key: cord-0806266-mo1225fk
authors: Chouikha, Anissa; Lagare, Adamou; Ghedira, Kais; Diallo, Amadou; Njouom, Richard; Sankhe, Safietou; Derrar, Fawzi; Victoir, Kathleen; Dellagi, Koussay; Triki, Henda; Diagne, Moussa Moise
title: SARS-CoV-2 Lineage A.27: New Data from African Countries and Dynamics in the Context of the COVID-19 Pandemic
date: 2022-05-09
journal: Viruses
DOI: 10.3390/v14051007
sha: 7c355a8b23d0bdb4805501ea9fc618e7110ad050
doc_id: 806266
cord_uid: mo1225fk

SARS-CoV-2 is constantly evolving with lineages emerging and others eclipsing. Some lineages have an important epidemiological impact and are known as variants of interest (VOIs), variants under monitoring (VUMs) or variants of concern (VOCs). Lineage A.27 was first defined as a VUM since it holds mutations of concern. Here, we report additional lineage A.27 data and sequences from five African countries and describe the molecular characteristics, and the genetic history of this lineage worldwide. Based on the new sequences investigated, the most recent ancestor (tMRCA) of lineage A.27 was estimated to be from April 2020 from Niger. It then spread to Europe and other parts of the world with a peak observed between February and April 2021. The detection rate of A.27 then decreased with only a few cases reported during summer 2021. The phylogenetic analysis revealed many sub-lineages. Among them, one was defined by the substitution Q677H in the spike (S) gene, one was defined by the substitution D358N in the nucleoprotein (N) gene and one was defined by the substitution A2143V in the ORF1b gene. This work highlights the importance of molecular characterization and the timely submission of sequences to correctly describe the circulation of particular strains in order to be proactive in monitoring the pandemic.

Since its onset, the SARS-CoV-2 pandemic has been characterized by the emergence of several viral lineages. Some of them rapidly spread over the world due to their increased transmissibility and displaced older lineages. Others were associated with increased virulence and/or their ability to escape the host immune responses. Tracking the circulation of variants and lineages is, currently, one of the most important pillars of SARS-CoV-2 surveillance worldwide. As of November 2021, the WHO (World Health Organization) had identified four variants of concern (VOCs): B.1.1.7 (Alpha) which originated from the United Kingdom [1] , B.1.351 (Beta) from South Africa, P.1 (Gamma) from Brazil and B.1.617.2 (Delta) from India [2] . At that time, the Delta variant displaced all the other variants [3, 4] and was subdivided into sub-lineages from AY.1 to AY.133 in the Pango lineage designation system [5] . Recently, in November 2021, a new VOC, B.1.1.529 (Omicron), was described in Botswana and South Africa, which has rapidly spread all over the world [6] . These VOCs have shown sustained regional community transmission followed by quick worldwide dispersal. The Alpha variant was the first detected with an international spread; it was displaced by the Delta variant, and presently, Omicron is the most detected variant all over the world [2] .

In addition, other variants presenting mutations potentially associated with altered phenotypes are labeled as variants of interest (VOIs) or variants under monitoring (VUMs) according to the WHO classification, or variants under investigation (VUIs) according to the European Centre for Disease Prevention and Control (eCDC) or Public Health England (COG-UK) classifications [2, 7, 8] . These variants are subject to attentive surveillance, to better understand how easily they might be transmitted and the effectiveness of currently authorized vaccines against them. The A.27 lineage was first classified as a VUM by the eCDC and the COG-UK [7, 8] and as a VOI by the French Society of Microbiology [9] since it shows a mutational pattern similar to VOCs and VOIs, with several amino acid substitutions along the viral genome, especially in the S gene [10] [11] [12] [13] . Among these amino acid substitutions, L18F, L452R and N501Y are located in the N-terminal (NTD) or receptor binding domain (RBD) regions and have been suggested to result in immune escape and higher transmissibility [14] [15] [16] [17] . In contrast, A.27 does not have the D614G mutation, typical of the SARS-CoV-2 B lineage and which is now predominant worldwide. At the time of writing, the A.27 lineage was first detected in Denmark in mid-December 2020 and then reported in 38 countries: 17 from Europe, and 16 from Africa, together with the USA, India, Indonesia, Iceland and Australia. However, almost half of the sequences reported in the GISAID database are from France [18] .

