in July, continue to rise
through August, and be stable thereafter in concordance
with observations in many countries including Switzer-
land, Denmark, France, Germany or the Netherlands (see
Fig. 4 B).
While the shape of the expected frequency trajectories
from imports in Fig. 4 B is consistent with observations,
this naive import model underestimates the final ob-
served frequency of 20E (EU1) by between 1- and 11-fold
depending on the country, see Fig. S9. This discrepancy
might be to due to either intrinsically faster transmission
of 20E (EU1) or due to underestimation of introductions.
Underestimates might be due to country-specific report-
ing and behavioural factors such as the relative ascertain-
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