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Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2
Katherine Laiton-Donato1*, Carlos Franco-Muñoz1*, Diego A. Álvarez-Díaz1, Hector Alejandro
Ruiz-Moreno1, José A. Usme-Ciro1,2, Diego Andrés Prada1, Jhonnatan Reales-González1 Sheryll
Corchuelo1, María T. Herrera-Sepúlveda1, Julian Naizaque1, Gerardo Santamaría1, Jorge Rivera1,
Paola Rojas1, Juan Hernández Ortiz3, Andrés Cardona3, Diana Malo4, Franklin Prieto-Alvarado4,
Fernando Ruiz Gómez5, Magdalena Wiesner1, Martha Lucia Ospina Martínez6, Marcela
Mercado-Reyes1.
* These authors contributed equally.
Authors details
1 Group Genomics of Emerging Microorganisms. Dirección de Investigación en Salud Pública,
Instituto Nacional de Salud, Bogotá, Colombia.
2 Centro de Investigación en Salud para el Trópico - CIST, Facultad de Medicina, Universidad
Cooperativa de Colombia, Santa Marta, Colombia.
3 Laboratorio Genómico One Health. Medellín, Colombia.
4 Dirección de Vigilancia en Salud Pública, Instituto Nacional de Salud, Bogotá, Colombia.
5 Ministerio de Salud y Protección Social de Colombia. Bogotá Colombia.
6 Dirección General, Instituto Nacional de Salud, Bogotá, Colombia.
Corresponding author: Katherine Laiton-Donato. Group Genomics of Emerging Microorganisms,
Department of Public Health Research, Instituto Nacional de Salud, Av. Calle 26 No 51-20,
Bogotá 111321, Colombia . Email: kdlaitond@unal.edu.co
Highlights
Monitoring the emergence of new variants of SARS-CoV-2 in real time is a worldwide
priority.
Emerging variants of SARS-CoV-2 may have high impact biological implications for
public health
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
The SARS-CoV-2 B.1.621 variant of interest was characterized by several
substitutions: T95I, Y144T, Y145S, ins146N, R346K, E484K, N501Y and P681H in
spike protein.
Summary
The genetic diversity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the
potential to impact the virus transmissibility and the escape from natural infection- or vaccine-
elicited neutralizing antibodies. Here, representative samples from circulating SARS-CoV-2 in
Colombia between January and April 2021, were processed for genome sequencing and lineage
determination following the nanopore amplicon ARTIC network protocol and PANGOLIN
pipeline. This strategy allowed us to identify the emergence of the B.1.621 lineage, considered a
variant of interest (VOI) with the accumulation of several substitutions affecting the Spike
protein, including the amino acid changes T95I, Y144T, Y145S and the insertion 146N in the N-
terminal domain, R346K, E484K and N501Y in the Receptor-binding Domain (RBD) and
P681H1 in the S1/S2 cleavage site of the Spike protein. The rapid increase in frequency and
fixation in a relatively short time in Magdalena, Atlántico, Bolivar, Bogotá D.C, and Santander
that were near the theoretical herd immunity suggests an epidemiologic impact. Further studies
will be required to assess the biological and epidemiologic roles of the substitution pattern found
in the B.1.621 lineage.
Keywords: SARS-CoV-2, variant of interest (VOI), evolution, emergence
1. Introduction
In September 2020, SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI)
started to be reported, with more distinctive substitutions than expected from the characteristic
clock-like molecular evolution of this virus evidenced during the first year pandemic (Abdool
Karim and de Oliveira, 2021; ECDE, 2021). In the following months, with data from clinical and
genomic surveillance worldwide, the US government interagency group and Centers for Disease
Control and Prevention (CDC) proposed a hierarchical variant classification scheme with three
classes of SARS-CoV-2 variants where the higher classes include the possible attributes of lower
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classes; 1) Variant of Interest, 2) Variant of Concern and 3) Variant of High Consequence
(VOHC). A VOI is characterized by a set of Spike protein substitutions associated with increased
infectivity, resistance to post-vaccinal/infection antibodies, possible increase in transmissibility
and worse clinical outcome. VOC, besides the possible attributes of VOI, there is evidence of
impact on diagnostics, treatments, or vaccines, increased transmissibility, and disease severity.
