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Emergence of new combinations of SARS-CoV-2 peak receptor binding domain variants in Senegal

The ongoing viral evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens the effectiveness of our strongest defenses against coronavirus disease 19 (COVID-19): vaccines, therapeutics and diagnostics. To keep pace with continued viral diversification, molecular surveillance serves as an essential alert system to identify new strains to be assessed for potential immune or diagnostic escape. More recently, the identification of lines of concern for SARS-CoV-2, B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta), immediately preceded their increase in prevalence and global spread1,2,3,4. Subsequent reports have demonstrated that increased transmissibility and immune evasion are linked to these lines, which are defined by mutations in the spike receptor binding domain (RBD), including N501Y, K417N / T, L452R and E484K. Notably, it has already been shown that the E484K and L452R mutations in RBD confer immune evasion in cell culture selection experiments.5, which is consistent with their increasing prevalence6.7, probably due to better viral fitness8.9. Therefore, vigilant monitoring of circulating strains for these mutations is of critical importance to potentially prevent their spread.

The SARS-CoV-2 pandemic in Senegal has increased in several waves occurring in March-November 2020 (wave 1), December 2020-March 2021 (wave 2) and July-September 2021 (wave 3). The first variant of concern that was reported in Senegal was B.1.1.7, which was first identified in a patient diagnosed on December 30, 2020 in the second wave.ten. To compare the strains of SARS-CoV-2 circulating during the first two waves of the pandemic in Senegal, a panel of 150 nasopharyngeal samples of the first wave and 150 s in viral transport medium (VTM) was collected in a study. approved by the Ethical Commission of the Senegalese Ministry of Health (000129 / MSAS / CNERS). VTM samples were sequenced by Next Generation Sequencing (NGS) using a metagenomic approach with probe enrichment (xGen) and analysis on an Illumina HiSeq11. The genomes were assembled using BLAST and the sequence NC_045512 as a reference, followed by clade assignment and mutation analysis with the NextClade tool (clades.nextstrain.org) and lineage assignments with the tool. Pangolin12. Genome coverage> 60% was obtained for N = 213 specimens (N = 96 first wave, N = 117 s), with an average depth of coverage of 43,006x (GISAID accession numbers EPI_ISL_1630259-1630270). The genomes of the first wave were divided into 3 clades: 19B (N = 3), 20A (N = 78) and 20B (N = 15), similar to the composition of strains in other countries at the same period13. In the Pangolin nomenclature14, nine lines were present in the first wave, which was dominated by B.1.416 (57/96, 59.4%, Fig. 1A). Viral diversity increased sharply during the second wave with the genomes of 9 clades present: 19A (N = 1), 19B (N = 11), 20A (N = 108), 20B (N = 81), 20C (N = 3), 20D (N = 1), 20E (N = 1), 20G (N = 1) and 20I (N = 1). An increased diversity of Pangolin lineages was also observed in the second wave, with 20 lineages identified, the majority of which were not present in the first wave (Fig. 1A). Most notable among the new strains found exclusively in wave two, variant B.1.1.7 accounted for 5% of all infections of the second wave (6/117) and was present in four different cities (Dakar, Tivaoune, Diamnadio and Thies, Fig. 1B), confirming an extensive distribution in western Senegal. The first B.1.1.7 infection in this study was diagnosed on December 21 in Thiès, which predates the first case previously identified in Senegal.ten. The December 21 patient was a patient who had been tested due to contact with an infected person, suggesting that B.1.1.7 was already circulating in Senegal in early December. The remaining 5 B.1.1.7 cases were all diagnosed in early January during the exponential phase of the peak of the second wave of cases.

Figure 1

Molecular surveillance of SARS-CoV-2 in Senegal. In the panel (A), the number of sequences classified into the indicated lines present in waves one and two are displayed in proportion to the total number of sequences generated with genomic coverage> 60% of each wave, as indicated in the total numbers under each plot. An * indicates the lines that were present in both waves. In the panel (B), sample sequence and metadata are listed for each strain carrying a spike mutation of concern at position 501, 484, or 452. All sequences in this panel are from wave two. In the panel (VS), the line defining the amino acid mutations (compared to the reference genome NC_045512) for the new strains identified in this study are presented. (A) Identified lines, (B) Summary of variants, (VS) Escape the variant lines.

Escape mutations in the spike protein were absent from the first wave, but were present in 4% (5/117) of all infections from the second wave (Fig. 1B, Supplementary Table 1). Additional details for all genomes with mutations of concern at positions 501, 484 and / or 452 in the RBD peak are shown in Figure 1B. When classified by clade, all L452R mutations were exclusively found in genomes of clade 19B, while the L452M mutation appears to have emerged in the second wave of clade 20A (Supplementary Table 1). In addition to strains individually carrying L452R, variant strains carrying a combination of L452R + N501Y (3/117, 2.6%) were also identified. The N501Y mutation confers a higher affinity for the ACE2 receptor and is present in several variants of concern (alpha, beta, gamma) while L452R is a characteristic escape mutation found in the delta and epsilon lines which also increases infectivity.4,8,9,15,16. The combination of these two mutations in one strain is of concern for the potential rapid spread of an immune escape variant. The three genomes carrying the L452R / N501Y combination belonged to the A.27 line (clade 19B) and do not encode the D614G mutation which today predominates in most infections worldwide. Likewise, the other lineage-defining mutations for the variants of concern were absent in the A.27 genomes, with the exception of L18F and H655Y, both of which are present in the gamma lineage (Fig. 1C). While 13 common single nucleotide polymorphisms (SNPs) were identified for this line, each individual genome also had unique SNPs, suggesting that these were not cases related to inheritance. The three patients with A.27 infections were diagnosed in the Almadie district of Dakar in December 2020 and were between 36 and 55 years old (Fig. 1B).

In addition to the L452R + N501Y double mutant, a single genome was identified which carried a unique combination of E484K + N501T peak RBD mutations in a lineage B.1 genome (clade 20C) with D614G also present. This line has been provisionally named B.1.501T.V1 (Fig. 1C). The patient infected with this variant strain was a patient diagnosed in December 2020 in Diamniadio (Fig. 1B). While E484K provides a loophole for neutralizing antibodies17.18, the N501T mutation improves the affinity of the spike receptor binding domain (RBD) for ACE2 in vitro and is expected to improve transmissibility, similar to N501Y19.20. Strains harboring N501T first appeared in August 2020 in northern Italy6 and the N501T mutation was recently discovered in an emerging Brazilian line that differs from B.1.501T.V121. Alarmingly, N = 2122 N501T strains were published on GISAID from samples collected in the months following the identification of this sample in Senegal (January-April 2021) in countries in Africa, Europe. , Asia, North America and South America (GISAID, accession date April 18, 2021)6. Taken together, these trends suggest that convergent evolution around the world leads to mutations at the leading positions E484 and N501 in many lineages, suggesting a possible increased suitability for viruses carrying these mutations.