Emergence of SARS-CoV-2 through Recombination and Strong Purifying Selection

Abstract

COVID-19 has become a global pandemic caused by a novel coronavirus SARS-CoV-2. Understanding the origins of SARS-CoV-2 is critical for deterring future zoonosis and for drug discovery and vaccine development. We show evidence of strong purifying selection around the receptor binding motif (RBM) in the spike gene and in other genes among bat, pangolin and human coronaviruses, indicating similar strong evolutionary constraints in different host species. We also demonstrate that SARS-CoV-2's entire RBM was introduced through recombination with coronaviruses from pangolins, possibly a critical step in the evolution of SARS-CoV-2's ability to infect humans. Similar purifying selection in different host species and frequent recombination among coronaviruses suggest a common evolutionary mechanism that could lead to new emerging human coronaviruses.

One sentence summary:
Extensive Recombination and Strong Purifying Selection among coronaviruses from different hosts facilitate the emergence of SARS-CoV-2.

Full text

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Emergence of SARS-CoV-2 through Recombination
and Strong Purifying Selection
Short Title: Recombination and origin of SARS-CoV-2
One Sentence Summary: Extensive Recombination and Strong Purifying Selection among
coronaviruses from different hosts facilitate the emergence of SARS-CoV-2
Xiaojun Li1†, Elena E. Giorgi2†, Manukumar Honnayakanahalli Marichann1, Brian Foley2,
Chuan Xiao3, Xiang-Peng Kong4, Yue Chen1, Bette Korber2 and Feng Gao1*
1Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
2Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM 87544,
USA.
3Department of Chemistry and Biochemistry, The University of Texas at El Paso, El Paso, TX
79968, USA.
4Department of Biochemistry and Molecular Pharmacology, Grossman School of Medicine, New
York University, New York, NY 10016
†These authors contributed equally.
*Correspondence to: fgao@duke.edu
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Abstract
COVID-19 has become a global pandemic caused by a novel coronavirus SARS-CoV-2.
Understanding the origins of SARS-CoV-2 is critical for deterring future zoonosis and for drug
discovery and vaccine development. We show evidence of strong purifying selection around the
receptor binding motif (RBM) in the spike gene and in other genes among bat, pangolin and
human coronaviruses, indicating similar strong evolutionary constraints in different host species.
We also demonstrate that SARS-CoV-2’s entire RBM was introduced through recombination
with coronaviruses from pangolins, possibly a critical step in the evolution of SARS-CoV-2’s
ability to infect humans. Similar purifying selection in different host species and frequent
recombination among coronaviruses suggest a common evolutionary mechanism that could lead
to new emerging human coronaviruses.
Introduction
The severe respiratory disease COVID-19 was first noticed in late December 2019 (1). It rapidly
became epidemic in China, devastating public health and finance. By mid-March, COVID-19
had spread to ~150 countries and infected over 150,000 people (2). On March 11, 2020, the
World Health Organization (WHO) officially declared it a pandemic.
A complete genome sequence of the etiological agent of COVID-19 (3), severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) (4), identified it as a new member of the genus
Betacoronavirus, which include a diverse reservoir of coronaviruses (CoVs) isolated from bats
(5-7). While genetically distinct from the betacoronaviruses that cause SARS and MERS in
humans (8, 9), SARS-CoV-2 shares the highest level of genetic similarity (96.3%) with CoV
RaTG13, sampled from a bat in Yunnan in 2013 (8). Recently, CoV sequences closely related to
SARS-CoV-2 were obtained from confiscated Malaya pangolins in two separate studies (10, 11).
Pangolin SARS-like CoVs (Pan_SL-CoV) form two distinct clades corresponding to their
collection location. Pan_SL-CoV_GD from Guangdong (GD) province in China and are
genetically more similar to SARS-CoV-2 (91.2%) than Pan_SL-CoV_GX from Guangxi (GX)
province (85.4%).