VOHC, although currently there are no SARS-CoV-2 identified within this class to date, it is
expected that besides the possible attributes of a VOC, a VOHC has strong evidence of
diagnostic failure, a significant reduction in vaccine effectiveness, approved therapeutics, and
more severe clinical disease and increased hospitalizations (Centers for Disease Control and
Prevention, 2021).
Despite mutations spanning the whole genome, an interesting feature of these emerging variants
has been the presence of several amino acid substitutions falling in the Spike protein, the viral
protein responsible for receptor binding and membrane fusion and also the main target for
neutralizing antibodies (NAb) (Greaney et al., 2021) . Monitoring the emergence of new variants
of SARS-CoV-2 is a priority worldwide, as the presence of certain non-synonymous
substitutions and Insertion–deletion mutations (INDELs) could be related to biological
properties, such as altering the ligand-receptor affinity, the efficiency of neutralization by
naturally acquired polyclonal immunity or post-vaccination antibodiesand transmission capacity
(Davies et al., 2021; Jeyanathan et al., 2020; Rees-Spear et al., 2021).
In Colombia, the National Genomic Characterization Program led by the Instituto Nacional de
Salud has carried out real-time monitoring of the SARS-CoV-2 lineages since the beginning of
the pandemic through next generation sequencing and following the Pan American Health
Organization (PAHO) guidance for SARS-CoV-2 samples selection for genomic characterization
and surveillance (INS, 2021; Laiton-Donato et al., 2020).Until December 2020, over thirty
lineages were circulating inside the country without evidence of VOC and VOI importation.
However, a lineage turnover accompanied the third epidemic peak during March and April 2021,
involving the emergence of B.1 lineage descendants with high mutation accumulation (B.1.621
and the provisionally assigned B.1+L249S+E484K) (PAHO, n.d.), as well as the introduction of
the B.1.1.7, P.1 and VOI in Magdalena, Atlántico, Bolivar, Bogotá D.C, and Santander.
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In this study, we reported the emergence and spread of the novel B.1.621 lineage of SARS-CoV-
2, a new VOI with the insertion 146N and several amino acid substitutions in the Spike protein
(T95I,Y144T, Y145S, R346K, E484K, N501Y and P681H).
2. Materials and methods
A total of 471 nasopharyngeal swab specimens from patients with positive real time RT-PCR for
SARS-CoV-2, were collected between January 1st and April 30th, 2021were selected from
routine surveillance in all departments based on the representativeness and virologic criteria
(PAHO, n.d.). Samples were processed by using the amplicon sequencing protocol v3
(https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye). The
assembly of raw NGS data was performed by following the pipeline described for Oxford
Nanopore Technologies (ONT) platform (https://artic.network/ncov-2019/ncov2019-
bioinformatics-sop.html).
Lineage assignment started by filing a new issue in the pango-designation repository
(https://github.com/cov-lineages/pango-designation/issues/57) followed by designation as
B.1.621 lineage by the Pangolin curation team and PangoLEARN model training for subsequent
automatic lineage assignment.
Dataset was aligned using the MAFFT v.7 software and maximum likelihood tree reconstruction
was performed with the GTR+F+I+G4 nucleotide substitution model using IQTREE. Branch
support was estimated with an SH-like approximate likelihood ratio test (SH-aLRT) and ultrafast
bootstrap. Recombination detection was performed using RDP4 software with RDP,
GENECONV, Bottscan, Maxchi, Chimaera, SiSscan, and 3Seq tests (P-value <0.05). Dataset
1 included Colombian SARS-CoV-2 sequences representative of the different lineages and
dataset 2 included sequences previously reported as VOC or VOI. Adaptive evolution analysis at
the codon level was estimated by Hyphy using stochastic evolutionary models. The detection of
individual sites was performed with methods such as MEME (Mixed Effects Model of
Evolution), and FEL (Fixed Effects Likelihood) (P-value <0.3).