The present work, implemented in the frame of a collaborative monitoring conducted by a consortium of 10 African institutes of the Pasteur Network (REPAIR consortium), reports additional lineage A.27 sequence data from 5 countries in Africa (Niger, Tunisia, Senegal, Algeria and Cameroon), describes the molecular characteristics of these sequences and their classifications in comparison with other A.27 public available data and traces the dynamic evolutionary history of this lineage worldwide.

A total of 85 new SARS-CoV-2 sequences belonging to lineage A. 27 were identified and included in the present study; they originated as follows: 46 from Niger, 23 from Tunisia, 14 from Senegal, 1 from Cameroon and 1 from Algeria (Table 1) . 

Whole genome sequencing was performed for the 85 samples using Illumina or Nanopore (MinION) technologies. Viral RNA was extracted from a 140 µL nasopharyngeal sample with the Qiamp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. SARS-CoV-2 detection was performed using real-time PCR corresponding to the SARS-CoV-2 detection kit (The LightMix ® E-gene kit (TIB Molbiol, Berlin, Germany)). SARS-CoV-2-positive samples with a Ct value < 30 were selected for NGS sequencing. Genomes were extracted by an amplicon-based approach using the United States Food and Drug Administration (US FDA)-approved Illumina COVIDSeq kit [19] , a modified version of the ARTIC protocol. The extracted RNAs were reverse transcribed to single-strand cDNA as per the manufacturer's instructions, and SARS-CoV-2 PCR was performed with two specific primer pools combined with proven Illumina sequencing technology, which allowed obtaining 98 tiled 400 bp amplicons covering the whole genome. For each sample, PCR products were combined, and libraries were prepared using the Illumina Nextera DNA UD Indexes as per the manufacturer's instructions. Libraries were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA), and the concentration was measured using a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Library pool validation and mean fragment size were determined using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) as per the manufacturer's instructions. The 400 bp library pool was diluted to 4 nM. Libraries were pooled, denatured and diluted to 1.4 pM and then sequenced on a NextSeq550 instrument with a V2.5 High Output Cartridge 2 × 150 bp run kit (Illumina, San Diego, CA, USA). Sequences' raw data were processed using fastqc version 0.11.9 for quality control (https: //www.bioinformatics.babraham.ac.uk/projects/fastqc/) (accessed on 12 November 2021). Low-quality reads and adapters were filtered using trimmomatic version 0.39 with a Phred quality score of 30 as the threshold. Filtered reads were mapped against the SARS-CoV-2 reference (NC_045512) using the Burrows-Wheeler Aligner MEM algorithm (BWA-MEM) (v0.7.7). SAM tools were used to sort BAM files, to generate alignment statistics and to obtain coverage data [20] . The obtained whole genome sequences are listed in Table 1 .

The genomes were assembled using the EDGE COVID-19 pipeline which is based on the fully open source EDGE Bioinformatics software [21] . FaQCs was used to control the quality of the reads [22] . Low-quality regions of reads were trimmed and filtered if the reads failed a quality threshold of 20 or minimum length of 50bp. The reads that passed the QC process were then aligned with the original Wuhan-Hu-1 complete reference genome (RefSeq accession: NC_045512.2) using BWA-MEM [23] . Various parameters were defaulted: (i) minimum depth coverage (5x) to support a variant site, (ii) alternate base threshold (0.5) to support an alternative for the consensus base to be changed, (iii) indel threshold (0.5) to support an INDEL for the consensus base to be changed and (iv) minimum mapping quality [24] .

A total of 597 SARS-CoV-2 whole genome sequences belonging to the A.27 lineage originating from 34 countries and available in GISAID at the time of writing were retrieved. The number of sequences per country was as follows: Australia (n = 1), Austria (n = 7), Belgium (n = 21), Benin (n = 21), Burkina Faso (n = 15), Cote d'Ivoire (n = 25), Denmark (n = 2), England (n = 16), France (n = 276), Gabon (n = 1), Germany (n = 34), Greece (n = 1), Guinea (n = 1), Iceland (n = 1), India (n = 3), Indonesia (n = 1), Ireland (n = 2), Italy (n = 9), Luxembourg (n = 4), Mayotte (n = 13), the Netherlands (n = 5), Nigeria (n = 1), Rwanda (n = 1), Scotland (n = 1), Senegal (n = 3), Slovenia (n = 41), Spain (n = 10), Sudan (n = 1), Sweden (n = 13), Switzerland (n = 23), Togo (n = 23), Tunisia (n = 1), Turkey (n = 9) and the USA (n = 8). 