Understanding the origin of SARS-CoV-2 may help resolve strategies to deter future cross-
species transmissions and to establish appropriate animal models. Viral sequences nearly
identical to SARS and MERS viruses were found in civets and domestic camels, respectively
(12, 13), demonstrating that they originated from zoonotic transmissions with intermediate host
species between the bat reservoirs and humans—a common pattern leading to CoV zoonosis (5).
Viruses nearly identical to SARS-CoV-2 have not yet been found. In this paper we demonstrate,
through localized genomic analysis, a complex pattern of evolutionary recombination between
CoVs from distinct host species and cross-species infections that likely originated SARS-CoV-2.
Results
Acquisition of receptor binding motif through recombination
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Phylogenetic analysis of 43 complete genome sequences from three clades (SARS-CoVs and
bat_SL-CoVs; SARS-CoV-2, bat_SL-CoVs and pan_SL-CoVs; and two divergent bat_SL-
CoVs) within the Sarbecovirus group (9) confirms that RaTG13 is overall the closest sequence to
SARS-CoV-2 (fig. S1). It is followed by Pan_SL-CoV_GD viruses next, and then Pan_SL-
CoV_GX. Among the bat-CoV sequences in clade 2 (fig. S1), ZXC21 and ZC45, sampled from
bats in 2005 in Zhoushan, Zhejiang, China, are the most divergent, with the exception of the
beginning of the ORF1a gene (region 1, fig. 1A). All other Bat_SL-CoV and SARS-CoV
sequences form a separate clade 3, while clade 1 comprises BtKY72 and BM48-31, the two most
divergent Bat_SL-CoV sequences, in the Sarbecovirus group (fig. S1). Recombination in the first
SARS-CoV-2 sequence (Wuhan-Hu-1) with other divergent CoVs has been previously observed
(3). Here, to better understand the role of recombination in the origin of SARS-CoV-2 among
these genetically similar CoVs, we compare Wuhan-Hu-1 to six representative Bat_SL-CoVs,
one SARS-CoV, and the two Pan_SL-CoV_GD sequences using SimPlot analysis (14). RaTG13
has the highest similarity across the genome (8), with two notable exceptions where a switch
occurs (fig. 1A). In phylogenetic reconstructions, SARS-CoV-2 clusters closer to ZXC21 and
ZC45 than RaTG13 at the beginning of the ORF1a gene (region 1, fig. 1B), and, as reported (10,
15), to a Pan_SL-CoV_GD in region 2 (fig.s 1C and S2), which spans the receptor angiotensin-
converting enzyme 2 (ACE2) binding site in the spike (S) glycoprotein gene. Comparing Wuhan-
Hu-1 to Pan_SL-CoV_GD and RaTG13, as representative of distinct host-species branches in the
evolutionary history of SARS-CoV-2, using the recombination detection tool RIP (16), we find
significant recombination breakpoints before and after the ACE2 binding site (fig. S2A),
suggesting that SARS-CoV-2 carries a history of cross-species recombination between the bat
and the pangolin CoVs.
Pan_SL-CoV sequences are generally more similar to SARS-CoV-2 than CoV sequences, other
than RaTG13 and ZXC21, but are more divergent from SARS-CoV-2 at two regions in
particular: the beginning of the ORF1b gene and the highly divergent N terminus of the S gene
(regions 3 and 4, respectively, fig. 1A). Within-region phylogenetic reconstructions show that
Pan_SL-CoV sequences become as divergent as BtKY72 and BM48-31 in region 3 (fig. 1D),
while less divergent in region 4, where Pan_SL-CoV_GD clusters with ZXC21 and ZC45 (fig.
1E). Together, these observations suggest ancestral cross-species recombination between
pangolin and bat CoVs in the evolution of SARS-CoV-2 at the ORF1a and S genes. Furthermore
the discordant phylogenetic clustering at various regions of the genome among clade 2 CoVs
also supports extensive recombination among these viruses isolated from bats and pangolins.