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3. Results
The routine genomic surveillance of SARS-CoV-2 in Colombia was reinforced in January 2021
for a higher sensitivity monitoring of the potential importation of VOC. By May 7, 2021, a total
of 908 sequences from Colombia were available in the GISAID database. Lineage B.1 is the
best-represented lineage (with 229 records) due to its higher frequency from the beginning of the
pandemic. The recently designated B.1.621 lineage has been increasingly detected since January
11, 2021 (collection date of the first genome belonging to the lineage) to date (77 records),
occupying the fifth place in frequency (Figure 1A), and rapidly becoming fixed in some
departments located in the North of the country or co-circulating with other lineages in Bogotá
D.C. and Santander (Figure 1B).
The original assignment through the Pangolin algorithm for this monophyletic group was the B.1
lineage. The genetic background of the B.1.621 lineage includes some convergent amino acid
changes in the RBD of the Spike protein which have appeared independently in several VOI and
VOC: N501Y, present in B.1.1.7, B.1.351, and P.1 lineages; and E484K present in B.1.351 and
P.1 (Harvey et al., 2021) as well as P681H in the S1/S2 furin cleavage site in B.1.1.7 and
B.1.1.50 + P681H Variant (Zuckerman et al., 2021). However, a distinctive profile of
synonymous and non-synonymous substitutions was found in the Spike protein of B.1.621,
including T95I, Y144T, Y145S in the N-terminal domain in addition to substitutions R346K,
E484K and N501Y in the RBD, P681H in the S1/S2 furin cleavage site (Table 1) and the
insertion 146N in the N-terminal domain (NTD) (supplementary table 1).
The close phylogenetic relationship of the SARS-CoV-2 sequences belonging to the B.1.621
lineage with other sequences from representative lineages circulating worldwide and those
circulating in Colombia suggested a recent origin from the parental lineage B.1 (Figure 2a),
which was corroborated through the lineage designation (https://github.com/cov-lineages/pango-
designation/issues/57 ) (supplementary table 2). B.1.621 lineage has recently spread to fourteen
departments, with a major representation in the Caribbean region of Colombia (Figure 2b)
(https://microreact.org/project/5CAiK3qCMaEgE4vYkKVpZW/b7113efc). No recombination
events were found throughout the whole genome (data not shown). At least 9 codons in the Spike
protein displayed a signal suggestive of positive selection (supplementary table 3).
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4. Discussion
All genomes belonging to the B.1.621 lineage available until the end of April 2021 were
included in this study, with the earliest collection date on January 11th. 2021, corresponding to a
sample collected in the department of Magdalena, Colombia (EPI_ISL_1220045). While a very
high and unexplained genetic distance is found between every B.1.621 sequence and all closely
related sequences of the parental B.1 lineage, the whole branching pattern and intra-lineage
distance suggest low diversification that can be explained by its recent origin. The spread in the
country early during the third peak of the pandemic could be explained by a combination of
factors, including social exhaustion as well as the genetic background of the emerging lineage,
leading to changes in transmission.
In Colombia, the current strategy for SARS-CoV-2 genomic surveillance includes sampling in
principal and border cities, in special groups of interest, in patients with distinctive clinical
features and severity, and finally in community transmission with an unusual increase in cases
(https://www.ins.gov.co/Noticias/Paginas/coronavirus-genoma.aspx). The high frequency
observed of the emerging B.1.621 lineage also could be related to the strengthening of the
SARS-COV-2 genomic surveillance during the third peak of the pandemic in Colombia through
the implementation of the National Laboratory Network for SARS-CoV-2 sequencing.