Phylogenetic analysis was performed on 280 whole genomes (among the 597 retrieved from GISAID and those generated in the frame of the REPAIR consortium). This subset includes sequences from 35 different countries. All sequences with more than 15% of ambiguous nucleotides (Ns) throughout the whole genome were not included. Additionally, for countries with a large number of sequences such as France, only a subset of sequences selected randomly was included. Sequence alignment was performed with MAFFT using default parameters [25] . The resulting alignment was used to build a maximum likelihood phylogenetic tree using the IQ-TREE web server, supported by 1000 bootstrap replicates (http://iqtree.cibiv.univie.ac.at/) (accessed on 12 November 2021). The phylogenetic tree was then visualized using Figtree software (http://tree.bio.ed.ac.uk/software/figtree/) (accessed on 12 November 2021). The tree was rooted using the midpoint rooting method. The number of sequences per country was as follows: Algeria (n = 1), Australia (n = 2), Austria (n = 3), Belgium (n = 7), Benin (n = 11), Burkina Faso (n = 3), Cote d'Ivoire (n = 7), Denmark (n = 1), England (n = 13), France (n = 90), Gabon (n = 1), Germany (n = 9), Ghana (n = 1), Greece (n = 1), Guinea (n = 1), India (n = 2), Indonesia (n = 1), Ireland (n = 2), Italy (n = 5), Luxembourg (n = 2), Mayotte (n = 1), the Netherlands (n = 6), Niger (n = 30), Nigeria (n = 1), Rwanda (n = 1), Scotland (n = 1), Senegal (n = 11), Slovenia (n = 19), Spain (n = 5), Sweden (n = 2), Switzerland (n = 14), Togo (n = 7), Tunisia (n = 8), Turkey (n = 8) and the USA (n = 4).

Phylogeography analysis was based on 330 S gene sequences among the 597 whole genome sequences; only those with zero ambiguous nucleotides in the S gene were included. They were collected from 27 countries as follows: Australia (n = 1), Austria (n = 2), Belgium (n = 10), Denmark (n = 2), England (n = 13), France (n = 157), Germany (n = 17), Greece (n = 1), Ireland (n = 1), Italy (n = 8), Luxembourg (n = 4), Mayotte (n = 10), the Netherlands (n = 6), Nigeria (n = 1), Niger (n = 21), Rwanda (n = 1), Scotland (n = 1), Senegal (n = 4), Slovenia (n = 22), Spain (n = 4), Sweden (n = 2), Switzerland (n = 16),Togo (n = 8), Tunisia (n = 6), Turkey (n = 8) and the USA (n = 4).