The SARS-CoV-2 S glycoprotein mediates viral entry into host cells and therefore represents a
prime target for drug and vaccine development (17, 18). While SARS-CoV-2 sequences share
the greatest overall genetic similarity with RaTG13, this is no longer the case in parts of the S
gene. Specifically, amino acid sequences of the receptor binding motif (RBM) in the C terminal
of the S1 subunit are nearly identical to those in two Pan_SL-CoV_GD viruses, with only one
amino acid difference (Q498H)—although the RBM region has not been fully sequenced in one
of Guangdong pangolin virus (Pan_SL-CoV_GD/P2S) (fig. 2A). Pangolin CoVs from Guangxi
are much more divergent. Phylogenetic analysis based on the amino acid sequences of this
region shows three distinct clusters of SARS-CoV, SARS-CoV-2 and bat-CoV only viruses,
respectively (fig. 2B). Interestingly, while SARS-CoV and SARS-CoV-2 viruses use ACE2 for
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viral entry, all CoVs in the third cluster have a 5-aa deletion and a 13-14-aa deletion in RBM
(fig. 2A) and cannot infect human target cells (5, 19).
Although both SARS-CoV and SARS-CoV-2 use the human ACE2 as their receptors (8, 20)
they show a high level of genetic divergence (figs. 1 and S1). However, structures of the S1 unit
of the S protein from both viruses are highly similar (21-23), with the exception of a loop, not
proximal to the binding site, that bends differently (fig. 2C). This suggests that viral entry
through binding of ACE2 is structurally constrained to maintain the correct conformation.
Among 17 distinct amino acids between SARS-CoV-2 and RaTG13 (fig. 2A), five contact sites
are different, likely impacting RaTG13’s binding to ACE2 (fig. 2D and Table S1). The single
amino acid difference (Q or H at position 498) between SARS-CoV-2 and Pan_SL-CoV_GD is
at the edge of the ACE2 contact interface; neither Q or H at this position form hydrogen bonds
with ACE2 residues (fig. 2E). Thus, a functional RBM nearly identical to the one in SARS-CoV-
2 is naturally present in Pan_SL-CoV_GD viruses. The very distinctive RaTG13 RBM suggests
that this virus is unlikely to infect human cells, and that the acquisition of a complete functional
RBM by a RaTG13-like CoV through a recombination event with a Pan_SL-CoV_GD-like virus
enabled it to use ACE2 for human infection.
Three small insertions are identical in SARS-CoV-2 and RaTG13 but not found in other CoVs in
the Sarbecovirus group (24). The RaTG13 sequence was sampled in 2013, years before SARS-
CoV-2 was first identified. It is unlikely that both SARS-CoV-2 and RaTG13 independently
acquired identical insertions at three different locations in the S gene. Thus, it is plausible that an
RaTG13-like virus served as a progenitor to generate SARS-CoV-2 by gaining a complete
human ACE2 binding RBM from Pan_SL-CoV_GD-like viruses through recombination. Genetic
divergence at the nucleic acid level between Wuhan-Hu-1 and Pan_SL-CoV_GD viruses is
significantly reduced from 13.9% (fig. 1E) to 1.4% at the amino acid level (fig. 2B) in the RBM
region, indicating recombination between RaTG13-like CoVs and Pan_SL-CoV_GD-like CoVs.
Furthermore, SARS-CoV-2 has a unique furin cleavage site insertion (PRRA) not found in any
other CoVs in the Sarbecovirus group (24), although similar motifs are also found in MERS and
more divergent bat CoVs (25) (Fig. S3). This PRRA motif makes the S1/S2 cleavage in SARS-
CoV-2 much more efficiently than in SARS-CoV and may expand its tropism and/or
enhance its transmissibility (23). A recent study of bat CoVs in Yunnan, China, identified a
three-amino acid insertion (PAA) at the same site (26). Although it is not known if this PAA
motif can function like the PRRA motif, the presence of a similar insertion at the same site
indicates that such insertion may already be present in the wild bat CoVs. The more efficient
cleavage of S1 and S2 units of the spike glycoprotein (25) and efficient binding to ACE2 by
SARS-CoV-2 (22, 27) may have allowed SARS-CoV-2 to jump to humans, leading to the rapid
spread of SARS-CoV-2 in China and the rest of the world.
