key: cord-0332328-ttygo77i authors: Umair, M.; Ikram, A.; Rehman, Z.; Haider, S. A.; Badar, N.; Ammar, M.; Ali, Q.; Ahad, A.; Suleman, R.; Salman, M. title: Genomic surveillance reveals the emergence of SARS-CoV-2 Lineage A from Islamabad Pakistan date: 2021-12-25 journal: nan DOI: 10.1101/2021.12.24.21268367 sha: f17f6df75b3d2394df69f811540ff9bc184b4804 doc_id: 332328 cord_uid: ttygo77i The lineage A of SARS-CoV-2 has been around the world since the start of the pandemic. In Pakistan the last case of lineage A was reported in April, 2021 since then no case has been reported. In November, 2021 during routine genomic surveillance at National Institute of Health we have found 07 cases of lineage A from Islamabad, Pakistan. The study reports two novel deletions in the spike glycoprotein. One 09 amino acid deletion (68-76 a.a) is found in the S1 subunit while another 10 amino acid deletion (679-688 a.a) observed at the junction of S1/S2 referred as furin cleavage site. The removal of furin cleavage site may result in impaired virus replication thus decreasing its pathogenesis. The actual impact of these two deletions on the virus replication and disease dynamics needs to be studied in detail. Moreover, the enhanced genomic surveillance will be required to track the spread of this lineage in other parts of the country. fatalities. In Pakistan, however, 1,291,737 individuals were infected, resulting in 28,882 deaths [4] . Moreover, Pakistan is hit by four waves: the first from May to July 2020, the second from October 2020 to January 2021, and the third from March to May 2021 [5] . The fourth wave, triggered by the delta variant, commenced in July 2021 [6] . Considering the upsurge in cases of SARS-CoV-2, Pakistan's government is especially concerned about the impact and control measures of the circulating variants, which have evolved rapidly over the last two years [7, 8] US states have reported a total of 2,783 cases of the A lineage [9] . The US accounts for 27%, the UAE has 13%, China has 9%, Germany has 8%, and Japan has 5% of the lineage A cases [10] . Lineage A of SARS-CoV-2, which is still in circulation, is the root of the pandemic, subsequently divided into sublineages [11, 12] . Of note, Alpha and Omicron share two important deletions ("H69/V70") of the spike gene with lineage A. These deletions are used as a marker in PCR tests [13] . Additionally, H69/V70del compensates for immune escape mutations that impair infectivity [14] [15] [16] . Hence, understanding the virus's ongoing evolution, epidemiology, circulating lineages, as well as evaluating the effects of spike protein mutations on COVID-19 transmission and vaccine performance are all essential for COVID-19 mitigation and control, which can be assisted by genomic surveillance studies [17] . As a result, the current finding of lineage A (clade S) cases in . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The Illumina DNA Prep Kit (Illumina, Inc, USA) was used to prepare the paired-end (2x150 bp) sequencing library according standard protocol. The prepared libraries were pooled and subjected to sequencing on Illumina platform, iSeq, using sequencing reagent, iSeq 100 i1 Reagent v2 (300cycle) (Illumina, Inc, USA). The quality of sequencing reads were assessed through FastQC tool (v0.11.9) [18] . The low quality low-quality base calls (< 30) and adapter sequences were removed using Trimmomatic (v0.39). The alignment of filtered reads were performed through Burrows-Wheeler Aligner's (BWA, . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The multiple sequence alignment of all the study isolates were performed with the Wuhan-Hu-01 (GISAID ID: EPI_ISL_402125) using Clustal W. The homology model of spike glycoprotein was built through Modeller V9.2 using pdb ID:6VSB as template and 100 models were generated. These models were subjected to model evaluation using Ramachandran Plot and Q-mean score. The structure of spike protein of wild type SARS-CoV-2 was superimposed on the spike structure with the deletions. The detailed structural analysis revealed that two (68-76 and 679-688) loops are missing in the lineage A spike protein (Figure 3) . The structural comparison have also shown RMSD value of 1.7 Å from the native structure that further verified conformational changes in the structure. This is the first study to report the emergence of lineage A with two large deletions (68-76 and 679-688del) in the spike protein from Pakistan. These deletions have to be seen in light of the functional significance. Typically the