Meta-Analysis of the Dynamics of the Emergence of Mutations and Variants of SARS-CoV-2

TL;DR: In this article, the authors analyzed the appearance and prevalence trajectory of mutations that appeared in all SARS-CoV-2 genes from December 2019 to January 2021, and analyzed the structural properties of the spike glycoprotein of the B.1.7, B.351 and P.1 (Brazil) variants of concern.

Abstract: The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late December 2019 in Wuhan, China, and is the causative agent for the worldwide COVID-19 pandemic. SARS-CoV-2 is a 29,811 nucleotides positive-sense single-stranded RNA virus belonging to the betacoronavirus genus. Due to inefficient proofreading ability of the viral RNA-dependent polymerase complex, coronaviruses are known to acquire new mutations following replication, which constitutes one of the main factors driving the evolution of its genome and the emergence of new genetic variants. In the last few months, the identification of new B.1.1.7 (UK), B.1.351 (South Africa) and P.1 (Brazil) variants of concern (VOC) highlighted the importance of tracking the emergence of mutations in the SARS-CoV-2 genome and their impact on transmissibility, infectivity, and neutralizing antibody escape capabilities. These VOC demonstrate increased transmissibility and antibody escape, and reduce current vaccine efficacy. Here we analyzed the appearance and prevalence trajectory of mutations that appeared in all SARS-CoV-2 genes from December 2019 to January 2021. Our goals were to identify which modifications are the most frequent, study the dynamics of their spread, their incorporation into the consensus sequence, and their impact on virus biology. We also analyzed the structural properties of the spike glycoprotein of the B.1.1.7, B.1.351 and P.1 variants. This study offers an integrative view of the emergence, disappearance, and consensus sequence integration of successful mutations that constitute new SARS-CoV-2 variants and their impact on neutralizing antibody therapeutics and vaccines. IMPORTANCESARS-CoV-2 is the etiological agent of COVID-19, which has caused > 2 million deaths worldwide as of January, 2021. Mutations occur in the genome of SARS-CoV-2 during viral replication and affect viral infectivity, transmissibility and virulence. In early March 2020, the D614G mutation in the spike protein emerged, which increased the viral transmissibility and is now found in >90% of all SARS-CoV-2 genomic sequences in GISAID database. Between October and December 2020, B.1.1.7 (UK), B.1.351 (South Africa) and P.1 (Brazil) variants of concern (VOCs) emerged, which have increased neutralizing antibody escape capabilities because of mutations in the receptor binding domain of the spike protein. Characterizing mutations in these variants is crucial because of their effect on adaptive immune response, neutralizing antibody therapy, and their impact on vaccine efficacy. Here we tracked and analyzed mutations in SARS-CoV-2 genes over a twelve-month period and investigated functional alterations in the spike of VOCs.

Summary

INTRODUCTION

• In late December 2019, a new betacoronavirus known as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) emerged in the city of Wuhan in the province of Hubei, China (1).
• Coronaviruses are therefore expected to evolve through genetic drift much slower than other RNA viruses that do not have this ability, such as influenza viruses (8,10).
• Genetic variants are therefore the rare successful offshoots of the reference strain.
• In addition to the N501Y mutation, both the South African and Brazil variants possesses RBD mutations K417N(T) and E484K, which are also associated with further increased Nabs escape capabilities (24,25).

MATERIALS AND METHODS

• Data collection and mutational analysis Genomes uploaded to the GISAID EpiCoVTM server database were analyzed from December 1st, 2019, to December 31st, 2020, and selected viral sequences with submission dates from December 1st, 2019 to January 6, 2021.
• The authors filtered through 309,962 genomes for the analysis of selected mutations.
• For the analysis of the mutations in B.1.1.7, B.1.351, and P.1 variants, the authors used the GISAID EpiCoVTM server database.
• Only complete SARS-CoV-2 genomes (28 to 30 Kbps) isolated from human hosts were analyzed.

Structural modeling

• Mutations in the spike protein in complex with hACE2 were analyzed using a mutagenesis tool for PyMOL (PDB: 7A94).
• Figures and rendering were prepared with PyMOL.