Strong purifying selection among SRAS-CoV-2 and closely related viruses
Recombination from Pan_SL-CoV_GD at the RBM and at the unique furin cleavage site
insertion prompted us to examine the SARS-CoV-2 sequences within these regions. Amino acid
sequences from SARS-CoV-2, RaTG13, and all Pan_SL-CoV viruses are identical or nearly
identical before, between, and after the RBM and the furin cleavage site, while all other CoVs
are very distinctive (fig. 3A and S3). The average of all pairwise dN/dS ratios (w) among SARS-
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CoV-2, RaTG13, and Pan_SL-CoV viruses at the 3’-end of the S gene (after the furin cleavage
site) is 0.013, compared to w =0.05 in the S region preceding the furin cleavage site, and to w
=0.04 after the site for all other CoVs. The much lower w value at the 3’-end of the S gene
among the SARS-CoV-2, RaTG13, and Pan_SL-CoV viruses indicates that this region is under
strong purifying selection within these sequences (fig. 3A). A plot of synonymous and
nonsynonymous substitutions relative to Wuhan-Hu-1 highlights the regional differences across
the region before and after the RBM and the furin cleavage site (fig. 3A): the 3’ end of the region
is highly conserved among the SARS-CoV-2, RaTG13, and Pan_SL-CoV viruses (Group A),
while far more nonsynonymous mutations are observed in the rest of the CoV sequences (Group
B). The shift in selective pressure in the 3’ -end of the gene among these related viruses versus
other CoVs begins near codon 368 (fig. 3B), and such a shift was not evident among other
compared CoVs (fig. 3B-D).
We observe similar patterns of purifying selection pressure in other parts of the genome,
including the E and M genes, as well as the partial ORF1a and ORF1b genes (fig. 4).
Interestingly, the purifying selection pressure varies among different viruses depending on which
genes are analyzed. The broadest group includes SARS-CoV-2, RaTG13, all Pan_SL-CoV and
the two bat CoVs (ZXC21 and ZC45) for both E and M genes (figs. 4 and S5). The second group
includes SARS-CoV-2, RaTG13, and all Pan_SL-CoV only for the 3’ end of the S gene. The
narrowest selection group only contains SARS-CoV-2, RaTG13, and pangolin CoVs from
Guangdong for the partial regions of ORF1a and ORF1b (figs. 4 and S6). Consistently low ω
values and strong purifying selection pressure on SARS-CoV-2, RaTG13 and Pan_SL-CoV_GD
viruses suggest that these complete and partial genes are under similar functional/structural
constraints among the different host species. In two extreme cases, amino acid sequences of the
E gene and the 3’ end of ORF1a are identical among the compared CoV sequences, although
genetic distances are quite large among these viruses at the nucleic acid level. Such evolutionary
constraints across viral genomes, especially at functional domains in the S gene, which plays an
important role in cross-species transmission (5, 17), coupled with frequent recombination, may
facilitate cross-species transmissions between RaTG13-like bat and/or Pan_SL-CoV_GD-like
viruses.
Frequent recombination between SARS-CoVs and bat_SL-CoVs
Previous studies using more limited sequence sets found that SARS-CoVs originated through
multiple recombination events between different bat-CoVs (10, 17, 19, 28, 29). Our phylogenetic
analyses of individual genes show that SARS-CoV sequences tend to cluster with YN2018B,
Rs9401, Rs7327, WIV16 and Rs4231 (group A) for some genes and Rf4092, YN2013, Anlong-
112 and GX2013 (group B) for others (fig. S7). SimPlot analysis using both groups of bat_SL-
CoVs and the closely related bat CoV YNLF-34C (29) shows that SARS-CoV GZ02 shifts in
similarity across different bat SL-CoVs at various regions of the genome (fig. 5A). In particular,
phylogenetic reconstruction of the beginning of ORF1a (region 1) confirms that SARS-CoVs
cluster with YNLF-34C (29), and this region is distinctive comparing to all other CoVs (fig. 5B).
