key: cord-0318281-ws53645o authors: Umair, M.; Ikram, A.; Rehman, Z.; Haider, A.; Badar, N.; Ammar, M.; Ahad, A.; Suleman, R.; Salman, M. title: Genomic diversity of SARS-CoV-2 in Pakistan during fourth wave of pandemic date: 2021-10-03 journal: nan DOI: 10.1101/2021.09.30.21264343 sha: 4c76598921c4520f14571f51333c49f89052b12d doc_id: 318281 cord_uid: ws53645o The emergence of different variants of concern of SARS-CoV-2 has resulted in upsurges of COVID positive cases around the globe. Pakistan is also experiencing fourth wave of COVID-19 with increasing number of positive cases. In order to understand the genomic diversity of circulating SARS-CoV-2 strains during fourth wave of pandemic in Pakistan, the current study was designed. The samples from 89 COVID-19 positive patients were subjected to whole genome sequencing using GeneStudio S5. The results showed that 99% (n=88) of isolates belonged to delta variant and only one isolate belonged to alpha variant. Among delta variant cases 26.1% (n=23) isolates were showing B.1.617.2 while 74% of isolates showing AY.4 lineage. Islamabad was found to be the most affected city with 54% (n=48) of cases, followed by Karachi (28%, n=25), and Rawalpindi (10%, n=9). AY.4 has slight difference in mutation profile compared to B.1.617.2. E156del, G142D and V26I mutations in spike and T181I in NSP6 were present in B.1.617.2 but not in AY.4. Interestingly, A446V mutation in NSP4 has been only observed in AY.4. The current study highlights the circulation of primarily delta variant (B.1.617.2 and AY.4) during fourth wave of pandemic in Pakistan. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) belongs to family coronaviridae that cause moderate to severe respiratory illness. As of September 27, 2021, 232,612,493 people have been infected worldwide with 4,726,175 fatalities [1] . Since the commencement of the COVID-19 pandemic, different mutations have emerged that ultimately lead to various lineages of SARS-CoV-2 [2] . These lineages were then classified as Variants of Concern (VOCs) and Variants of Interest (VOIs). There are currently four VOCs (Alpha, Beta, Gamma, and Delta), two VOIs (Lambda and Mu) and four variants are under investigation (Eta, Theta, Iota, and Kappa) [3] . Among the VOCs, delta was found to be the highly transmissible variant after alpha and first identified from India in September 2020. It is currently the second most prevalent VOC, which is now spread in more than 100 countries [4] . In Pakistan the first case of delta variant was detected on May 16, 2021. It has been reported that delta variant is 60% more transmissible than alpha. It also has the potential for reduction in neutralization by some EUA monoclonal antibody treatments and post-vaccination sera [5] . The Pfizer-BioNTech vaccine appeared to give 79% protection against delta variant infection after 14 days of getting a second dose, compared to 92% protection against the alpha variant [6] . In Pakistan, 1,250,425 individuals have been infected with 27,597 deaths as of September 29, 2021 [1] . Pakistan has experienced three waves of COVID-19 pandemic and currently is under the fourth wave. The first wave was from May-July 2020, the second was from October, 2020-January, 2021, the third wave began in March, 2021 and ended in May, 2021 while the fourth wave begins in July 2021 [7] . The average positivity rate in the start of fourth wave was 25% and now drops to 5% (as of September, 29, 2021). Despite such a high positivity rate during the fourth wave, the data regarding the circulating strains of SARS-CoV-2 is not available. Hence, in order to explore the genomic diversity of SARS-CoV-2 during fourth wave of pandemic in Pakistan whole genome sequencing of SARS-CoV-2 positive samples were performed. The oropharyngeal swab specimens of subjects who tested positive during clinical RT-PCR testing were retrospectively collected (n=89) and used in the present analysis. The study was approved by ethical review committee of National Institute of Health, Islamabad. Total RNA was extracted from the specimens using KingFisher TM Flex Purification System (ThermoFisher Scientific, US). Clinical RT-PCR testing was performed using TaqPath TM COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, Waltham, US) for SARS-CoV-2 at National Institute of Health, Islamabad, Pakistan. The COVID-19 positive specimens with low . 