RESULTS

• Identification of emerging mutations in various SARS-CoV-2 genes.
• The authors also demonstrate that most genes in SARS-CoV-2 have mutations with overall frequencies lower than 10% (Fig.1).
• Therefore, mutations presented here vastly underrepresent the global landscape of mutation frequency dynamics.
• These results allow us to better understand the frequencies, localization, and interactions of mutations in the S protein of the B.1.1.7 variant.

DISCUSSION

• The emergence of new genetic variants that are more transmissible, virulent and resistant to antibody neutralization have highlighted the importance of studying the function of mutations in the viral genome.
• The Oxford-AstraZeneca vaccine has displayed compromised efficacy against the B.1.351 variant with only 10% vaccine efficacy (36).
• The combination of D614G with other mutations in S can enable Nabs escape (19).
• The global frequency of mutations and variants in the database is therefore biased to represent the genetic landscape of the virus in the countries doing the most testing, sequencing, and data sharing.
• A global approach to analyzing both transmitted variants and non-transmitted sub-variants and quasispecies could provide a better understanding of the effects of SARS-CoV-2 mutations.

FIGURE LEGENDS

• Variations in mutations and mutation frequencies in SARS-CoV-2 genes, also known as Figure 1.
• Graphs were generated using RStudio and Biorender.
• G) Genome of the SARS-CoV-2 P.1 variant with identified nucleotides substitution, deletions, and insertions.

Figures

Table 2: Synonymous, non-synonymous and deletion mutations in the B.1.1.7 variant

Table 3: Synonymous, non-synonymous mutations and deletions in the B.1.351 variant

Table 1: Non-synonymous mutations in SARS-CoV-2 genes with a worldwide frequency ≥0.01%.

Table 6: Resistance of SARS-CoV-2 Variants to Vaccine-Induced Antibody Neutralization

Table 5: Efficacy of Vaccines Against SARS-CoV-2 Variants

Table 4: Synonymous, non-synonymous and deletions in the P.1 variant.

Full text

Meta-Analysis and Structural Dynamics of the Emergence of
Genetic Variants of SARS-CoV-2
Nicolas Castonguay
1
, Wandong Zhang
2,3
and Marc-Andre Langlois
1,4*
1
Department of Biochemistry, Microbiology & Immunology, Faculty of Medicine, University of
Ottawa, Ontario, Canada K1H 8M5.
2
Department of Cellular & Molecular Medicine, Faculty of Medicine, University of Ottawa,
Ontario, Canada K1H 8M5.
3
Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa,
Canada K1A 0R6
4
uOttawa Center for Infection, Immunity and Inflammation (CI3).
Running title: Genome Evolution of SARS-CoV-2.
*Correspondence should be addressed to: langlois@uottawa.ca
Key words: SARS-CoV-2, COVID-19, Variants of concern, B.1.1.7, B1.351, P.1, D614G
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 15, 2021. ; https://doi.org/10.1101/2021.03.06.21252994doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

Castonguay et al., 2021 Genome Evolution of SARS-CoV-2
2
ABSTRACT
The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late December
2019 in Wuhan, China, and is the causative agent for the worldwide COVID-19 pandemic. SARS-CoV-2
is a positive-sense single-stranded RNA virus belonging to the betacoronavirus genus. Due to the error-
prone nature of the viral RNA-dependent polymerase complex, coronaviruses are known to acquire new
mutations at each cycle of genome replication. This constitutes one of the main factors driving the evolution
of its relatively large genome and the emergence of new genetic variants. In the past few months, the
identification of new B.1.1.7 (UK), B.1.351 (South Africa) and P.1 (Brazil) variants of concern (VOC) have
highlighted the importance of tracking the emergence of mutations in the SARS-CoV-2 genome that impact
transmissibility, virulence, and immune and neutralizing antibody escape. Here we analyzed the appearance
and prevalence trajectory over time of mutations that appeared in all SARS-CoV-2 genes from December,
2019 to April, 2021. The goal of the study was to identify which genetic modifications are the most frequent
and study the dynamics of their propagation, their incorporation into the consensus sequence, and their
impact on virus biology. We also analyzed the structural properties of the spike glycoprotein of the B.1.1.7,
B.1.351 and P.1 variants for its binding to the host receptor ACE2. This study offers an integrative view of
the emergence, disappearance, and consensus sequence integration of successful mutations that constitute
new SARS-CoV-2 variants and their impact on neutralizing antibody therapeutics and vaccines.
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 15, 2021. ; https://doi.org/10.1101/2021.03.06.21252994doi: medRxiv preprint