YNLF-34C is more divergent from SARS-CoV than other bat-CoV viruses before and after this
region, confirming the previously reported complex recombinant nature of YNLF-34C (29) (fig.
5A). At the end of the S gene (region 2), SARS-CoVs cluster with group A CoVs, forming a
highly divergent clade (fig. 5C). In region 3 (ORF8), SARS-CoVs and group B CoVs, together
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with YNLF-34C, form a very divergent and distinctive cluster (fig. 5D). To further explore the
recombinant nature of SARS-CoVs, we compared GZ02 to representative bat CoV sequences
using the RIP recombination detection tool (16). We identified four significant breakpoints (at
99% confidence) between the two parental lineages (fig. S8A), further supported by phylogenetic
analysis (fig. S8B-S8D). In addition, the two aforementioned groups of bat CoVs (shown in light
brown and light blue in the trees) show similar cluster changes across the five recombinant
regions, suggesting multiple events of historic recombination among bat SL-CoVs. These results
demonstrate that SARS-CoV shares a recombinant history with at least three different groups of
bat-CoVs and confirms the major role of recombination in the evolution of these viruses.
Of the bat SL-CoVs that contributed to the recombinant origin of SARS-CoV, only group A
viruses bind to ACE2. Group B bat SL-CoVs do not infect human cells (5, 19) and have two
deletions in the RBM (figs. 1E and 2A). The short deletion between residues 445 and 449, and in
particular the loss of Y449, which forms three hydrogen bonds with ACE2, will significantly
affect the overall structure of the RBM (figs. 2F and 2E). The region encompassing the large
deletion between residues 473 and 486 contains the loop structure that accounts for the major
differences between the S protein of SARS-CoV and SARS-CoV-2 (fig. 2C). This deletion
causes the loss of contact site F486 and affects the conserved residue F498’s hydrophobic
interaction with residue M82 on ACE2 (fig. 2F). These two deletions will render RBM in those
CoVs incapable to bind human ACE2. Therefore, recombination may play a role in enabling
cross-species transmission in SARS-CoVs through the acquisition of an S gene type that can
efficiently bind to the human ACE2 receptor.
ORF8 is one of the highly variable genes in coronaviruses (5, 17, 29) and its function has not yet
been elucidated (5, 17, 30). Breakpoints within this region show that recombination occurred at
the beginning and the end of ORF8 (fig. S9), where nucleic acid sequences are nearly identical
among both SARS-CoVs and group B bat CoVs. Moreover, all compared viruses form three
highly distinct clusters (fig. 5D), suggesting that the ORF8 gene may be biologically constrained
and evolves through modular recombination. The third recombination region at the beginning of
ORF1a is where SARS-CoV-2 also recombined with other bat CoVs (region 1, fig. 1A). This
region is highly variable (5, 17) and recombination within this part of the genome was also found
in many other CoVs, suggesting that it may be a recombination hotspot and may factor into
cross-species transmission.
Discussion
There are three important aspects to betacoronavirus evolution that should be carefully
considered in phylogenetic reconstructions among more distant coronaviruses. First, there is
extensive recombination among all of these viruses (10, 17, 19, 28, 29) (figs. 1 and 5), making
standard phylogenetic reconstructions based on full genomes problematic, as different regions of
the genome have distinct ancestral relationships. Second, between more distant sequences,
synonymous substitutions are often fully saturated, which can confound analyses of selective
pressure and add noise to phylogenetic analysis. Finally, there are different selective pressures at
work in different lineages, which is worth consideration interpreting trees.
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The currently sampled pangolin CoVs are too divergent from SARS-CoV-2 for them to be
SARS-CoV-2 progenitors, but it is noteworthy that these sequences contain an RBM that can
most likely bind to human ACE2. While RaTG13 is the most closely related CoV sequence to
SARS-CoV-2, it has a distinctive RBM, which is not expected to bind to human ACE2. SARS-
CoV-2 has a nearly identical RBM to the one found in the pangolin CoVs from Guangdong.
