Coronavirus (SARS-CoV-2) Mutations

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Coronavirus (SARS-CoV-2) Mutations Oct 31, 2020 5:53:33 GMT

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Post by Admin on Oct 31, 2020 5:53:33 GMT

Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020

A variant of SARS-CoV-2 emerged in early summer 2020, presumably in Spain, and
has since spread to multiple European countries. The variant was first observed in
Spain in June and has been at frequencies above 40% since July. Outside of Spain,
the frequency of this variant has increased from very low values prior to 15th July
to 40-70% in Switzerland, Ireland, and the United Kingdom in September. It is also
prevalent in Norway, Latvia, the Netherlands, and France. Little can be said about
other European countries because few recent sequences are available. Sequences in this
cluster (20A.EU1) differ from ancestral sequences at 6 or more positions, including the
mutation A222V in the spike protein and A220V in the nucleoprotein. We show that this
variant was exported from Spain to other European countries multiple times and that
much of the diversity of this cluster in Spain is observed across Europe. It is currently
unclear whether this variant is spreading because of a transmission advantage of the
virus or whether high incidence in Spain followed by dissemination through tourists is
sufficient to explain the rapid rise in multiple countries.

INTRODUCTION
Following its emergence in Wuhan in late 2019 (WHO
Emergency Committee, 2020; Zhu et al., 2020), SARSCoV-2 has
caused a global pandemic resulting in unprecedented efforts to
reduce transmission and develop therapies and vaccines. The
spread of the virus across the world has been tracked with phylogenetic
analysis of viral genome sequences (Worobey et al., 2020; Hadfield et al.,
2018; Pybus et al., 2020) which were and still are generated at
a rate far greater than for any other pathogen.
More than 157,000 full genomes are available in GISAID
as of October 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 antigenic
properties of the virus. One mutation in particular, D614G in
the spike protein, has received much attention. This variant
(Nextstrain clade 20A) seeded large outbreaks in Europe in early
2020 and subsequently dominated the outbreaks in the Americas,
thereby largely replacing previously circulating lineages.
This rapid rise has led to the suggestion that this variant is
more transmissible (Korber et al., 2020; Volz et al., 2020).

While the virus spread globally in early 2020 before borders were
closed and viral variants circulating were distributed across the
world, intercontinental travel remained suppressed through the
summer of 2020.

The paucity of intercontinental travel allowed continent
specific variants to emerge. Within Europe, however,
travel resumed in the summer of 2020. Here we report
on a novel SARS-CoV-2 variant 20A.EU1 (S:A222V) that
emerged in early summer 2020, presumably in Spain,
and subsequently spread to multiple locations in Europe.

Over the summer, it rose in frequency in parallel in multiple
countries. As we report here, this variant, 20A.EU1,
and a second variant 20A.EU2 with mutation S:S477N
in the spike protein account for the majority of recent
sequences in Europe. It is unclear at present whether
the rapid spread of either variant is due to association
with particular demographics, properties of the virus, or
chance but the dynamics of both should be carefully monitored.

Last Edit: Oct 31, 2020 5:56:07 GMT by Admin

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Coronavirus (SARS-CoV-2) Mutations Oct 31, 2020 22:43:33 GMT

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Post by Admin on Oct 31, 2020 22:43:33 GMT

METHODS
Phylogenetic analysis
We use the Nextstrain pipeline for our phylogenetic analyses
github.com/nextstrain/ncov/
(Hadfield et al., 2018). Briefly, we align sequences using
mafft (Katoh et al., 2002), subsample sequences (see below),
add sequences from the rest of the world for phylogenetic
context based on genomic proximity, reconstruct
a phylogeny using IQ-Tree (Minh et al., 2019) and
infer a time scaled phylogeny using TreeTime (Sagulenko
et al., 2018). For computational feasibility, ease of
interpretation, and to balance disparate sampling efforts
between countries, the Nextstrain-maintained runs subsample
the available genomes across time and geography,
resulting in final builds of ∼4,000 genomes each.