The epidemic history of the A.27 lineage was investigated with a coalescent-based strategy inferred by Bayesian Markov chain Monte Carlo (MCMC) analysis implemented in BEAST software version 1.8.4 (http://beast.bio.ed.ac.uk) (accessed on 12 November 2021). For this analysis, a parametric model of coalescent population growth corresponding to the coalescent Bayesian Skygrid under the uncorrelated lognormal relaxed clock was used, allowing the evolutionary rate to vary among taxa. To find the best substitution model to use with BEAST, "Find Best DNA/Protein Models (ML)" in MEGAX was used. The HKY nucleotide substitution model with gamma-distributed rates among sites was identified as one of the best models based on Bayesian information criterion (BIC) and Akaike information criterion (AIC) criteria and was used under the relaxed molecular clocks. The gamma distribution allows for several gamma rate categories to be applied to the substitution rate, allowing gamma rate heterogeneity. MCMC was run using default parameters, with a chain length of 100,000,000 generations for the SARS-CoV-2 A.27 sequences in order to achieve an effective sample size (ESS) ≥ 200, sampled every 100 thousand steps with 10% of the initial runs excluded. The BEAST output log file was analyzed with the TRACER v1.6 program (available from http://tree.bio.ed.ac.uk/software/tracer/) (accessed on 12 November 2021). A tree was generated using the MCMC algorithm, being part of the BEAST package (version 1.8.4). The TreeAnnotator program was used to summarize and construct the maximum clade credibility tree (MCC) from 1000 trees. Briefly, the burnin (as states) was fixed to 100, equivalent to 10% of the total number of trees generated by BEAST. The posterior probability limit was fixed at 0.7. Posterior summaries were only calculated for nodes in the target tree that have a posterior probability greater than the specified limit. Nodes with very little support were not considered. Phylogeographic analysis was performed using the BEAST package to estimate the diffusion process of the SARS-CoV-2 A.27 lineage in the world, and particularly in Africa. The capital of each country was used as a discrete location state, and the sampling date was used to calibrate the time scale. The MCC tree was uploaded into SPREAD to generate a keyhole markup language file (kmL) (Supplementary Material S1). The selected sequences were assigned to a total of 27 distinct geographic groups corresponding to each country. In order to provide a spatial projection, the migration routes indicated by the tree were visualized using Google Earth Pro version 7.3.4 to identify significant migrations.

A total of 682 sequences belonging to the A.27 lineage were included in the present study: 85 are from Niger, Tunisia, Senegal, Cameroon and Algeria, and 597 are from other countries and were retrieved from the GISAID database as of 12 November 2021.

The demographic and clinical data related to the 85 A.27 samples reported as part of the present work are shown in Table 1 . The sequences were isolated from 60 males and 25 females. Out of the 60 patients for whom the clinical form was provided, 35 were asymptomatic (58.3%), 23 presented with mild infection (38.3%) and only 2 (3.3%) had a severe infection ( Table 1 ). The age ranged from less than 1 to 85 years (mean age = 41.5 ± 19.2 years). Most cases were due to an apparent indigenous transmission except for two travelers who came to Tunisia and were detected positive upon arrival (one was arriving from Libya and the other from Côte d'Ivoire). In contrast, most of the cases reported from Senegal were detected in Chinese outgoing travelers who seemingly were infected in Senegal. As of November 2021, the A.27 variant was reported in 38 different countries from the 5 different continents: most of them were in Europe (n = 18 countries), and from Africa (n = 16 countries); the variant was also reported in the USA, India, Indonesia and Australia. The worldwide geographic distribution of the A.27 sequences including those previously reported in GISAID and the ones reported for the first time in the present work is shown in Figure 1. than the specified limit. Nodes with very little support were not considered.

Phylogeographic analysis was performed using the BEAST package to estimate the diffusion process of the SARS-CoV-2 A.27 lineage in the world, and particularly in Africa. The capital of each country was used as a discrete location state, and the sampling date was used to calibrate the time scale. The MCC tree was uploaded into SPREAD to generate a keyhole markup language file (kmL) (Supplementary Material S1). The selected sequences were assigned to a total of 27 distinct geographic groups corresponding to each country. In order to provide a spatial projection, the migration routes indicated by the tree were visualized using Google Earth Pro version 7.3.4 to identify significant migrations.

A total of 682 sequences belonging to the A.27 lineage were included in the present study: 85 are from Niger, Tunisia, Senegal, Cameroon and Algeria, and 597 are from other countries and were retrieved from the GISAID database as of 12 November 2021.

The demographic and clinical data related to the 85 A.27 samples reported as part of the present work are shown in Table 1 . The sequences were isolated from 60 males and 25 females. Out of the 60 patients for whom the clinical form was provided, 35 were asymptomatic (58.3%), 23 presented with mild infection (38.3%) and only 2 (3.3%) had a severe infection ( Table 1 ). The age ranged from less than 1 to 85 years (mean age = 41.5 ± 19.2 years). Most cases were due to an apparent indigenous transmission except for two travelers who came to Tunisia and were detected positive upon arrival (one was arriving from Libya and the other from Côte d'Ivoire). In contrast, most of the cases reported from Senegal were detected in Chinese outgoing travelers who seemingly were infected in Senegal. As of November 2021, the A.27 variant was reported in 38 different countries from the 5 different continents: most of them were in Europe (n = 18 countries), and from Africa (n = 16 countries); the variant was also reported in the USA, India, Indonesia and Australia. The worldwide geographic distribution of the A.27 sequences including those previously reported in GISAID and the ones reported for the first time in the present work is shown in Figure 1 . last reported sequences at the time of writing were from Turkey, with a collection date of 5 August 2021. Figure 2 shows the number of reported sequences according to their date of isolation. The histogram is based on 643 out of the 682 A.27 sequences, reported in GISAID and in the present work (the remaining sequences were published with no or incomplete date of isolation and could not be included in the graph).