spike glycoprotein of SARS-Cov-2 has been proven to be involved in entry of virus into host cells by binding with the angiotensin-converting enzyme 2 (ACE2) [19, 20] . The spike protein is composed of two subunits, an N-terminal S1 subunit responsible for receptor binding and a C-terminal S2 subunit responsible for virus fusion with cell membrane [21, 22] . Previously, from Pakistan, the first three cases of lineage A have been reported Interestingly, only two of the isolates (GISAID ID: EPI_ISL_2886419; EPI_ISL_2832924) from Germany in April, 2021 harbors the site I deletion (68-76 amino acids) in their genomes while these isolates devoid of the site II deletion. Instead of site II deletion these two isolates possess 676-680 amino acid deletion in the spike glycoprotein. This study has also identified 68-76 amino . CC-BY-ND 4.0 International license It 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) preprint The copyright holder for this this version posted December 25, 2021. ; https://doi.org/10.1101/2021.12.24.21268367 doi: medRxiv preprint acid deletion in the S1 subunit. Previously the two amino acid deletion (69-70del) has been studied more extensively and found to have an impact on increasing infectivity as well as increasing the susceptibility to neutralising antibodies by a conformational change thus favoring a more open spike conformation [14] The other major deletion identified in the studied samples spanned in the 679-688 amino acid region. Functionally, at the junction of S1 and S2, the specific sequence motif 682-689 amino acid region is recognized and cleaved by furin proteases during viral packaging thus promoting virus infectivity and pathogenicity. This site has been identified in MERS-CoV and SARS-CoV-2 however, it is absent in other coronaviruses of the same family [23, 24] . It has been reported previously, that deletion of furin cleavage site results in attenuated viral replication thus reducing the pathogenesis and ablating the disease. Thus presence of furin cleavage site is crucial for viral replication. The loss of furin cleavage site may also result in altered antibody neutralization profile [25] [26] [27] [28] . The current study studied samples collected from the Capital Territory Region Islamabad where the positivity rate drop has dropped to <0.5% where now potential emergence of lineage A of SARS-CoV-2 with novel deletions in the spike glycoprotein is being noted. As the sequencing has been limited to a single city, in order to study the spread of this variant into different parts of the country, enhanced genomic surveillance will be required. Moreover, currently the country has increased vaccination rate with 27% population being fully vaccinated and 12% being partially vaccinated. Whether the emerging new lineages of SARS CoV2 can affect the already vaccinated individuals other than the non-vaccinated ones, requires a watchful eye to follow and therefore require a more robust genomic surveillance encompassing larger cohort and cities. All the authors declared no conflict of interest. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-ND 4.0 International license It 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) preprint The copyright holder for this this version posted December 25, 2021. ; https://doi.org/10.1101/2021.12.24.21268367 doi: medRxiv preprint . CC-BY-ND 4.0 International license It 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) preprint The copyright holder for this this version posted December 25, 2021. ; https://doi.org/10.1101/2021.12.24.21268367 doi: medRxiv preprint What's important to know about SARS-CoV-2 variants of concern? Genetic Variants of SARS-CoV-2-What Do They Mean? 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Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69-V70, France Genomic surveillance to combat COVID-19: challenges and opportunities FastQC: a quality control tool for high throughput sequence data Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Structural basis of receptor recognition by SARS-CoV-2 Mechanisms of coronavirus cell entry mediated by the viral spike protein Flexible, Functional, and Familiar: Characteristics of SARS-CoV-2 Spike Protein Evolution. Front Microbiol Author Correction: SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects All the sequences generated in the current study are submitted to the GISAID that are available at "https://www.gisaid.org/login/" under the accession numbers: EPI_ISL_7542800-EPI_ISL_7542804, EPI_ISL_7571414-EPI_ISL_7571415.