Tracking down B.1.351 SARS‐CoV‐2 variant in Pakistan through genomic surveillance

To the Editor, The emergence of severe acute respiratory syndrome 2 (SARS‐CoV‐ 2) variants of concern (VOC) have posed a serious threat to the control of the disease worldwide. Of note are the B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma) that were first reported from the United Kingdom, South Africa, and Brazil, respectively. The B.1.351 contains characteristic mutations in the receptor‐binding domain of spike glycoprotein (S) protein: K417N, E484K, and N501Y that have functional significance. As of May 7, 2021, 14 543 sequences of B.1.351 lineage have been reported from 85 countries around the world. Since the start of the pandemic, the National Institute of Health, Pakistan has been involved in the surveillance of SARS‐CoV‐2 and although the B.1.1.7 cases have been detected, the whole‐ genome sequence of B.1.351 has not been reported in Pakistan. We hereby report the genomic diversity of the first two sequences of the B.1.351 variant detected from Pakistan. On April 24 and 26, 2021, two oropharyngeal samples were received at the Department of Virology, National Institute of Health for the detection of SARS‐CoV‐2 as part of routine surveillance. Briefly, viral RNA was extracted using the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit on the KingFisher FlexTM Purification System (Thermo Fisher Scientific). The TaqPathTM COVID‐19 CE‐IVD RT‐PCR kit (Thermo Fisher Scientific) was used for the detection of SARS‐CoV‐2. These two samples were selected for whole‐genome sequencing due to amplification of spike gene onTaqPathTM assay and having low Ct value (<22 for S gene). For whole‐genome sequencing, the cDNA synthesis and amplification were performed according to the Primal‐Seq Nextera XT protocol (version 2) using SuperScriptTM IV VILOTM Master Mix (Invitrogen) and Q5 High‐Fidelity 2X Master Mix (New England BioLabs) respectively, with the ARTIC nCoV‐2019 Panel (Integrated DNA Technologies, Inc.). Illumina DNA Prep Kit (Illumina, Inc.) was used for library preparation and subjected to sequencing on Illumina iSeq 100 (Illumina, Inc.). The read quality of sequenced files was assessed using the FastQC tool (v0.11.9). The data were processed and analyzed as per recommended guidelines of the Centers for Diseases Control and Prevention. On real‐time PCR, the sample NIH‐S6 (EPI_ISL_1969999) showed a Ct value of 24 for N‐gene, 23 for ORF1ab, and 21 for S‐gene. The patient was a 28‐year‐old female who attended a funeral ceremony in her neighborhood on April 20, 2021, before infection. The patient had mild symptoms of having body aches, loss of taste and smell, and had no travel history. Similarly, the Ct values of sample NIH‐S12 (EPI_ISL_1969995) were 14 for N‐gene, 16 for ORF1ab, and 14 for S‐gene. The patient was a 30‐year‐old male with low‐grade fever and body aches and had no travel history. Whole‐genome sequencing results showed the detection of B.1.351 in both cases having characteristic mutations in the spike: D80A, D215G, D614G, E484K, K417N, N501Y, and A701V (Table 1). Based on phylogenetic analysis, Pakistani sequences were closely related to the B.1.351 (Figure 1). As Pakistan fights against the COVID‐19 pandemic, a total of 864 557 cases and 19 106 deaths have been reported until May 10, 2021, with a high number of cases (n = 283 192) seen during the third wave (https://covid.gov.pk/stats/pakistan). Based on our laboratory data (spike gene target failure cases using COVID‐19 TaqPathTM kit; Thermo Fisher Scientific), the recent surge in cases correlates with the detection of the B.1.1.7 variant in the country. Conversely, no data is available on the prevalence of other VOCs including B.1.351,