Castonguay et al., 2021 Genome Evolution of SARS-CoV-2
3
IMPORTANCE
SARS-CoV-2 is the etiological agent of COVID-19, which has caused > 3.4 million deaths worldwide as
of April, 2021. Mutations occur in the genome of SARS-CoV-2 during viral replication and affect viral
infectivity, transmissibility, and virulence. In early March 2020, the D614G mutation in the spike protein
emerged, which increased viral transmissibility and is now found in over 90% of all SARS-CoV-2 genomic
sequences in GISAID database. Between October and December 2020, B.1.1.7 (UK), B.1.351 (South
Africa) and P.1 (Brazil) variants of concern (VOCs) emerged, which have increased neutralizing antibody
escape capabilities because of mutations in the receptor binding domain of the spike protein. Characterizing
mutations in these variants is crucial because of their effect on adaptive immune responses, neutralizing
antibody therapy, and their impact on vaccine efficacy. Here we tracked and analyzed mutations in SARS-
CoV-2 genes since the beginning of the pandemic and investigated their functional impact on the spike of
these three VOCs.
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 15, 2021. ; https://doi.org/10.1101/2021.03.06.21252994doi: medRxiv preprint

Castonguay et al., 2021 Genome Evolution of SARS-CoV-2
4
Graphical Abstract
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 15, 2021. ; https://doi.org/10.1101/2021.03.06.21252994doi: medRxiv preprint

Castonguay et al., 2021 Genome Evolution of SARS-CoV-2
5
INTRODUCTION
In late December 2019, a new betacoronavirus known as Severe Acute Respiratory Syndrome Coronavirus
2 (SARS-COV-2) emerged in the city of Wuhan in the province of Hubei, China (1). SARS-CoV-2 is the
etiological viral agent for the worldwide COVID-19 pandemic resulting in more than 162 million infected
and 3.4 million deaths worldwide as of April, 2021 (2,3). SARS-CoV-2 is an enveloped, positive-sense
single-stranded RNA (+ssRNA) virus with a genome length of 29,811 nucleotides (4). The mutation rates
of RNA viruses are generally higher than that of DNA viruses because of the low fidelity of their viral RNA
polymerases (5,6). Mutations occur when viral replication enzymes introduce errors in the viral genome
resulting in the creation of premature termination codons, deletions and insertions of nucleotides that can
alter open reading frames and result in amino acid substitutions in viral proteins. These mutations combined
with the selective pressure of the human immune system lead to the selection and evolution of viral genomes
(6,7). However, coronaviruses are one of the few members of the RNA virus family that possess limited
but measurable proofreading ability via the 3'-to 5'- exoribonuclease activity of the non-structural viral
protein 14 (nsp14) (8,9). Coronaviruses are therefore expected to evolve through genetic drift much slower
than other RNA viruses that do not have this ability, such as influenza viruses (8,10). Additionally, SARS-
CoV-2 and other coronaviruses have low known occurrences of recombination between family members
(i.e., genetic shift), and therefore are mostly susceptible to genetic drift (11).
SARS-CoV-2 has reached pandemic status due to its presence on every continent and has since maintained
a high level of transmissibility across hosts of various ethnical and genetic backgrounds (2, 12). Moreover,
SARS-CoV-2 infections have been reported to naturally infect minks, ferrets, cats, tiger, and dogs, which
allows the virus to replicate in completely new hosts and mutate to produce new variants and possibly new
strains (13,14). In March 2020, the now dominant D614G mutation first emerged in the spike protein (S)
of SARS-CoV-2. The S protein is present as a trimer at the surface of the viral envelope and is responsible
for attachment of the virus to the human angiotensin converting enzyme 2 (hACE2), the entry receptor for
SARS-CoV-2 into human cells (15). Published evidence has now shown that D614G increases viral fitness,
transmissibility and viral load but does not directly affect COVID-19 pathogenicity (16,17,18,19).
Additionally, emerging evidence indicates that D614G may have epistatic interactions that exacerbate the
impact of several other independent mutations (19). Mutations in the S protein, and particularly in the
receptor binding domain (RBD), are of very high concern given that they can directly influence viral
infectivity, transmissibility, and resistance to neutralizing antibodies and T cell responses.
New mutations are frequently and regularly detected in the genome of SARS-CoV-2 through whole genome
sequencing; however, very few of these mutations make it into the transmitted viral consensus sequence.
The reference strain is generally regarded as the dominant transmitted strain at a given time. Its sequence
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 15, 2021. ; https://doi.org/10.1101/2021.03.06.21252994doi: medRxiv preprint

Frequently asked questions

Q1. What have the authors contributed in "Meta-analysis and structural dynamics of the emergence of genetic variants of sars-cov-2" ?

