ABSTRACT
Here, we report the nearly complete genome sequences of nine severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with the D614G mutation. These viruses were detected from various infected individuals with different levels of severity from Pahang, Malaysia. In addition, this study described the presence of lineage B.1.351 as a type of variant of concern (VOC) and lineages B.1.466.2 and B.1.524 as local variants.
ANNOUNCEMENT
The current pandemic of coronavirus disease 19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the viral family Coronaviridae and genus Betacoronavirus (1). The COVID-19 D614G mutation was associated with higher risk of infection (2). Here, we report nine nearly complete genome sequences of variants of concern (VOC) from the Beta B.1.351 lineage and several unassigned variants that belong to local lineages (3). The clinical specimens of nine patients with various clinical presentations in Sultan Ahmad Shah Medical Centre @IIUM (SASMEC @IIUM) were collected directly from combined oropharyngeal and nasopharyngeal swabs in April 2020 and April 2021. These individuals were detected as COVID-19-infected individuals through reverse transcriptase PCR (RT-PCR) (threshold cycle [CT] value, <30) and traced from active contact tracing during severe acute respiratory infection (SARI) surveillance. The study was approved by the International Islamic University Malaysia Research Ethics Committee (IREC 2021-080).
The total RNA was extracted using a Maxwell HT simplyRNA kit (Promega, USA) and converted into cDNA using SuperScript IV reverse transcriptase (Invitrogen) with some modifications; a hexamer annealing and extension step of 25°C for 2 min was performed, followed by cDNA synthesis at 42°C for 50 min. A portion (1:10 volume) of the cDNA from sample IIUM91 was used as the template for multiplex PCR using Q5 high-fidelity DNA polymerase (New England BioLabs [NEB], USA) and the Artic v3 primer pools. The amplicons for more recent samples were generated using the commercially available NEBNext ARTIC SARS-CoV-2 companion kit (NEB). Equal volumes of PCR products obtained from the two primer pools were mixed; pool 1 and pool 2 were mixed according to the designated protocols (4) and purified using AMPure XP for PCR purification (Beckman Coulter Life Sciences, USA). The purified PCR products were quantified using a double-stranded DNA (dsDNA) high-sensitivity assay (DeNovix, Inc., USA), and 50 ng was used to construct an Illumina library using the NEB UltraII library preparation kit as described previously (5). The constructed library was sequenced on an iSeq 100 sequencing system (Illumina, Inc., USA) with run configuration of 1 × 300 bp or 1 × 250 bp. On average, 125,319 single-end reads were generated from each sample (minimum, 68,787; maximum, 423,112). These raw reads were used to reconstruct the SARS-CoV-2 genome using a combination of bioinformatic tools as listed at https://github.com/CDCgov/SARS-CoV-2_Sequencing/tree/master/protocols/BFX-UT_ARTIC_Illumina. Briefly, the raw reads were aligned to the reference strain WuHan-Hu-1 genome (GenBank accession number MN908947) using the Burrows-Wheeler Aligner MEM algorithm (BWA-MEM) v0.7.17-r1188 (6) and subsequently trimmed to remove the primer binding region, and a consensus genome was generated from the filtered alignment using iVar v1.2.2 (7). Details regarding the reported genomes are summarized in Table 1.
TABLE 1.
Summary of SARS-CoV-2 strains sequenced from Pahang, Malaysia
| Sequence name | Genome size (bp) | Lineage | WHO label | Cladeb | GC content (%) | No. of reads | BioSample no. |
|---|---|---|---|---|---|---|---|
| hCoV-19/Malaysia/UMP-IIUM5480/2021 | 29,764 | B.1.351 | Beta (β) | GH | 38.0 | 91,342 | SAMN19778019 |
| hCoV-19/Malaysia/IIUM5556/2021 | 29,764 | B.1.351 | Beta (β) | GH | 37.9 | 113,985 | SAMN19778013 |
| hCoV-19/Malaysia/IIUM5676/2021 | 29,782 | B.1.524 | No WHO label; Malaysian lineagea | G | 38.0 | 83,614 | SAMN19778016 |
| hCoV-19/Malaysia/IIUM5754/2021 | 29,764 | B.1.351 | Beta (β) | GH | 38.0 | 104,861 | SAMN19778014 |
| hCoV-19/Malaysia/IIUM5755/2021 | 29,764 | B.1.351 | Beta (β) | GH | 38.0 | 83,456 | SAMN19778015 |
| hCoV-19/Malaysia/IIUM5763/2021 | 29,782 | B.1.466.2 | No WHO label; Indonesian lineagea | GH | 37.9 | 68,787 | SAMN19778017 |
| hCoV-19/Malaysia/IIUM5770/2021 | 29,764 | B.1.351 | Beta (β) | GH | 38.0 | 70,583 | SAMN19778018 |
| hCoV-19/Malaysia/IIUM6472/2021 | 29,764 | B.1.351 | Beta (β) | GH | 37.9 | 88,134 | SAMN19778020 |
| hCoV-19/Malaysia/IIUM91/2020 | 29,701 | B.1.468 | No WHO label; Indonesia/Singapore lineagea | GH | 38.0 | 423,112 | SAMN16383837 |
These lineages were listed as neither variants of concern (VOC) nor variants of interest (VOI) by the World Health Organization. We determined these lineages using the PANGO Web server (3), which is available online at https://cov-lineages.org/index.html.
Members of clade G contain mutations C241T, C3037T, and A23403G, as well as S-D614G; members of clade GH contain mutations C241T, C3037T, A23403G, and G25563T, as well as S-D614G plus NS3-Q57H.
Of the nine strains sequenced, six were classified as the B.1.351 variant, which harbors the E484K and N501Y mutations commonly associated with increased transmission rate (8). In addition, this study also documented B.1.524 of Malaysian lineage and B.1.466.2 and B.1.468 of Indonesian lineages.
Data availability.
These sequences were deposited in GenBank under the accession numbers MW079428.1 and MZ443817.1 to MZ443824.1. The accession numbers in the NCBI Sequence Read Archive (SRA) are SRP286590 and SRP324679. The sequences in the GISAID database are as follows: EPI_ISL_455313, EPI_ISL_2622006, EPI_ISL_2622007, EPI_ISL_2622045, EPI_ISL_2622046, EPI_ISL_2622047, EPI_ISL_2622079, EPI_ISL_2622088, and EPI_ISL_2622089.
ACKNOWLEDGMENTS
We thank the Ministry of Higher Education Malaysia and Universiti Malaysia Pahang (UMP) for supporting this work via FRGS/1/2019/WAB13/UMP/03/1 and RDU190364, respectively.
We humbly acknowledge the COVID-19 task forces from SASMEC @IIUM.
Contributor Information
Hajar Fauzan Ahmad, Email: fauzanahmad@ump.edu.my.
Simon Roux, DOE Joint Genome Institute.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
These sequences were deposited in GenBank under the accession numbers MW079428.1 and MZ443817.1 to MZ443824.1. The accession numbers in the NCBI Sequence Read Archive (SRA) are SRP286590 and SRP324679. The sequences in the GISAID database are as follows: EPI_ISL_455313, EPI_ISL_2622006, EPI_ISL_2622007, EPI_ISL_2622045, EPI_ISL_2622046, EPI_ISL_2622047, EPI_ISL_2622079, EPI_ISL_2622088, and EPI_ISL_2622089.