GENOME SEQUENCES

Genome Sequences of 15 SARS-CoV-2 Sublineage B.1.617.2

Strains in Bangladesh
Mokibul Hassan Afrad,a Manjur Hossain Khan,b Sadia Isfat Ara Rahman,a Omar Hamza Bin Manjur,c Mohabbat Hossain,c
Ahmed Nawsher Alam,b Farzana Islam Khan,b Nawroz Afreen,b Farsim Tarannum Haque,b Nicholas R. Thomson,d,e Tahmina Shirin,b
Firdausi Qadria,c

a International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh

b Institute of Epidemiology, Disease Control, and Research, Dhaka, Bangladesh
c Institute for Developing Science and Health Initiatives, Dhaka, Bangladesh
d Wellcome Sanger Institute, Hinxton, Cambridgeshire, United Kingdom
e London School of Hygiene and Tropical Medicine, London, United Kingdom

ABSTRACT We report the coding-complete genome sequences of 15 severe acute respi-

ratory syndrome coronavirus 2 (SARS-CoV-2) sublineage B.1.617.2 strains that were obtained
from Bangladeshi individuals with a history of recent travel to India and from the
Bangladeshi community. Genomic data were generated by Nanopore sequencing using
the amplicon sequencing approach developed by the ARTIC Network.

B angladesh reported the first confirmed case of severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) (family Coronaviridae, genus Betacoronavirus) on 8 March
2020; as of 31 May 2021, there were 798,830 confirmed coronavirus disease 2019 (COVID-19)
cases (1). Bangladesh has been observing a surge of confirmed SARS-CoV-2 cases, dominated
by the SARS-CoV-2 beta variant B.1.351, since the third week of March 2021 (2). However,
the rate of confirmed SARS-CoV-2 cases nationwide gradually decreased to less than 10%

Downloaded from https://journals.asm.org/journal/mra on 22 September 2021 by 203.190.254.97.

(SARS-CoV-2 test positivity rate) in mid-May 2021. Recently (third week of May 2021), there
was a substantial increase in the COVID-19 detection rate in the bordering districts
(Chapainawabgonj, Khulna, and Satkhira) in the northwestern part of Bangladesh.
We investigated whether this sudden increase was due to SARS-CoV-2 variants that might
have been imported into Bangladesh through population movement in the Bangladesh-India
border regions.
We obtained the SARS-CoV-2-positive samples (n = 15) through the nationwide
COVID-19 surveillance network maintained by the Institute of Epidemiology, Disease
Control, and Research (IEDCR), a mandated institute for disease outbreak investigation
and response in Bangladesh. This genomic surveillance is an ongoing activity that was Citation Afrad MH, Khan MH, Rahman SIA, Bin
Manjur OH, Hossain M, Alam AN, Khan FI,
approved by the institutional review board of the IEDCR (protocol IEDCR/IRB/2020/11). Afreen N, Haque FT, Thomson NR, Shirin T,
These 15 samples include samples from (i) randomly selected SARS-CoV-2-positive Qadri F. 2021. Genome sequences of 15 SARS-
CoV-2 sublineage B.1.617.2 strains in
community patients (n = 7) with COVID-19 symptoms who were residing in the border
Bangladesh. Microbiol Resour Announc 10:
district of Chapainawabgonj and (ii) Bangladeshi individuals (n = 8) with a history of e00560-21. https://doi.org/10.1128/MRA.00560-21.
recent travel to India who were found to be SARS-CoV-2 positive by reverse transcrip- Editor Jelle Matthijnssens, KU Leuven
tase (RT) PCR, irrespective of COVID-19 symptom appearance (3). The patients’ ages Copyright © 2021 Afrad et al. This is an open-
access article distributed under the terms of
ranged from 13 to 52 years; four of the patients were asymptomatic, while others pre- the Creative Commons Attribution 4.0
sented mild to moderate symptoms. Except for one individual, none had been vacci- International license.
nated prior to infection. Viral RNA was extracted from nasopharyngeal swab samples Address correspondence to Firdausi Qadri,
fqadri@icddrb.org.
using the QIAamp viral RNA minikit (Qiagen). Sequencing libraries were prepared using
Received 3 June 2021
the multiplex PCR amplicon sequencing approach developed by the ARTIC Network (4, Accepted 18 June 2021
5). Libraries were multiplexed and sequenced on a FLO-MIN106D (R9.5) flow cell for at Published 15 July 2021
least 12 h. Raw reads were base called and demultiplexed with MinKNOW v21.02.1.

Volume 10 Issue 28 e00560-21 mra.asm.org 1

 Afrad et al.

