SARS-CoV-2 in the Republic of Guinea: Fragment and Whole-Genome Sequencing, Phylogenetic Analysis

Genetic diversity of SARS-CoV-2 isolates circulating in the Republic of Guinea in May and June 2020, as well as in March 2021, has been demonstrated using fragment (S gene) and whole genome sequencing of 14 strains. Analysis of nucleotide sequences and phylogenetic constructs make it possible to divide the studied strains into 3 groups. Comparison of the obtained data with the already available epidemiological data proves the initial importation of COVID-19 from Western European countries, and also demonstrates four independent import routes in two time periods (March 2020 and no later than March 2021).


Introduction
The pandemic of the new coronavirus infection, COVID-19, the etiological agent of which is the SARS-CoV-2 coronavirus, has affected absolutely all states of the world. As of May 27, 2021,169,094,726 laboratory-confirmed cases of the disease were registered in the world, 3,512,510 of which were fatal [1]. In African states, 4,830,593 cases were reported, including 129,620 deaths. The Republic of Guinea accounted for 23,080 cases, including 160 deaths.
Having spread around the world, since December 2019, the etiological agent of COVID-19 has undergone various changes in the genome in the course of its evolution, producing many strain genovariants [2,3,4]. It is believed that strains with changes in the gene of the spike glycoprotein ("S gene"), especially in the region encoding the receptor binding domain (RBD), are of epidemic significance. [5,6] Changes in the structure of the spike protein are primarily associated with an increase in the infectiousness of the causative agent of and also contribute to the escape of the virus from the immune response [7,8,9].
Among the whole variety of genetic variants of SARS-CoV-2, as of May 31, 2021, the World Health Organization recognized the following variants of the virus as epidemically significant [10], for which an increase in transmissibility, severity of the disease, as well as a decrease in sensitivity to established therapy regimens was proven [11,12,13,14]: • Alpha -B.1.1.7 (501Y.V1) was first detected in samples collected in the UK in September 2020. The Alpha variant has been registered in 114 countries.
• Beta -B 1.351 (501.V2), first described in South Africa in December, 2020. This strain has become widespread in 92 countries of the world.
• Gamma -P1 (501Y.V3). It was first registered in Brazil in December, 2020. It has spread in 49 states.
Today, intensive work is being carried out on sequencing and bioinformatic analysis of the genomes of SARS-CoV-2, in order to identify new gene variants as well as to establish pathways for the spread of existing ones, since epidemically significant gene variants pose a serious threat to public health. However, for developing countries, sequencing of the detected strains of SARS-CoV-2 is problematic due to economic reasons, poor logistics, and other factors [15,16]. In this regard, the Republic of Guinea is no exception. Within the framework of the Russian-Guinean scientific and technical cooperation fragment and whole genome sequencing of SARS-CoV-2 genovariants circulating in the territory of the Republic of Guinea has been performed.
The main task of our study was to investigate the nucleotide sequences of the S gene, as well as the complete genomes of SARS-CoV-2 strains isolated in the Republic of Guinea at different time periods, in order to study their genetic diversity, as well as to establish their origin.
To achieve this goal, the following objectives were identified: 1. Fragment sequencing (S gene).
3. Construction of phylogenetic trees based on the sequencing of both the S gene and complete genomes.