Sequences were downloaded from GISAID using the
nextstrain/ncov workflow. A table acknowledging the
invaluable contributions by many labs is available as a
supplement. We focus in particular on the epidemics in
the UK, Switzerland (dataset described in Nadeau et al.
(2020)) and Spain for which we have most sequencing
data.

Defining the 20A.EU1 Cluster
The cluster was initially identified as a monophyletic
group of sequences stemming from the larger 20A clade
with amino acid substitutions at positions S:A222V,
ORF10:V30L, and N:A220V or ORF14:L67F (overlapping
reading frame with N), corresponding to nucleotide mutations
C22227T, C28932T, and G29645T. In addition, sequences in
20A.EU1 differ from their ancestors by the
synonymous mutations T445C, C6286T, and C26801G.
There are currently 7,906 sequences in the cluster by this
definition.

The sub-sampling of the standard Nextstrain analysis means
that we are not able to visualise the true size
or phylogenetic structure of the cluster in question. To
specifically analyze this cluster using all available sequences,
we designed a specialized build which focuses
on cluster-associated sequences and their most genetically
similar neighbours.

We identify sequences in the cluster based on the
presence of nucleotide substitutions at positions 22227,
28932, and 29645 and use this set as a ‘focal’ sample
in the nextstrain/ncov pipeline. This selection will exclude
any sequences with no coverage or reversions at
these positions, but the similarity-based sampling during the
Nextstrain run will identify these, as well as any

Variant Representative Mutations Spike Substitution
20A.EU1 C22227T, C28932T, G29645T A222V
20A.EU2 C4343T, G5629T, G22992A S477N
S:S98F C21855T, A25505G, G25996T S98F
S:D80Y C3099T, G21800T, G27632T D80Y

TABLE I Representative mutations of 20A.EU1 (the focus
of this study) and other notable variants.

other nearby sequences, and incorporate them into the
dataset. We used these three mutations as they included
the largest number of sequences that are distinct to the
cluster. By this criterion, there are currently 7,864 sequences
in the cluster – slightly fewer than above because
of missing data at these positions.

To visualise the changing prevalence of the cluster over
time, we plotted the proportion of sequences identified by
the four substitutions described above as a fraction of the
total number of sequences submitted, per ISO week. Frequencies
of other clusters are identified in an analogous
way. Case data for Fig S2 were obtained from ECDC
(European Center for Disease Control, 2020).

Phylogeny and Geographic Distribution
The size of the cluster and number of unique mutations among
individual sequences means that interpreting overall patterns
and connections between countries
is not straightforward. We aimed to create a simplified
version of the tree that focuses on connections between
countries and de-emphasizes onward transmissions
within a country. As our focal build contains ‘background’
sequences that do not fall within the cluster,
we used only the monophyletic clade containing the four
amino-acid changes and three synonymous nucleotide
changes that identify the cluster. Then, subtrees that
only contain sequences from one country were collapsed
into the parent node. The resulting phylogeny contains
only mixed-country nodes and single-country nodes that
have mixed-country nodes as children. Nodes in this tree
thus represent ancestral genotypes of subtrees: sequences
represented within a node may have further diversified
within their country, but share a set of common mutations.
We count all sequences in the subtrees towards
the geographic distribution represented in the pie-charts
in Fig. 3.

This tree allows us to infer lower bounds for the number
of introductions to each country, and to identify plausible
origins of those introductions. It is important to
remember that, particularly for countries other than the
United Kingdom, the full circulating diversity of the variant
is probably not being captured, thus intermediate
transmissions cannot be ruled out. In particular, the
closest relative of a particular sequence will often have
been sampled in the UK simply because sequencing efforts
in the UK exceed most other countries by orders of
magnitude. It is, however, not our goal to identify all
introductions but to investigate large scale patterns of
spread in Europe.