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) The copyright holder for this preprint this version posted October 3, 2021. ; https://doi.org/10.1101/2021.09.30.21264343 doi: medRxiv preprint cycle threshold (Ct) values (≤ 27) followed by selection based on their geographical location, i.e., representing the entire country were selected for whole genome sequencing. The cDNA synthesis was performed using SuperScript™ IV VILO™ Master Mix (Invitrogen, USA). The library was prepared using the Ion Ampliseq™ Library Kit Plus (Thermofisher Scientific, US) and Ion AmpliSeq™ SARS-CoV-2 research assay panel (Thermofisher Scientific, US) according to the manufacturer's instructions. The prepared library underwent template preparation and enrichment with the Ion Chef system. The enriched templates were then loaded onto an Ion 540 chip for semiconductor sequencing on the Ion GeneStudio S5 machine using the Ion S5 sequencing reagents (Thermofisher Scientific, US) as per manufacturer's protocols. The Fastq files were exported using File Exporter plugin from Torrent Suite 5.14.0 and then processed for quality assessment using the FastQC tool (v0.11.9) [8] . Trimmomatic (v0.39) [9] was employed to eliminate artifacts and technical biases by removing adapter sequences and low-quality base calls (< 20). The filtered reads were aligned using the Burrows-Wheeler Aligner's (BWA, v0.7.17) [10] and available reference genome (Wuhan-Hu-1, Accession number: MN908947.3). According to Centers for Disease Control and Prevention (CDC, USA) guidelines, variants were identified and consensus sequences for all genomes were generated [11] . Pangolin v3.1.11 and pangoLEARN model dated 09-08-2021 were used to classify the assembled genomes into PANGO lineages [12] . During July 19, 2021 to August 24, 2021 a total of 89 whole genomes of SARS-CoV-2 were sequenced at National Institute of Health, Islamabad. The age range of patients were 3-90 years with median age of 32 years. The male to female ratio was 53:36. Out of the 89 isolates, 88 were . 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 copyright holder for this preprint this version posted October 3, 2021. (Table 1 ). In addition, we have also identified some unique mutations in the studied sequences. In one . 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 copyright holder for this preprint this version posted October 3, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 3, 2021. ; https://doi.org/10.1101/2021.09.30.21264343 doi: medRxiv preprint cases in India was also due to slow vaccination rate. A study by Public Health England (PHE) states that although the delta variant makes the vaccine less effective but still prevent hospitalization in more than 90% of cases [14] . The same observed in Pakistan, where we have increased number of cases but have less deaths and hospitalization with case fatality rate of 2%. The dynamics of 4 th wave of COVID-19 pandemic in Pakistan has shown interesting epidemic modifying pattern which holds importance in national decision making for handling the pandemic. The decrease in hospitalization even with a highly transmissible variant of concern AY.4 suggest that vaccination coverage might help in suppressing future waves of the COVID-19 in Pakistani population. Furthermore, the disease also highlights the continued effectiveness of genomic surveillance of SARS CoV2 variants. All the authors declared no conflict of interest. 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_3462498-EPI_ISL_3462527, EPI_ISL_3553499-EPI_ISL_3553528, EPI_ISL_3827529-EPI_ISL_3827557. 13. https://outbreak.info/situation-reports/delta?loc=IND&loc=GBR&loc=USA&selected 14. https://qz.com/india/2036052/delta-variant-is-causing-a-surge-in-covid-cases-globally/ . 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) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 3, 2021. ; https://doi.org/10.1101/2021.09.30.21264343 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) The copyright holder for this preprint this version posted October 3, 2021. ; https://doi.org/10.1101/2021.09.30.21264343 doi: medRxiv preprint COVID Live Update: 233,105,342 Cases and 4,769,835 Deaths from the Coronavirus -Worldometer SARS-CoV-2 Lineages and Sub-Lineages Circulating Worldwide: A Dynamic Overview Delta to dominate world SARS-CoV-2 Variant Classifications and Definitions Five things we know about the Delta variant (and two things we don't) | Gavi, the Vaccine Alliance COVID-19 Health Advisory Platform by Ministry of National Health Services Regulations and Coordination FastQC: a quality control tool for high throughput sequence data Trimmomatic: a flexible trimmer for Illumina sequence data Fast and accurate long-read alignment with Burrows-Wheeler transform Rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome coronavirus 2 Pangolin: lineage assignment in an emerging pandemic as an epidemiological tool