Materials and methods
Specimens -nasopharyngeal swabs were collected from suspected cases and contact persons as part of the public health emergency response of the Ministry of Health in the Republic of Guinea.
Isolation of RNA from specimens was carried out using a kit for the isolation of nucleic acids "RiboPrep" ("InterLabService", Russia). Detection of the SARS-CoV-2 genetic material was carried out using the RT-PCR kits: Sequencing of complete genomes was carried out using semiconductor sequencing technology on the Ion S5TM XL System platform (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. The primary data processing was carried out using the Torrent Suite Software version 5.12.1 (Thermo Fisher Scientific,USA). The genome was assembled through mapping the filtered reads to the reference genome (Wuhan-Hu-1 (NC_045512.2)) using the BWA software version 07.17. S-gene sequencing was carried out according to the Sanger method on an AB 3500xl (Applied Biosystems, USA) using Data Collection v3.1 software.
Alignment of the nucleotide sequences of both complete genomes and the S gene was carried out using the MAFFT v.7 program (https://mafft.cbrc.jp/alignment/server/). The search for single nucleotide polymorphisms (SNPs) in the compared genomes was performed using the Snippy 2.0 program (https://github.com/tseemann/snippy). Comparative phylogenetic analysis was conducted using the BioNumerics 7.6 software package (Applied Maths NV, Belgium) and the Maximum Parsimony algorithm.
In total, 14 samples obtained in different time periods and from different regions of the Republic of Guinea were analyzed. Whole genome sequencing was carried out for 4 samples, in other 10 samples only the S gene was sequenced. The obtained nucleotide sequences were deposited in the international databases NCBI GenBank. Data on the studied samples are presented in Table 1.