TABLE 1 Data for each Bangladeshi SARS-CoV-2 sublineage B.1.617.2 isolate

Sampling Patient Recent GC GenBank
date Sampling age travel Genome Coverage content accession SRA
Strain name (day/mo/yr) locationa (yr) Symptomsb Vaccinatedc to India size (bp) (%)d (%) no. accession no.

Volume 10 Issue 28 e00560-21

IEDCR-AFIP-001 29/4/2021 Dhaka 41 NA No Yes 29,769 99.55 42.4 MZ377102 SRR14800278
IEDCR-DJ-002 29/4/2021 Dinajpur 46 1 Yes Yes 29,769 99.55 40.5 MZ377103 SRR14800279
IEDCR-OIS-0003 1/5/2021 Khulna 45 – No Yes 29,769 99.55 40.5 MZ377104 SRR14800272
IEDCR-V-11 13/5/2021 Chapainawabgonj 21 1 No No 29,769 99.55 39.9 MZ377105 SRR14800271
IEDCR-V-13 13/5/2021 Chapainawabgonj 13 1 No No 29,769 99.55 40.4 MZ377106 SRR14800270
IEDCR-V-16 13/5/2021 Chapainawabgonj 31 1 No No 29,769 99.55 40 MZ377107 SRR14800268
IEDCR-V-15 13/5/2021 Chapainawabgonj 27 1 No No 29,769 99.55 40.5 MZ377114 SRR14800269
IEDCR-V-18 13/5/2021 Chapainawabgonj 27 1 No No 29,769 99.55 40.1 MZ377115 SRR14800267
IEDCR-OIS-0079 13/5/2021 Khulna 50 NA NA Yes 29,769 99.55 40.9 MZ377113 SRR14800273
IEDCR-V-20 17/5/2021 Chapainawabgonj 30 1 No No 29,769 99.55 40 MZ377108 SRR14800266
IEDCR-V-22 17/5/2021 Chapainawabgonj 52 1 No No 29,770 99.56 40.2 MZ377116 SRR14800265
IEDCR-OIS-0049 18/5/2021 Bagerhat 43 – No Yes 29,769 99.55 40.8 MZ377111 SRR14800275
IEDCR-OIS-0070 18/5/2021 Pirojpur 46 NA NA Yes 29,769 99.55 42.1 MZ377112 SRR14800274
IEDCR-OIS-0036 22/5/2021 Gaibandha 28 – No Yes 29,769 99.55 40.5 MZ377109 SRR14800277
IEDCR-OIS-0039 22/5/2021 Jhenaidah 49 – No Yes 29,772 99.56 41 MZ377110 SRR14800276
a The Khulna, Chapainawabgonj, and Jhenaidah districts are located in the western border area of Bangladesh at the Bangladesh-India border.
b 1, present (fever, cough, or mild weakness); –, absent; NA, information not available.
c Yes, vaccinated with one dose of AstraZeneca COVID-19 vaccine.

d With reference to the Wuhan Hu-1 genome (GenBank accession NC_045512.2).

mra.asm.org 2
Downloaded from https://journals.asm.org/journal/mra on 22 September 2021 by 203.190.254.97.
Microbiology Resource Announcement

FIG 1 Phylogenetic analysis of coding-complete SARS-CoV-2 sublineage B.1.617.2 genomes. The orange box represents samples obtained from the Downloaded from https://journals.asm.org/journal/mra on 22 September 2021 by 203.190.254.97.
bordering districts. A total of 50 viral genomes are displayed, including (i) 15 genomes from Bangladesh (highlighted in red), (ii) the SARS-CoV-2 Wuhan
Hu-1 reference genome, and (iii) randomly selected genomes from GISAID representing different lineages, as well as recently isolated B.1.617.2 genomes
from India. For each viral genome, the strain name, place and date of sampling, GISAID accession number, and SARS-CoV-2 lineage are indicated.

Processed reads were assembled using the ARTIC guppyplex script with Medaka v1.4 using
the ARTIC EPI2ME v3.3.0 SARS-CoV-2 pipeline (FastQ quality control plus ARTIC plus NextClade)
(https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html). In total, 4,324,431 reads
were obtained (range, 79,314 to 734,341 reads per sample; average length, 507 bp).
Table 1 summarizes the information for each sample. Nucleotide variations were con-
firmed by visual inspection using CLC Genomics Workbench v21.0 (Qiagen). Compared to
the reference Wuhan Hu-1 genome, a set of 10 amino acid substitutions (shared among all
15 strains) was identified, including the five signature amino acid changes (T19R, L452R,
T478K, P681R, and D950N) in the spike protein corresponding to the genetic markers of sub-
lineage B.1.617.2. Sequences were aligned using MAFFT v7.4 (6). A phylogenetic tree (Fig. 1)
was constructed with IQ-TREE v2 (7) with an ultrafast bootstrap value of 1,000 (8) and with