The earliest isolates of A.27 were detected in November 2020 (weeks 48 and 49) were from Niger, reported here for the first time. The number of reported sequence creased up to February/March 2021 (weeks 5-2021 to 9-2021) and then decreased. The reported sequences at the time of writing were from Turkey, with a collection date August 2021. Figure 2 shows the number of reported sequences according to their date of isola The histogram is based on 643 out of the 682 A.27 sequences, reported in GISAID an the present work (the remaining sequences were published with no or incomplete da isolation and could not be included in the graph). The sequences from Turkey hold minor aa mutations, namely, A2460V, L3495F and S3675F in the orf1a gene, L1701F in the ORF1b gene and T223I in the ORF3a gene, and the Italian sequence hold the mutations T95I, T572S and H1083Y in the S gene. For the phylogeographic analysis, 330 sequences corresponding to the S gene and belonging to A.27 genomes were investigated in the present study; a total of 300 were retrieved from the GISAID database as of 24 August 2021, and 30 with zero ambiguous nucleotides in the S gene are part of the 85 WGSs reported for the first time in the present study.

The tMRCA of the progenitor virus that ultimately gave rise to all SARS-CoV-2 A.27 strains circulating in the world was estimated around 20 April 2020 with an HPD (highest posterior density) of 95% (January 2020, August 2020). The sequences from Turkey hold minor aa mutations, namely, A2460V, L3495F and S3675F in the orf1a gene, L1701F in the ORF1b gene and T223I in the ORF3a gene, and the Italian sequence hold the mutations T95I, T572S and H1083Y in the S gene.

For the phylogeographic analysis, 330 sequences corresponding to the S gene and belonging to A.27 genomes were investigated in the present study; a total of 300 were retrieved from the GISAID database as of 24 August 2021, and 30 with zero ambiguous nucleotides in the S gene are part of the 85 WGSs reported for the first time in the present study.

The tMRCA of the progenitor virus that ultimately gave rise to all SARS-CoV-2 A.27 strains circulating in the world was estimated around 20 April 2020 with an HPD (highest posterior density) of 95% (January 2020, August 2020).

To gain insight into the spatial temporal dynamics of the diffusion process of the SARS-CoV-2 A.27 variant in Africa and in the world, the spatial estimates annotated in the S gene region MCC tree were mapped on Google Earth Pro version 7.3.4 (http://www.google. com/earth/download/ge/) (accessed on 12 November 2021). This mapping allows one to visualize the virus's geographic spread process over time. The links between different geographic regions represent branches in the MCC tree on which virus migration occurs, and the circle areas reflect the number of branches maintaining a particular location at that time point (Supplementary Material S1). Figure 4 shows the temporal dynamics of A.27's spatial dispersal processes in the world. 