Results and discussion
The first laboratory-confirmed case of new coronavirus infection in the Republic of Guinea was registered on March 12, 2020 in the capital city, Conakry, in a citizen of the European Union, who arrived from France aboard the airplane shortly before the registration of the disease [17]. In total, in March 2020, until the closure of international traffic (in accordance with the Order of the Government of Guinea, dated March 23, 2020 air traffic was suspended for an indefinite period, and since March 27, the land borders [18] were also closed) 22 cases of the disease, 9 of which were reliably imported, were reported. Among those 9 cases, 8 were related to import from France, and one from Italy [17]. The subsequent spread was associated with community transmission of the virus, up to the opening of the borders in August 2020. It should be noted that, apart from import by aircraft, the likelihood of bringing the virus in by crew members of sea vessels was also not excluded, in view of the presence of large port cities in Guinea -Conakry, Boke and Boffa, the importance of which as a "COVID-19 entrance gates" was emphasized in "National COVID-19 Response Plan" [19].
Throughout the entire period, specialists from the Russian-Guinean Research Center for companies involved in the extraction of minerals. The sub-prefecture of Kindia, from where the rest of the studied samples came, is a major economic center as well as a key transport hub [21].
It is through Kindia that the internal migration of the population from the capital to the inner regions of the country and vice versa, as well as to neighboring states, is carried out. In total, 107 laboratory-confirmed cases of COVID-19 were detected in the Kindia sub-prefecture in March 2021, so we examined 9,3% of isolates from this territory over March.
Bioinformatic analysis showed that the studied samples were not genetically homogeneous. Analysis of the nucleotide sequence of the S gene, carried out for all 14 samples, allows them to be divided into 3 groups. The first includes samples (C1, C2, C3, C4) taken in May-June 2020 in the prefectures of Boke (Kamsar, Kolaboui, Sangaredi) and Kindia (Kindia).
All of them have the D614G mutation, and the C3 isolate also has an additional Cys1235Phe mutation, but they do not belong to the known genovariants of epidemic significance. Strains of the same genotype have been described for the states of Western Europe, Asia, and North America. Importantly, sample C1 is one of the first laboratory-confirmed cases of COVID-19 in the Kindia sub-prefecture.  1.617.2); however, other differences do not allow assigning the studied isolates to the Delta genovariant. Samples E12 and E13 were utterly identical to each other and were obtained from settlement Damakania (sub-prefecture of Kindia). Sample E20, which differs from E12 and E13 by 4 SNPs, but is in the same group with them, was isolated in another settlement, Sambaya. Strains of a genotype similar to the third group in the international NCBI GenBank database are represented by only a few specimens from France, Luxembourg and the United Kingdom.
It is important to note that the isolates of the second and third groups, despite their genetic heterogeneity, circulated in the territory of a common sub-prefecture (Kindia) in the same time period (biomaterial samples were taken between March 2 and March 20, 2021), which indicates independent pathways of importation of infection into the territory. The results of Sgene sequencing of isolates of all groups are presented in Table 2.
Phylogenetic analysis using the nucleotide sequence of the S gene showed that the samples of the first group (obtained in 2020) were greatly distanced from the isolates of 2021, and were in the same clade with the isolates circulating in 2020 in many countries of the world, without a clearly defined territorial affiliation, which confirms the data on the importation of COVID-19 into Guinea from abroad. The genovariants of the second group also formed a separate clade, being located together with the typical variants of the Alpha line. Samples of the third group formed a separate clade, spaced from the isolates of the second group, being the closest to the samples from France, Luxembourg, and United Kingdom. The tree constructed according to the results of phylogenetic analysis of the S gene is shown in Figure 1.
Since four samples dated 2020, did not have any characteristic features by the nucleotide sequence of the S gene that would allow us to infer their origin, genome-wide sequencing was carried out, followed by the construction of a phylogenetic tree. Mutations in the genome of these strains are shown in Table 3.
The results of sequencing the complete genomes showed that the samples of 2020 belonged to two phylogenetic clades -GR and G. The first clade includes samples C1 and C2 taken in May 2020 in Kolaboui and Sangaredi (Boke prefecture). Both samples belonged to the GR cluster, which does not have a clear geographical confinement. The strains of this cluster were isolated on the territory of the states of Western Europe, North America and Asia. Samples C3 and C4 (June 2020) belonged to clade G and showed high phylogenetic affinity for the strains circulating in Western Europe. So, in phylogenetic construct, these isolates were located in the clade consisting exclusively of genovariants registered in France and / or Switzerland (Figure 2).
Given the high genetic heterogeneity, as well as the different time setting, we can talk about at least four independent imports of coronavirus infection into the territory of the Republic of Guinea. Since the isolates of 2020 demonstrated affinity to two clusters, GR and G, it can be assumed that (before the border closure) at least two independent imports of SARS-CoV-2 genovariants took place in March 2020. And since immediately after the announcement of the COVID-19 outbreak the borders of Guinea became closed, then the internal transmission of infection before the reopening of the borders was precisely through those imported strains.
In confirmation of the epidemiological data, the phylogenetic closeness of the C3 and C4 samples (cluster G) to the samples from France and Switzerland indicates the importation of coronavirus infection from the countries of Western Europe. The strains of the GR cluster (samples C1 and C2) circulating in Guinea at that time were also mainly distributed in Western Europe. Thus, based on the results of our analysis, we have shown that the initial importation of COVID-19 into Guinea took place along at least two independent routes, which are most likely associated with the countries of Western Europe.
After the recommencing of international traffic, despite the anti-epidemic measures being taken, the import of new types of genovariants could not be avoided. So, the Alpha variant already circulated in Guinea at least since the beginning of March 2021 (the date of sampling of E10 is March 02, 2021). Thereat, at the same time, strains phylogenetically distant from the Alpha variant circulated in the same territory (sub-prefecture of Kindia).
Thus, the identification of four SARS-CoV-2 variants, phylogenetically close to the strains circulating outside Guinea, but at the same time phylogenetically distant from each other, suggests at least four independent COVID-19 imports to Guinea -two of which were carried out in March 2020, and two no later than March 2021.

Conclusions
Based on the above laid out data, the following conclusions can be formulated: 1. The SARS-CoV-2 isolates circulating in the Republic of Guinea are genetically heterogeneous and belong to different phylogenetic groups.
2. Modern genovariants are replacing the originally imported ones.
3. The importation of COVID-19 into the territory of the Republic of Guinea was carried out at least through 4 independent routes.
4. The initial routes of import are connected with the countries of Western Europe.

Ethical statement
Ethical approval to conduct the study was not needed. Biological specimens were collected as part of the public health emergency response of the Ministry of Health in the Republic of Guinea; therefore, consent for sample collection was waived. Personal information was anonymized.