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Coronavirus (SARS-CoV-2) Mutations Nov 1, 2020 7:01:02 GMT

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Post by Admin on Nov 1, 2020 7:01:02 GMT

RESULTS
Figure 1 shows a time scaled phylogeny of sequences
sampled in Europe and their global context colored by
the amino-acids at position 222, 477, and 614 in the
spike protein. 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
mutation S:A222V colored in blue. We designate this cluster
as 20A.EU1. Another prominent cluster
(20A.EU2) with the substitution S:S477N in the spike
protein is common in France, but appears less prevalent
than 20A.EU1 in other countries where both variants
have circulated (Fig. S3).

Our analysis here focuses on the variant 20A.EU1 with
substitution S:A222V. The substitution S:A222V is in the
spike protein’s domain A (also referred to as the NTD),
and is not in a portion of spike that is known to play a
direct role in receptor binding or membrane fusion, though
mutations can sometimes mediate long-range effects on
protein conformation or stability. However, 20A.EU1 has
increased rapidly in size over the summer and now accounts
for a large fraction of sequences in several European countries.

When all lineages circulating in Switzerland since 1 May
are considered, the notable rise and
expansion of 20A.EU1 is clear (see Fig S4).
Updated phylogenies of SARS-CoV-2 of Europe
and individual European countries are provided at
nextstrain.org/groups/neherlab. The page also includes
links to analysis of the individual clusters discussed in
here (the cluster build for 20A.EU1 can be found at
nextstrain.org/groups/neherlab/ncov/20A.EU1).
Earliest Sequences in the 20A.EU1 cluster
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 (England) on the 18th July, with a Swiss sequence s
ampled on the 22nd and an Irish sampled on
the 23rd. By the end of July, samples from Spain, the
UK (England, Northern Ireland), Switzerland, Ireland,
Belgium, and Norway were identified as being part of
the cluster. By the 22nd August, the cluster also included
sequences from France, more of the UK (Scotland, Wales),
Germany, Latvia, Sweden, and Italy. Two
sequences from Hong Kong and nine sequences from New
Zealand, presumably exports from Europe, were first detected
in mid-August and mid-September, respectively.
The proportion of sequences from each country which
fall into the cluster, by ISO week, is plotted in Fig 2.

Here, we included countries with at least 20 sequences
from the cluster. By plotting the weekly proportion of
sequences submitted by a country that fall into the cluster,
we can see how the cluster-associated sequences have
risen in frequency (Fig 2). As might be expected from
the much earlier first sample date, the cluster first rises
in frequency in Spain, initially jumping to around 60%
prevalence within a month of the first sequence being
detected. In the United Kingdom, France, Ireland, and
Switzerland we observe a gradual rise starting in midJuly.

In Wales and Scotland the variant was at 80% in
mid-September, whereas frequencies in Switzerland and
England were around 50% at that time. In contrast,
Norway observed a sharp peak in early August, but few
sequences are available for later dates. The date ranges
and number of sequences observed in this cluster are
summarized in Table II.

To quantify the growth of this cluster more precisely,
we fit a logistic growth curve to the observed frequency of
20A.EU1 in Switzerland, Spain, England, Scotland and
Wales, see Fig. S1. The estimated growth rate varies between
around s = 30−40% per week in all five countries.

Country First Observation # Sequences Last Observation Frequency in Sept & Oct
Netherlands 2020-06-20 49 2020-09-28 0.21
Spain 2020-06-20 256 2020-09-20 0.88
United Kingdom 2020-07-18 7207 2020-10-13 0.43
England 2020-07-18 4732 2020-10-13 0.36
Northern Ireland 2020-07-21 41 2020-09-23 0.14
Scotland 2020-08-01 926 2020-10-11 0.66
Wales 2020-08-03 1508 2020-10-10 0.74
Switzerland 2020-07-22 179 2020-09-10 0.37
Ireland 2020-07-23 56 2020-09-16 0.51
Norway 2020-07-29 59 2020-08-19 0
Belgium 2020-07-29 10 2020-09-23 0.11
France 2020-08-01 21 2020-09-16 0.12
Sweden 2020-08-14 3 2020-09-16 0.17
Hong Kong 2020-08-15 2 2020-08-17 0
Germany 2020-08-17 2 2020-08-19 0
Latvia 2020-08-22 10 2020-08-25 0
Italy 2020-08-25 1 2020-08-25 0
New Zealand 2020-09-22 9 2020-10-08 0.16