To gain insight into the spatial temporal dynamics of the diffusion process of the SARS-CoV-2 A.27 variant in Africa and in the world, the spatial estimates annotated in the S gene region MCC tree were mapped on Google Earth Pro version 7.3.4 (http://www.google.com/earth/download/ge/) (accessed on 12 November 2021). This mapping allows one to visualize the virus's geographic spread process over time. The links between different geographic regions represent branches in the MCC tree on which virus migration occurs, and the circle areas reflect the number of branches maintaining a particular location at that time point (Supplementary Material S1). Figure 4 shows the temporal dynamics of A.27's spatial dispersal processes in the world. Based on the investigated A.27 S gene sequences, the phylodynamic and phylogeographic analysis revealed that the virus started its spread from Niger in Africa. The virus faced a big spread in March and April 2020 and then migrated from Niger to France starting from April 2020. Once in France, the virus migrated rapidly to Germany in the same month when the virus started to spread in Europe. At the same time, the virus migrated from Niger to reach Togo in June 2020. In parallel, the A.27 lineage continued its spread in Niger in July and August 2020. From August 2020, the A.27 lineage saw a big spread in Africa and Europe. Indeed, starting from July 2020, the virus migrated from Germany to reach Austria in August 2020, and from France to the United Kingdom and Belgium in September 2020, to Luxembourg in December 2020 and to Senegal in early 2021. The virus migrated from Niger to Mayotte around August 2020 and to Senegal in December 2020, and then to Turkey around January 2021. The virus seemed to migrate from Senegal to reach Rwanda in April 2021. The virus migrated from the United Kingdom around September 2020 to reach Tunisia where it spread in December 2020. The A.27 variant spread worldwide in 2021, with two distinct introductions in the USA from Niger around February 2021 and in France by April 2021, as well as migration from Germany to reach Australia by March 2021. Based on the investigated A.27 S gene sequences, the phylodynamic and phylogeographic analysis revealed that the virus started its spread from Niger in Africa. The virus faced a big spread in March and April 2020 and then migrated from Niger to France starting from April 2020. Once in France, the virus migrated rapidly to Germany in the same month when the virus started to spread in Europe. At the same time, the virus migrated from Niger to reach Togo in June 2020. In parallel, the A.27 lineage continued its spread in Niger in July and August 2020. From August 2020, the A.27 lineage saw a big spread in Africa and Europe. Indeed, starting from July 2020, the virus migrated from Germany to reach Austria in August 2020, and from France to the United Kingdom and Belgium in September 2020, to Luxembourg in December 2020 and to Senegal in early 2021. The virus migrated from Niger to Mayotte around August 2020 and to Senegal in December 2020, and then to Turkey around January 2021. The virus seemed to migrate from Senegal to reach Rwanda in April 2021. The virus migrated from the United Kingdom around September 2020 to reach Tunisia where it spread in December 2020. The A.27 variant spread worldwide in 2021, with two distinct introductions in the USA from Niger around February 2021 and in France by April 2021, as well as migration from Germany to reach Australia by March 2021.

A.27 is a SARS-CoV-2 variant that was reported in late 2020 [7] [8] [9] [10] [11] [12] . Its genome holds several mutations along the viral genome, some of which are shared with other VOCs and VOIs [7, 8] . The present study on the COVID-19 pandemic in Africa was conducted in the frame of a collaborative investigation led by the Pasteur Network, the REPAIR program, which is a multidisciplinary exploration in the context of the pandemic to determine, among others, the molecular epidemiology of SARS-CoV-2 in Africa.

We reported 85 new sequences from African countries and provided an overview of the A.27 lineage genetic characteristics and its epidemiology in Africa and worldwide, based on the new and previously reported sequences in the GISAID database.

Up to November 2021, sequences belonging to the A.27 lineage were reported from 38 different countries, most of them from Europe (N = 17) and Africa (n = 16). At the time of writing, and based on the sequences submitted in the GISAID database [18, 26] , the first reported sequences were from Denmark, dated to 14 December 2020, but we reported sequences from Niger, detected as early as November 2020. This lineage was described as a variant holding the N501Y substitution in the S gene, which increases the transmissibility of the virus [13] ; also note that the A.27 lineage has a G in amino acid residue 614 of the S protein, while most of the circulating lineages have the D614G change [26] . This lineage was then described in Mayotte which is a French territory [27] . Then, after, lineage A.27 was named the Henry Mondor variant since it caused many grouped cases in France [11] . Indeed, the highest number of sequences was reported in France (n = 276), and most of the cases were reported in February-March 2021; then, the frequency of detection of the variant declined [11] . The same situation was found in Germany where the first appearance of the lineage dated to early January; the frequency of A.27 then increased until the end of February and then declined concomitantly with the increase in the B.1.1.7 UK lineage [10, 28] . This lineage was also described in Cote d'Ivoire in January-February 2021 [11] . In our series, most of the A.27 sequences from Tunisia were detected in February and April 2021, and the lineage was then apparently displaced by the B.1.1.7 UK lineage that was introduced in February 2021 [29] . Most sequences from Niger were detected earlier, in November-December 2020, with only few sequences in January and February 2021. The indigenous sequences from Senegal dated to December 2020 to February 2021. The other sequences reported in this work were from outgoing Chinese travelers living in the same environment/district in Dakar city. Interestingly, some of these travelers were returning from a stay in China less than a week before the detection of the infection. This is even more interesting given that, to our knowledge, no A.27 strain has been identified in China thus far. In addition, although it seems that the detection of the A.27 lineage is becoming less frequent worldwide, it is noteworthy that new sequences from Turkey (July-August 2021) were recently reported, indicating recent active circulation, and they should be further monitored.

