key: cord-0809865-wk9nfmho authors: Ntoumi, Francine; Mapanguy, Claujens Chastel Mfoutou; Tomazatos, Alexandru; Pallerla, Srinivas Reddy; Linh, Le Thi Kieu; Casadei, Nicolas; Angelov, Angel; Sonnabend, Michael; Peter, Silke; Kremsner, Peter G; Velavan, Thirumalaisamy P. title: Genomic surveillance of SARS-CoV-2 in the Republic of Congo date: 2021-03-15 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.03.036 sha: a55d9139f519cb5995d9e073213af7b5561712a7 doc_id: 809865 cord_uid: wk9nfmho OBJECTIVE: We performed whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Congolese individuals sampled between April and July 2020. METHODS: We screened 96 samples for SARS-CoV-2 using RT-PCR, and 19 samples with Ct values <30 were sequenced using Illumina Next-Generation Sequencing (NGS). The genomes were annotated and screened for mutations using the web tool 'coronapp'. Subsequently, different SARS-CoV-2 lineages were assigned using PANGOLIN and Nextclade. RESULTS: Eleven SARS-CoV-2 genomes were successfully sequenced and submitted to the GSAID database. All genomes carried the spike mutation D614 G and were classified as part of the GH clade. The Congolese SARS-CoV-2 sequences belong to lineage B1 and nextclade 20A and 20C, which split into distinct clusters, indicating two separate introductions of the virus into the Republic of Congo. CONCLUSION: This first study provides valuable information on SARS CoV-2 transmission in the central African region, contributing to SARS CoV-2 surveillance on a temporal and spatial scale. With the first cases reported on 14 March 2020 (Ntoumi and Velavan, 2020) , Republic of Congo reports a total of 7794 cases with 117 deaths (as on 25 January 2021), and the transmission driven by community (WHO, 2021) . The first SARS-CoV-2 genome was described in January 2020, and since then several studies have tracked its evolution worldwide. Mutations in SARS-CoV-2 using next-generation sequencing methods are increasingly being studied to understand the potential association with transmission dynamics, pathogenicity, diagnostic performance, vaccine efficacy and immune evasion (ECDC, 2020) . There is a paucity of data on SARS-CoV-2 sequences from Central Africa, despite increasing submissions to databases from other regions of Africa. To date, no studies have reported on SARS-CoV-2 genomic lineages/strains in the Republic of Congo. This first study used samples collected between April and July 2020 and performed next-generation sequencing to understand the SARS-CoV-2 genomes that circulated during the early phases of the outbreak. Informed written consent was obtained from all participants. The study was approved by the ministry of scientific research and technological innovation, Republic of Congo (Approval Nr. 049/MRSIT-CAB) and by institutional ethics committee of the Congolese foundation for medical research (Approval Nr. 027/CIE/FCRM/2020). An epidemiological survey was conducted between April and July 2020 to assess the spread of SARS-COV-2 in the general population of Brazzaville (Batchi-Bouyou et al., 2020) . A total of 96 positive samples were randomly selected, and 19 samples (with Ct values <30) were subsequently sequenced using Illumina Next-Generation Sequencing (NGS) methodology. Viral RNA was extracted from nasopharyngeal samples using the QIAamp Viral RNA Mini Kit Libraries were prepared according to the COVID-19 ARTIC v3 Illumina library construction and sequencing protocol V.5 (DNA Pipelines R&D, 2020), using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs). The libraries were quantified (Qubit DNA BR, Thermo Scientific), normalized, pooled and sequencing was performed on an Illumina MiSeq v2 with 2 x 250 bp cycles. Viral genome assembly and variant calling was performed using nfcore/viralrecon pipeline (https://nf-co.re/viralrecon/1.1.0) (Ewels et al., 2020) . The SARS-CoV-2 genomes were screened for distinct mutations using online COVID-19 genome annotator "coronapp" (Mercatelli et al., 2020) . SARS-CoV-2 genomes from African countries were retrieved from GSAID (Shu and McCauley, 2017) . The eleven SARS-CoV-2 genomes were comparatively evaluated against reference NC_045512.2-Wuhan-Hu-1 and available SARS-CoV-2 genome sequences collected between April and July 2020, representing countries such as Ghana, Nigeria, Benin, Mali, Senegal, Côte d'Ivoire, Gabon, Democratic Republic of Congo, South Africa, and Kenya. Based on this comparative analysis, a maximum likelihood and phylogenetic tree were reconstructed. All the different SARS-CoV-2 genomes can be accessed in the GSAID database, with the respective IDs provided in the figure 1. The non-coding 3' and 5' regions were trimmed using Geneious Prime software and coding regions were aligned using a multiple sequence alignment (MAFTT) algorithm (Katoh et al., 2002) . A maximum likelihood tree was reconstructed with IQ-TREE server (Trifinopoulos et al., 2016) using the general time-reversible (GTR) model with rate heterogeneity (GTR+G). Branch support was calculated by ultrafast bootstrap consisting of 1000 alignments (Hoang et al., 2018) . SARS-CoV-2 genomes were classified into lineages using Phylogenetic Assignment of Named Global Outbreak LINeages (PANGOLIN) (Rambaut et al., 2020) and viral clades were assigned by Nextclade Beta (https://clades.nextstrain.org/) and Nextstrain (Hadfield et al., 2018) . The final dataset was displayed with the Interactive Tree of Life (iTOL) v4 (Letunic and Bork, 2019) . Of the 19 samples, 11 met the quality criteria for submission. All SARS-CoV-2 genome sequences were reported from different districts within Brazzaville, the capital of the Republic J o u r n a l P r e -p r o o f of Congo. All were symptomatic individuals with a median age of 45 years and 7 were male. Eleven SARS-CoV-2 genomes were deposited on the GISAID platform (Shu and McCauley, 2017 ) (https://www.gisaid.org/) (Accession numbers EPI_ISL_581455, 581462, 581472 and 581486 to 581493). Mutations annotated are summarized in Table 1 . The resulting SARS-CoV-2 genomes were compared with the reference NC_045512.2-Wuhan-Hu-1. The amino acid substitutions S (D614G) in the spike protein and NSP12b in the non-structural protein (NSP) occurred in all SARS-CoV-2 isolates. In addition, 10 of the 11 SARS-CoV-2 isolates carried the NSP2 (Y537Y), NSP3 (F106F) and ORF3a (Q57H) substitutions (Table 1) Genomics-based surveillance has helped researchers to assess transmission and evolutionary dynamics of the SARS-CoV-2 virus. This first study from the Republic of Congo aimed to provide crucial information on circulating strains during the early pandemic period (between April and July 2020), when sufficient diagnostic capacity is not yet available (Batchi-Bouyou et al., 2020) . All SARS-CoV-2 genomes carried the spike mutation D614G which is associated with efficient replication ex vivo and transmission in vivo (Hou et al., 2020) . The B.1 and B.1.1 lineage, as observed in this study, is also widespread in other African regions (Simulundu et al., 2021) . High SARS-COV2 IgG/IGM seroprevalence in asymptomatic Congolese in Brazzaville, the Republic of Congo Centre for Disease Prevention and Control. 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