Thus, it is plausible that RaTG13-like bat-CoV viruses may have obtained the RBM sequence
binding to human ACE2 through recombination with Pan_SL-CoV_GD-like viruses. We
hypothesize that this, and/or other ancestral recombination events between viruses infecting bats
and pangolins, may have had a key role in the evolution of the strain that lead to the introduction
of SARS-CoV-2 into humans.
All three human CoVs (SARS, MERS and SARS-2) are the result of recombination among
CoVs. Recombination in all three viruses involved the S gene, likely a precondition to zoonosis
that enabled efficient binding to human receptors (5, 17). Extensive recombination among bat
coronaviruses and strong purifying selection pressure among viruses from humans, bats and
pangolin may allow such closely related viruses ready jump between species and adapt to the
new hosts. Many bat CoVs have been found able to bind to human ACE2 and replication in
human cells (10, 19, 31-33). Serological evidence has revealed that additional otherwise
undetected spillovers have occurred in people in China living in proximity to wild bat
populations (34). Continuous surveillance of coronaviruses in their natural hosts and in humans
will be key to rapid control of new coronavirus outbreaks.
So far efforts have failed to find the original pathway of SARS-CoV-2 into humans by
identifying a coronavirus that is nearly identical to SARS-CoV-2, as those found for SARS and
MERS in civets and domestic camels respectively (12, 13). However, if the new SARS-CoV-2
strain did not cause widespread infections in its natural or intermediate hosts, such a strain may
never be identified. The close proximity of animals of different species in a wet market setting
may increase the potential for cross-species spillover infections, by enabling recombination
between more distant coronaviruses and the emergence of recombinants with novel phenotypes.
While the direct reservoir of SARS-CoV-2 is still being sought, one thing is clear: reducing or
eliminating direct human contact with wild animals is critical to preventing new coronavirus
zoonosis in the future.
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Acknowledgments: We thank all those who have contributed SARS-CoV-2 genome sequences
to the GISAID database (https://www.gisaid.org/) and Virological.org (http://virological.org/).
We also thank Dr. Xinquan Wang from Tsinghua University for sharing the PDB 6M0J structure
with us before its official release date. Funding: This work was supported by NIH Grants
(AI122909, AI118571, GM129525 and AI145655). EEG, BK, and BF acknowledge support by
the LDRD program at Los Alamos National Laboratory. Author contributions: Project
conceptualization: F.G., B.K., E.E.G; Structure analysis: C.X., X-P.K; Sequence analysis: F.G.,
B.K., X.L., E.E.G., M.H.M., Y.C.; Phylogenetic analysis: F.G. B.K., X.L., E.E.G., M.H.M.,
Y.C.; Recombination analysis: F.G., E.E.G., B.K., X.L., E.E.G., M.H.M.,; Manuscript writing:
F.G., B.K., E.E.G. Manuscript editing: F.G., B.K., E.E.G., X.L., C.X., X-P.K. F.G. and B.K.
supervised the project. Competing interests: All authors declare no competing interests. \ All data
is available in the main text or the supplementary materials.
Supplementary Materials:
Materials and Methods
Figures S1-S9
Table S1
References (35-46)
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Fig. 1. SARS-CoV-2 recombination with Pan_SL-CoV and Bat_SL-CoV. (A) SimPlot
genetic similarity plot between SARS-CoV-2 Wuhan-Hu-1 and representative CoV sequences,
using a 400-bp window at a 50-bp step and the Kimura 2-parameter model. (B-E) Phylogenetic
trees of regions of disproportional similarities, showing high similarities with ZXC21 (B) or
GD/P1La (C), or high divergences with both GD/P1La and GX/P4L (D) or GD/P1La (E). All
positions are relative to Wuhan-Hu-1.