We also studied the mutational profile and the phylogenetic grouping of the A.27 lineage based on the sequences reported in GISAID since the emergence of the lineage up to date. Derived from the 19B clade and with a D614 background, fourteen aa substitutions are found in almost all the A.27 sequences and can be considered as the A.27 lineage-defining mutations, namely, P286L, D2980G and N3651S in ORF1a, P1000L in ORF1b, L18F, L452R, N501Y, A653V, H655Y, D796Y and G1219V in the S protein, V50A in ORF3a, L84S in ORF8 and S202N in the N gene. Among the seven mutations in the S gene, three are also known from other VOCs and VOIs (L18F, L452R and N501Y) located in the NTD or RBD epitope regions of the S protein and were previously reported to result in immune escape and/or higher infectivity and transmissibility [14] [15] [16] [17] 30, 31] . The mutations A653V and H655Y, also in the RBD, are in proximity to the S1/S2 furin cleavage site, at position 681 that promotes infection and cell-cell fusion [32] . D795Y is close to the essential TMPRSS2 cleavage site [33] at position 815. The L18F replacement in the S protein is part of an antigenic supersite in the N-terminal domain of the S protein and also appears in VOCs that are associated with immune escape and reinfection, such as B.1.351 and P.1. However, data from the literature and from GISAID do not reveal a particular severity of this variant, which is also confirmed in our series. More than 90% of the sequences that we reported in this work are from asymptomatic patients or mild clinical forms.

In addition to these lineage-defining aa substitutions, other less frequent mutations are also found; some of them define special sub-clusters in the phylogenetic tree of A.27 sequences and are supported by high bootstrap values. Indeed, two major sub-clusters within the A.27 lineage are found: one includes 52 sequences holding the Q677H substitution in the S gene (Figure 4 , cluster in red), and the other includes 62 sequences all having the A2143V substitution in ORF1b (Figure 4, cluster in green) . The substitution Q677H in the S gene was previously described in other SARS-CoV-2 lineages from clades 20G, 20A and 20B. Located near the furin binding pocket, it was suggested that it might be of importance to S1/S2 cleavage [34] . Q677H and A2143V together with several minor mutations were mainly found up to April 2021, the period where A.27 was actively circulating. The few sequences detected from May to August 2021, including the most recent sequences in Turkey, have different mutational profiles in addition to the fourteen lineage-defining main mutations.

Lineage A.27 belongs to clade 19B which spread from the beginning of the pandemic as well as clade 19A; this lineage was overtaken by the viruses of clades 20A, 20B and 20C which carry the D614G mutation in the S gene [10] . SARS-CoV-2 viruses of clade 19B were rarely detected after the first wave of the COVID-19 pandemic and practically disappeared until its re-emergence in the last quarter of 2020, where several teams have observed the re-emergence of different variants of clade 19B, especially with the emergence of lineage A. 27 [9-11,26] . The phylodynamic analysis showed a high spread of the lineage starting from August 2020; this date was estimated by Calvignac-Spencer et al. as the most common ancestor's emergence date based on the sequences reported as of May 2021 [9]. The tMRCA was also estimated to have emerged in late September by the team of Kaleta et al. based on samples retrieved from GISAID as of August 2021, in addition to other sequences from Germany [26] . According to the sequences added in the present study, the most recent common ancestor of lineage A.27 dated from April 2020. This was followed by a big dynamic of lineage A.27, especially in February-March 2021, which reached the five continents with multiple introductions in Europe. Since April 2021, variants Alpha and Delta have emerged and displaced lineage A.27 [4] . Currently, lineage A.27 is no longer considered as a VOI, since it seems that it no longer poses significant added risk to global public health. However, its recent detection in Turkey with a slightly modified genetic profile of the isolates suggests the need to further monitor its circulation worldwide.