TABLE II Summary of sequences observed in the 20A.EU1 cluster

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Coronavirus (SARS-CoV-2) Mutations Nov 1, 2020 21:41:04 GMT

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Post by Admin on Nov 1, 2020 21:41:04 GMT

Cluster Source and Number of Introductions across Europe
Fig. 3 shows a collapsed phylogeny, as described in
Methods, indicating the observations of different genotypes
across Europe. The prevalence of early samples in
Spain, diversity of the Spanish samples, and prominence
of the cluster in Spanish sequences suggest Spain as the
likely origin for the cluster. Throughout July and August
2020, Spain had a higher per capita incidence than most
other European countries (see Fig S5) further supporting
Spain as origin of the cluster.

Since it is unlikely that diversity and phylogenetic patterns
sampled in multiple countries arose independently,
it is reasonable to assume that the majority of mutations
within the cluster arose once and were carried (possibly
multiple times) between countries. We use this rationale
below to provide lower bounds on the number of introductions
to different countries.

The 256 sequences in the cluster from Spain likely do
not represent the full diversity. Variants found only outside
of Spain may reflect diversity acquired since being
transmitted to another country, 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 but has not yet been sampled there. Despite
limitations in sampling, Fig. 3 clearly shows that
most major genotypes in this cluster were distributed to
multiple European countries.

Per-Country Inferences
From inspecting the phylogenetic relationships between
samples (Fig 3), we can estimate the number of
times the 20A.EU1 cluster has been introduced into each
country. In some cases countries have only one introduction,
but many countries have indications of multiple
separate introductions, and these we will cover in more
detail below.

There are only eleven non-European samples in the
cluster, from Hong Kong and New Zealand. Both are
likely exports from Europe: the Hong Kong sequences
indicate a single introduction, whereas the New Zealand
samples are from at least three separate transmissions
from Europe.

In order to estimate the number of introductions and
try to infer possible sources, the phylogenetic connections
between countries (Fig 3) were linked with the date
of the country’s first sample in the cluster and travel
restrictions. As well as the dates when countries reopened
their borders, we considered periods during which
quarantine-tree travel was possible and travel volume to
and from Spain (Fig. S8). Many EU and Schengenarea countries,
including Switzerland, the Netherlands,
and France, opened their borders to other countries in
the bloc on 15th June, though the Netherlands kept the
United Kingdom on their ‘orange’ list (NL Times, 2020;
Occupational Health & Safety and Environmental Protection
Unit at CERN, 2020; The Federal Council of the
Swiss Confederacy, 2020). Spain opened its borders to
EU member states (except Portugal, at Portugal’s request)
and associated countries on 21st June (McMurtry, 2020).

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Coronavirus (SARS-CoV-2) Mutations Nov 2, 2020 20:38:42 GMT

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Post by Admin on Nov 2, 2020 20:38:42 GMT

Norway, Latvia, Germany, Italy, Sweden: The
sequences from Norway, Latvia, Germany, and Italy all
indicate single introduction events, whereas Sweden’s
three sequences each indicate a separate introduction.

Italy, Germany, and Sweden have only a very small
number of sequences – one, two, and three, respectively
– meaning that many introductions might have been
missed. Norway and Latvia’s larger sequence counts
form two clear separate monophyletic groups within the
20A.EU1 cluster. The Norwegian samples seem likely
to be a direct introduction from Spain, as they cluster
tightly with Spanish sequences and the first sample (29th
July) was just after quarantine-free travel to Spain was
stopped. In Latvia, quarantine-free travel to Spain was
only allowed until the 17th July - a month before the first
sequence was detected on 22nd August. Latvia allowed
quarantine-free travel to other European countries for a
longer period, and this introduction may therefore have
come via a third country. Though the Latvian sequences
cluster most closely with diversity found in the UK, the
over-representation of UK sequences may explain this,
and the introduction may have come from unsampled
diversity in another country in Europe.