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Fig. 2. Impact of SARS-CoV-2 recombination on coreceptor binding. (A) AA sequences of
the receptor binding motif (RBM) in the spike (S) gene among Sarbecovirus CoVs compared to
Wuhan-Hu-1 (top). Dashes indicate identical aa’s, dots indicate deletions. ACE2 critical contact
sites highlighted in blue, two large deletions in green. (B) RBM aa phylogenetic tree, showing
three distinct clusters, with large deletions Bat-SL-CoVs in divergent cluster. (C) SARS-CoV
and SARS-CoV-2 receptor binding domains (RBD). Human ACE2 in green at the top and the S1
unit of the S-protein at the bottom; SARS-CoV S-protein (PDB 2AJF) in red, and SARS-CoV-2
S-protein in magenta with RBM in blue. All structure backbones shown as ribbons with key
residues at the interface shown as stick models, labeled using the same color scheme. (D) Impact
of different RBM aa between SARS-CoV-2 RaTG13 on ACE2 binding. (E) Impact of different
aa at position 498 (Q in SARS-CoV-2, top, and H in RaTG13, bottom) on ACE2 binding. Same
color-coding as in (C) with additional hydrogen bonding as light blue lines. (F) Impact of two
deletions on ACE2 binding interface in some bat-SL-CoVs, positions indicated in yellow, and
modeled structure with long deletion between residue 473 in light blue.
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Fig. 3. Strong purifying selection after furin cleavage in S gene among SARS-CoV-2 and
closely related viruses. (A) Phylogenetic tree (left) and Highlighter plot (right) of sequences
around the RBM and furin cleavage site compared to SARS-CoV-2 Wuhan-Hu-1 (na positions
22541-24391). ACE2 receptor binding motif (RBM) and furin cleavage site highlighted in light-
gray boxes. Mutations compared to Wuahn-Hu-1 are light blue for synonymous, red for non-
synonymous. Dominance of synonymous mutations within group A compared to group B
highlighted on the right. (B) Cumulative plots of each codon average behavior for all pairwise
comparisons for indels and synonymous (light blue) and non-synonymous (red) mutations, by
group. A vertical steps in group A at around codon 370 (na 1105) shows a shift in localized
accumulations; non-synonymous mutations end after the furin cleavage site. Group B instead
lacks this abrupt change in slope. w’s denote average ratios of the rate of nonsynonymous
substitutions per nonsynonymous site (dN/dS) for each group and region half. (C) Sequence
dS/dN ratios compared to Wuhan-Hu-1 within codons 1-370 (na 1-1104, green) and codons 371-
620 (na 1105-1893, dark blue). (D) Proportion of tree branches under positive and negative
selection (right and left respectively) per site for the two groups using the mixed effects model of
evolution (MEME) from datamonkey (www.datamonkey.org).
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Fig. 4. Strong purifying selection on complete and partial gene regions among SARS-CoV-
2, RaTG13 and Pan_SL-CoV viruses. Purifying selection pressure on complete and partial
genes within different viruses (red boxes) as evident by shorter branches in aa phylogenetic trees
compared to na trees. Distinct purifying selection patterns are observed among different viruses:
(A) SARS-CoV-2, RaTG13, Pan_SL-CoV and bat CoV ZXC21 and ZC45; (B) SARS-CoV-2,
RaTG13, all Pan_SL-CoV sequences; (C) SARS-CoV-2, RaTG13 and Pan_SL-CoV_GD.
Cumulative plots of the average behavior of each codon for all pairwise comparisons for
synonymous mutations, non-synonymous mutations and indels within each gene region. w’s
denote average ratios of the rate of nonsynonymous substitutions per nonsynonymous site
(dN/dS) for each group.
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Fig. 5. Multiple recombination of SARS-CoVs with different bat_SL-CoVs. (A) SimPlot
genetic similarity plot between SARS-CoV GZ02 and SARS_SL-CoVs, using a 400-bp window
at a 50-bp step and the Kimura 2-parameter model. Group A reference CoVs (YN2018B,
Rs9401, Rs7327, WIV16 and Rs4231) in blue, group B CoVs (Rf4092, YN2013, Anlong-112
and GX2013) in orange, YNLF-34C in green, and outlier control HKU3-12 in red. (B-D)
Phylogenetic trees for high similarity regions between GZ02 and YNLF-34C (B), group (C), and
group B (D). All positions are relative to Wuhan-Hu-1.
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