With this approach, we expect to characterize an approximate 1% of the cases and determine the
adjusted frequency of the lineage in the country and to evaluate the possible predominance and
the replacement of other lineages in the country. For this, intensified genomic characterization
will be carried out with a multi-stage sample design throughout the national territory.
Since the last trimester of 2020, several convergent substitutions have been evidenced in the
lineages of SARS-CoV-2 explained by a high rate of genetic variability by wide naive
population, the selection pressure by monoclonal antibody therapies (Liu et al., 2021; Wibmer et
al., 2021) and vaccination (Garcia-Beltran et al., 2021; Wang et al., 2021). Substitutions in the
spike protein are common, however some distinctive substitutions have relevant characteristics
for instance, the presence of E484K has been associated with lower neutralizing activity from
convalescent plasma (Liu et al., 2021). The 69/70 deletion spike together with the E484K and
N501Y substitutions decrease the ability to neutralize antibodies (Xie et al., 2021). Conversely,
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the P681H substitution in the S1/S2 furin cleavage site, although increase spike cleavage by
furin-like proteases does not significantly impact viral entry or cell-cell spread in vitro (Örd et
al., 2020) neither with higher infection rate or higher prevalence.
The insertion, 145N in the spike protein of B.1.621 is the first evidenced in this position in
SARS-CoV-2, this insertion could affect the S1 closed-open conformation and the subsequent
binding to the ACE2 (Berger and Schaffitzel, 2020), however its implications in terms of
infection, transmission and pathogenesis are still unknown.
Although B.1.621 does not meet all of the VOC classification criteria so far, the set of mutations
gathered the Spike protein could confer a synergistic impact on attributes such as reduction of
vaccine-induced protection from severe disease, increased transmission and disease severity
(Centers for Disease Control and Prevention, 2021).
Thus, laboratory characterization and enhanced routine genomic epidemiology studies are still
required in order to monitor the possible change of status of this variant, or the
emergence/introduction of new SARS-CoV-2 variants in the country.
Finally, the B.1.621 lineage has nucleotide substitutions already included in the VOC real-time
reverse transcription PCR (real-time RT-PCR) screening tests (Bal et al., 2021). This should be
considered in the analysis of these screening tests because the occurrence of these substitutions
in lineages not regarded as VOC could lead to overestimating the number of VOC cases.
The B.1.621 lineage has been identified so far in Colombia, USA, Spain, Netherlands, Denmark,
Mexico, Germany and Curacao. The study was limited to genomic and evolutionary
characterization. The public health implications must be to assess through the biological and
epidemiologic roles.
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Table1. Nucleotide and amino acid substitution pattern of B.1.621
Genomics coordinate with respect to
the reference genome (NC_045512.2)
Amino acid
Change Region/Protein
ORF1ab:
A3428G T1055A Non-structural protein 3
C4878T T1538I Non-structural protein 3
A11451G Q3729R Non-structural protein 6
C14408T P4715L RNA-dependent RNA polymerase
C17491T P5743S Helicase domain
ORF3a:
G25563T Q57H accessory protein ORF3a
deletion: 26158-26162 (4 nucleotides) V256- accessory protein ORF3a
frameshift N257X accessory protein ORF3a
ORF8:
A27924C T11K ORF8 immunoglobulin (Ig)
domain protein and related proteins
C28005T P38S ORF8 immunoglobulin (Ig)
domain protein and related proteins
C28093T S67F ORF8 immunoglobulin (Ig)
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domain protein and related proteins
Spike:
C21846T T95I N-terminal domain of S1 subunit
T21992A, A21993C Y144T N-terminal domain of S1 subunit
A21996C, C21997T Y145S N-terminal domain of S1 subunit
21998:AAC ins146N N-terminal domain of S1 subunit
G22599A R346K receptor binding domain
G23012A E484K receptor binding domain
A23063T N501Y receptor binding domain
C23604A P681H S1/S2 cleavage region
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Figure 1. Percent of variant B.1.621 in Spain, USA and Colombia.1A) Gisaid
registries of variant
B.1.621 in 2021. Since January the continuous record has been maintained in Colombia. 1B)
Lineage percentage and number of cases of COVID-19 in five departments with circulation of
B.1.621 variant and the capital city. B.1.1.7 and P.1 VOC lineages are shown, others lineages
circulating are represented as “others”.