Spain: Linked 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 cluster variant seems to have initially spread
among agricultural workers 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. This initial expansion of the 20A.EU1 cluster
via a suspected super-spreading event that spread into
the local population and moved across the country may
have been critical in increasing the cluster’s prevalence
in Spain just before borders re-opened.

Switzerland: Quarantine-free travel to Spain was
possible from 15th June to 10th August. The majority
of holiday return travel is expected from mid-July
to mid-August towards the end of school holidays. The
first sequence identified as part of the cluster was taken
on 22nd July.

To estimate introductions, we consider 13 nodes where
Swiss sequences share diversity with Spanish sequences,
or have parental nodes that contain Spanish sequences,
suggesting an introduction into Switzerland, possibly via
a third country. Additionally, we see 6 nodes where
a genotype was observed in Switzerland and in another
country, suggesting either an additional import
from Spain, a third country, or a transmission between
Switzerland and the other country. Three of the 19
putative introductions involve more than twenty sequences,
and seem to have grown rapidly, consistent with the
growth of the overall cluster.

For those nodes that don’t directly or through their
parents share diversity with Spanish sequences, the Swiss
sequences are most closely related to diversity found in
the UK, France, and the Netherlands, suggesting possible
transmission from other EU countries to Switzerland or
diversity in Spain that was not sampled.

In a second Switzerland-specific analysis we compared
the size of transmission chains started by sequences from
within the 20A.EU1 cluster to outside the 20A.EU1 cluster.

To do so, we identified introductions into Switzerland and
downstream Swiss transmission chains through
considering a tree of all available Swiss sequences
combined with foreign sequences with high similarity to
Swiss sequences (procedure described in Nadeau et al.
(2020)). Overall, we estimate between 5-71 independent
introductions of the 20A.EU1 variant into Switzerland
are plausible based on available sequences. Introductions
within the 20A.EU1 cluster tend to cause larger
transmission chains compared to introductions of non20A.EU1
strains (Fig. S7). In particular, although a
similar percentage of introductions of 20A.EU1 variants
and non-20A.EU1 strains give rise to local transmission
chains, 17-40% of 20A.EU1 variant transmission chains
contain more than 20 sequences, compared to 0-4% of
non-20A.EU1 transmission chains introduced around the
same time. The given ranges represent two different
definitions of a transmission chain
Belgium: Along with many European countries, Belgium
reopened to EU and Schengen Area countries on
the 15th June. Belgium employed a regional approach to
travel restrictions, meaning that while travellers
returning from some regions of Spain were subject to quarantine
from the 6th of August, it was not until the 4th September
that most of Spain was subject to travel restrictions.

Belgian sequences share diversity with sequences
from Spain, the UK, Switzerland, the Netherlands, and
France, and stem from at least three separate introductions.

France: France has had no restrictions on EU and
Schengen-area travel since it re-opened borders on the
15th of June. France’s 21 sequences have stemmed from
at least four introductions: two cluster directly with
Spanish sequences and one stems directly from a parent
with Spanish sequences. The remaining introduction is
genetically further from the diversity sampled in Spain,
and may indicate an introduction from another country,
possibly the United Kingdom or Switzerland.

Netherlands: The Netherlands began imposing a
quarantine on travellers returning from some regions of
Spain on the 28th July (Dutch News, 2020a), increasing
the areas from which travellers must quarantine until the
whole of Spain was included on the 25th August (Dutch
News, 2020b). The distribution of sequences from the
Netherlands suggests at least six introductions. Four
introductions share diversity with Spanish sequences,
suggesting direct importations from Spain. In the remaining
introductions, the sequences and their parent nodes
share no diversity with Spanish sequences, but instead
share genotypes with sequences from the UK, Ireland,
Switzerland, and Germany.