Despite its high spread in European countries, especially France and Germany, and the outbreak observed in Mayotte (East Africa), the origin of the A.27 lineage was estimated to be West Africa [9,10]; the present study can confirm the West African origin since the earliest cases were described in Niger.

This work brings additional genomic data and provides an overview on the epidemiology and genetic history of a SARS-CoV-2 lineage that emerged in late 2020 until August 2021. Despite the limitations related to the lack of onsite NGS technologies, the limited resources to test a large number of strains and thus the limited number of strains per country included in the study, the obtained results are not compromised at the African region level, and this work highlights the importance of networking and collaborations in monitoring variants. Although the observed trend worldwide suggests that A.27 has been outcompeted by the B.1.1.7. UK and B.1.617.2 Delta lineages, it may be necessary to continue monitoring the spread of A.27 at the international level, especially since the lineage circulation was relatively recent and the most recent sequences exhibit new mutation profiles that may contribute to its resurgence. Funding: This study was co-funded by the French Ministry for Europe and Foreign Affairs (MEAE, France) via the project "REPAIR COVID-19-Africa", coordinated by the Pasteur Network. We are also grateful for the support throughout the Africa Pathogen Genomic Initiative (PGI) from the Africa CDC, and the support from the Bill and Melinda Gates Foundation.

Institutional Review Board Statement: All original samples reported in this study were anonymized before analysis. Institute members of the Pasteur Network, participating in the REPAIR consortium, obtained ethical release for the conduct of this work and the use of the samples collected in their respective countries.

Data Availability Statement: Not applicable.

AMR focal point, Head of AMR reference-Lab/MoHTogo, Supervisor of National Reference Center for HIV &STIs, Tel.: +22-89-032-6506/msalou@univ-lome.tg

Laboratory of Clinical and Epidemiological Virology (Rega Institute), Rega-Herestraat 49-box 1040, 3000 Leuven • Emmanouil I

833189) Email: josemaria.marimonortizdez@osakidetza.eus • Serge Alain SADEUH

Centre National de Référence des Hépatites B, C et delta, INSERM U955, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny 94010 Créteil

Laboratoire de virologie

COVID-19: New coronavirus variant is identified in UK. BMJ 2020, 371, m4857

The World Health Organisation (WHO)

Rapid replacement of the Beta variant by the Delta variant in South Africa

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha

SARS-CoV-2 (hCoV-19) Mutation Reports

European Centre for Disease Prevention and Control. SARS-CoV-2 Variants of Concern

Investigation of SARS-CoV-2 Variants of Concern: Technical Briefings

Rise and Fall of SARS-CoV-2 Lineage A

Novel SARS-CoV-2 Variant Derived from Clade 19B

SARS-CoV-2 variants of concern, variants of interest and lineage A

Spreading of a new SARS-CoV-2 N501Y spike variant in a new lineage

Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies

The N501Y spike substitution enhances SARS-CoV-2 infection and transmission

Transmission, infectivity, and antibody neutralization of an emerging SARS-CoV-2 variant in California carrying a L452R spike protein mutation

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVID Seq next-generation sequencing

Twelve years of SAMtools and BCFtools. GigaScience 2021

Rapid evaluation and quality control of next generation sequencing data with FaQCs

Aligning sequence reads

A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology

MAFFT multiple sequence alignment software version 7: Improvements in performance and usability

Data, disease and diplomacy: GISAID's innovative contribution to global health

Early introductions and community transmission of SARS-CoV-2 variant B.1.1.7 in the United States

Potential New Lineage Causing a Cluster in Mayotte 2021

Antibody escape and global spread of SARS-CoV-2 lineage A

Sequencing Using a Two-Step Strategy Reveals High Genetic Diversity in the S Gene of SARS-CoV-2 after a High-Transmission Period in Tunis

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

Emergence in late 2020 of multiple lineages of SARS-CoV-2 Spike protein variants affecting amino acid position 677

The authors acknowledge the following teams for accepting our request to use their whole genome sequences in the phylogenetic analysis:

The authors declare no conflict of interest.