Volume 10 Issue 28 e00560-21 mra.asm.org 3

Afrad et al.

the GTR1F1I model selected as the best-fit model by the IQ-TREE Web server (9) and was
visualized using FigTree v1.4 (10). Phylogenetic analysis with Pangolin (github.com/cov
-lineages/pangolin) assigned 15 samples to clade G, lineage B.1.617.2. Here, we report the
early detection of SARS-CoV-2 delta variant B.1.617.2 in the Bangladeshi community,
which will be useful toward mitigation of a possible third wave of COVID-19 in Bangladesh,
as has been seen elsewhere.
Data availability. The data from this study can be found under GISAID accession
numbers EPI_ISL_1938477, EPI_ISL_2105565, EPI_ISL_2180455, EPI_ISL_2284894,
EPI_ISL_2284885, EPI_ISL_2284886, EPI_ISL_2284887, EPI_ISL_2304461, EPI_ISL_2304462,
EPI_ISL_2284888, EPI_ISL_2304463, EPI_ISL_2284890, EPI_ISL_2284891, EPI_ISL_2284892,
and EPI_ISL_2284893. The Sequence Read Archive (SRA) and GenBank accession num-
bers are listed in Table 1.

ACKNOWLEDGMENTS
The Bill and Melinda Gates Foundation funded the study. The International Centre
for Diarrhoeal Disease Research, Bangladesh (icddr,b) acknowledges with gratitude the
commitment of the Bill and Melinda Gates Foundation to its research efforts. icddr,b is
grateful to the governments of Bangladesh, Canada, Sweden, and the United Kingdom
for providing core/unrestricted support.
We acknowledge physicians and diagnostic testing staff members at the IEDCR and
the Institute for Developing Science and Health Initiatives who provided initial
diagnostic testing of the SARS-CoV-2 samples.

REFERENCES
1. World Health Organization. 2021. COVID-19 Bangladesh situation report-66, 31
May 2021. World Health Organization, Geneva, Switzerland. https://cdn.who.int/
media/docs/default-source/searo/bangladesh/covid-19-who-bangladesh
-situation-reports/who_covid-19-update_66_20210531.pdf?sfvrsn=1a6defec_9.
2. Saha S, Tanmoy AM, Hooda Y, Tanni AA, Goswami S, Sium SMA, Sajib MSI,
Malaker R, Islam S, Rahman H, Anik AM, Sarker N, Islam MS, Ghosh K,
Sarkar PK, Bipul MRA, Ahmed SS, Shahidullah M, Saha SK. 2021. COVID-19
rise in Bangladesh correlates with increasing detection of B.1.351 variant.
BMJ Glob Health 6:e006012. https://doi.org/10.1136/bmjgh-2021-006012.
3. Chinese Center for Disease Control and Prevention. 2020. Laboratory testing for
Nanopore. bioRxiv 2020.09.04.283077. https://doi.org/10.1101/2020.09
.04.283077.
6. Kuraku S, Zmasek CM, Nishimura O, Katoh K. 2013. aLeaves facilitates on-
demand exploration of metazoan gene family trees on MAFFT sequence
alignment server with enhanced interactivity. Nucleic Acids Res 41:
W22–W28. https://doi.org/10.1093/nar/gkt389.
7. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and
effective stochastic algorithm for estimating maximum likelihood phylogenies.
Mol Biol Evol 32:268–274. https://doi.org/10.1093/molbev/msu300.

Downloaded from https://journals.asm.org/journal/mra on 22 September 2021 by 203.190.254.97.

COVID-19. Chinese Center for Disease Control and Prevention, Beijing, China.
http://www.chinacdc.cn/en/COVID19/202003/P020200323390321297894.pdf.
4. Quick J. 2020. nCoV-2019 sequencing protocol v3 (LoCost). https://protocols.io/
view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye.
5. Tyson JR, James P, Stoddart D, Sparks N, Wickenhagen A, Hall G, Choi JH,
Lapointe H, Kamelian K, Smith AD, Prystajecky N, Goodfellow I, Wilson SJ,
Harrigan R, Snutch TP, Loman NJ, Quick J. 2020. Improvements to the
ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using
8. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. 2018.
UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol
35:518–522. https://doi.org/10.1093/molbev/msx281.
9. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS.
2017. ModelFinder: fast model selection for accurate phylogenetic esti-
mates. Nat Methods 14:587–589. https://doi.org/10.1038/nmeth.4285.
10. Rambaut A, Drummond AJ. 2012. FigTree: tree figure drawing tool, v1.4.2. Uni-
versity of Edinburgh, Institute of Evolutionary Biology, Edinburgh, Scotland.

Volume 10 Issue 28 e00560-21 mra.asm.org 4