Castonguay et al. this paper have shown that D614G increases viral fitness, transmissibility and viral load but does not directly affect COVID-19 pathogenicity. 

Q2. What is the etiological agent for the COVID-19 pandemic?

SARS-CoV-2 is the etiological viral agent for the worldwide COVID-19 pandemic resulting in more than 162 million infected and 3.4 million deaths worldwide as of April, 2021 (2,3). 

Q3. What are the mutations in the genome of SARS-CoV-2?

Mutations occur in the genome of SARS-CoV-2 during viral replication and affect viral infectivity, transmissibility, and virulence. 

Q4. What is the importance of tracking mutations and the evolution of the SARS-CoV-2?

Tracking mutations and the evolution of the SARS-CoV-2 genome is critical for the development and deployment of effective treatments and vaccines. 

Q5. What is the tool for tracking the emergence of mutations?

GISAID is a formidable tool for tracking the emergence of mutations, identifying the geographic region where it emerged, and tracking its spread around the globe. 

Q6. What is the caveat in analyzing sequences in the GISAID database?

Another caveat in analyzing sequences in the GISAID database is that consensus sequences are uploaded, but subsequences and quasispecies are generally not included. 

Q7. What is the effect of the K417T mutation on the C102 Nab?

The K417T mutation reduces interactions with neighboring residues in the C102 Nab and therefore the model predicts, as with K417N in B.1.1.7, an increased ability to escape neutralization (Fig. 7D). 

Q8. What is the efficacy of the vaccine against the B.1.351 variant?

The recently approved Johnson & Johnson adenovirus-based vaccine only requires a single dose, in comparison to two for the mRNA vaccines and Oxford-AstraZeneca vaccine, and has an efficacy of 66% against the original Wuhan reference strain, 52.0 to 64.0% against the B.1.351 variant and 66% against the P.1 variant (43). 

Q9. What is the effect of the P.1 vaccine on the immune system?

antibodies induced by the PfizerBioNTech and Moderna vaccines appear to display a 6.7-fold, and 4.5-fold decrease in neutralization efficacy against the P.1 variant (Table 6) (39). 

Q10. What is the effect of the N501Y mutation on the hACE2?

This mutation is reported to cause increased resistance to Nabs, increased transmissibility, and increased virulence in animal models (27). 

Q11. How many sequenced viral genomes were uploaded to the GISAID database?

The number of sequenced viral genomes uploaded to the GISAID database grew rapidly from 131,417 at the end of September, 2020 to 451,913 by January 30th, 2021 (28). 

Q12. What is the association between N501Y and hACE2?

The N501Y mutation is associated with an increased affinity to hACE2, along with an increase in infectivity and virulence (Table 2). 

Q13. What is the recent discovery of the B.1.1.7 variant in the UK?

A new variant was discovered in late 2020 in the UK that displayed increased affinity to hACE2, virulence and Nabs escape capabilities (Fig. 4) (24,25,30,31). 

Q14. What is the frequency of the mutations in the SARS-CoV-2 genome?

Here the authors present a retrospective metadata analysis of mutations throughout the SARS-CoV-2 genome that reached at least a 1% worldwide frequency between December 2019 and January 2021. 

Q15. What are the prevalent mutations in the B.1.1.7 variant?

The authors also observe the emergence of several mutations in S and N present in the B.1.1.7 variant, which now have a frequency higher than 65% (Fig. 2C, 2D and Table 2). 

Q16. What is the reason why new variants may go undetected?

Emergence of new variants may therefore go undetected until they leave their point of origin and enter countries with high testing and sequencing rates. 

Q17. What is the frequency of the recurring mutations in the SARS-CoV-2 genome?

The authors first selected recurring mutations that were present in more than 500 reported genomes by August 2020, and another selection was made in January 2021 to capture recurring mutations present in more than 4000 reported genomes in GISAID.