nt
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Figure 2. Phylogeny and distribution of SARS-CoV-2 b.1.621 variant in Colombia A)
Phylogenetic tree of the new lineage of SARS-CoV-2 emerging from B.1.621 lineage (pink
stars). The tree was reconstructed by maximum likelihood with the estimated GTR+F+I+G4
nucleotide substitution model for the dataset of 434 genomes. The interactive tree can be
accessed in the following link:
https://microreact.org/project/5CAiK3qCMaEgE4vYkKVpZW/b7113efc. B
) Map of distribution
of lineages across the country.
Funding
This work was funded by the Project CEMIN-4-2020 Instituto Nacional de Salud. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Disclosure statement
No conflict of interest was reported by the authors.
on
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Acknowledgements
The authors thank the National Laboratory Network for routine virologic surveillance of SARS-
CoV-2 in Colombia. We also thank all researchers who deposited genomes in GISAID’s EpiCoV
Database contributing to genomic diversity and phylogenetic relationship of SARS-CoV-2. We
thank Rotary International and Charlie Rut Castro for equipment’s donation. Finally, we thank
red RENATA and Universidad Industrial de Santander for the workstation bioinformatic support.
Data deposition
SARS-CoV-2 Colombian sequences belonging to the B.1.621 were deposited in GISAID under
accession numbers: EPI_ISL_1220045, EPI_ISL_1582980, EPI_ISL_1424054,
EPI_ISL_1424056, EPI_ISL_1582978, EPI_ISL_1820926, EPI_ISL_1424055,
EPI_ISL_1582993, EPI_ISL_1582979, EPI_ISL_1424057, EPI_ISL_1820929,
EPI_ISL_1820930, EPI_ISL_1820932, EPI_ISL_1820927, EPI_ISL_1424058,
EPI_ISL_1582984, EPI_ISL_1582986, EPI_ISL_1582991, EPI_ISL_1820958,
EPI_ISL_1582990, EPI_ISL_1582992, EPI_ISL_1582981, EPI_ISL_1582994,
EPI_ISL_1582988, EPI_ISL_1820959, EPI_ISL_1820928, EPI_ISL_1582996,
EPI_ISL_1632530, EPI_ISL_1582989, EPI_ISL_1820934, EPI_ISL_1582987,
EPI_ISL_1820955, EPI_ISL_1820935, EPI_ISL_1582997, EPI_ISL_1820925,
EPI_ISL_1820950, EPI_ISL_1820949, EPI_ISL_1820944, EPI_ISL_1820947,
EPI_ISL_1820943, EPI_ISL_1820946, EPI_ISL_1820954, EPI_ISL_1821882,
EPI_ISL_1820939, EPI_ISL_1820942, EPI_ISL_1820960, EPI_ISL_1820936,
EPI_ISL_1820948, EPI_ISL_1820931, EPI_ISL_1582977, EPI_ISL_1820965,
EPI_ISL_1821070, EPI_ISL_1821075, EPI_ISL_1821063, EPI_ISL_1820967,
EPI_ISL_1820968, EPI_ISL_1821062, EPI_ISL_1821064, EPI_ISL_1821069,
EPI_ISL_1821073, EPI_ISL_1821074, EPI_ISL_1821076, EPI_ISL_1821077,
EPI_ISL_1821071, EPI_ISL_1821072, EPI_ISL_1820962, EPI_ISL_1821065,
EPI_ISL_1821066, EPI_ISL_1821067, EPI_ISL_1821068, EPI_ISL_1824702,
EPI_ISL_1824703, EPI_ISL_1824706, EPI_ISL_1824711, EPI_ISL_1824712,
EPI_ISL_1824713, EPI_ISL_1824714.
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