# Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa

Wasfi Fares, Anissa Chouikha, Kais Ghedira, Meriam Gdoura, Dorra Rezig, Sondes Haddad Boubaker, Imen Ben Dhifallah, Henda Touzi, Walid Hammami, Zina Meddeb, et al.

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Wasfi Fares, Anissa Chouikha, Kais Ghedira, Meriam Gdoura, Dorra Rezig, et al. Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa. BMC Genomics, 2021, 22 (1), pp.540. 10.1186/s12864-021-07870-1. pasteur-03574784

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# Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa

Wasfi Fares¹*, Anissa Chouikha¹, Kais Ghedira², Meriam Gdoura¹, Dorra Rezig¹, Sondes Haddad Boubaker¹, Imen Ben Dhifallah¹, Henda Touzi¹, Walid Hammami¹, Zina Meddeb¹, Amel Sadraoui¹, Nahed Hogga¹, Imen Abouda¹, Aurélia Kwasiborski³, Véronique Hourdel³, Guillain Mikaty³, Valérie Caro³, Jean-Claude Manuguerra³, Nissaf Ben Alaya⁴,⁵ and Henda Triki¹,⁵

## Abstract

**Background:** In Tunisia a first SARS-CoV-2 confirmed case was reported in March 03, 2020. Since then, an increase of cases number was observed from either imported or local cases. The aim of this preliminary study was to better understand the molecular epidemiology and genetic variability of SARS-CoV-2 viruses circulating in Tunisia and worldwide.

**Methods:** Whole genome sequencing was performed using NGS approach on six SARS-CoV-2 highly positive samples detected during the early phase of the outbreak.

**Results:** Full genomes sequences of six Tunisian SARS-CoV-2 strains were obtained from imported and locally transmission cases during the COVID-19 outbreak. Reported sequences were non-identical with 0.1% nucleotide divergence rate and clustered into 6 different clades with worldwide sequences. SNPs results favor the distribution of the reported Tunisian sequences into 3 major genotypes. These SNP mutations are critical for diagnosis and vaccine development.

**Conclusions:** These results indicate multiple introductions of the virus in Tunisia and add new genomic data on SARS-CoV-2 at the international level.

**Keywords:** Whole genome sequencing, SARS-CoV-2, SNPs, Genotype, Tunisia

## Background

Human Coronaviruses (HCoV's) are members of the subfamily *Coronavirinae* in the family of *Coronaviridae* in the order of *Nidovirales*. Since 1960s, six different HCoV's were identified; two belongs to the genus *Alphacoronavirus* (229E, NL63) and four to the genus

*Betacoronavirus* (HKU1 and OC43, SARS-CoV, MERS-CoV). Similarly to other *coronaviruses*, SARS-CoV-2 is an enveloped, single-stranded positive-sense RNA (~ 29, 900 nt) with 5'-cap structure and 3'-poly-A tail [1].

In December 2019, a novel Human Coronavirus (HCoV) associated to severe acute respiratory syndrome was discovered in the city of Wuhan, Hubei province, China and was later named SARS-CoV-2 [2, 3]. The outbreak of the coronavirus disease (COVID-19) spread further worldwide and the World Health Organization

<footnote>* Correspondence: fwasfi@yahoo.fr
¹Laboratory of Clinical Virology, Institut Pasteur, University Tunis-El Manar, Tunis, Tunisia
Full list of author information is available at the end of the article</footnote>
(WHO) officially declared the pandemic aspect of the COVID-19 epidemic on March 12th, 2020 [4]. The WHO report dated on June 21st, 2020 confirmed 8,708, 008 cases of SARS-CoV-2 with 461715 deaths from 215 countries and territories [5].

The Tunisian government, through the Ministry of Health (MoH) and the National Observatory of New and Emerging Diseases, reviewed and initiated multisectoral measures to deal with the COVID-19 epidemic. The national strategy aims early detection of imported cases, isolation of confirmed cases as well as suspected cases. Moreover, the MoH commit to develop diagnostic and treatment procedures in order to limit the spread of the SARS-CoV-2 within the population and avoid exceeding of the health system capacity. These measures allowed the Tunisian authorities to identify the first SARS-CoV-2 in March 03, 2020 [6]. As of June 7th, a total of 1087 COVID-19 cases, including 286 imported and 801 locally transmitted cases, have been diagnosed and tested positive for SARS-CoV-2 with 49 deaths [7].

This work reports the first whole genome sequences of the SARS-CoV-2 viruses isolated in Tunisia during the early phase of the outbreak. Phylogenetic comparison with whole genome sequences reported from other countries and single-nucleotide polymorphism (SNPs) were assessed.

## Results

The six Tunisian sequences obtained were 29,541 to 29, 825 nucleotide-long and covered the whole coding region and more than 99.1% of the genome. The totality of complete genome sequences available at writing time, of

SARS-CoV-2 were downloaded from the GISAID (http://www.gisaid.org/) and the NCBI (http://www.ncbi.nlm.nih.gov/genbank) databases. All the repeated sequences as well as the sequences with missing regions have been excluded. The 268 selected sequences originated from 61 different countries worldwide. The phylogenetic tree in Fig. 1, shows that Tunisian SARS-CoV-2 sequences were scattered independently of each other in different clusters including strains mainly from Europe and Asia (Fig. 1a). Three out of six Tunisian sequences clustered with sequences from France: one case came from France (COV0425, Fig. 1g) and two were locally infected (COV1482 and COV1663, Fig. 1d and e). The sequence COV1339 was isolated from a case coming from Turkey, it grouped with sequences originated from Taiwan, Egypt and Finland (Fig. 1f). Sequence COV0010 was from a case with recent travel to Vietnam and clustered with sequences from Thailand, Vietnam, China and Japan (Fig. 1b). The last identified sequence COV0880 was from a locally infected individual and grouped with sequences from Turkey, Belarus and Kuwait (Fig. 1c).

The Tunisian SARS-CoV-2 retrieved full genome sequences alignment with the Wuhan reference sequences (NC045512) were used for the investigation of the SNPs and the resulting variable amino-acids composition throw genome detective web server (https://www.genomedetective.com/); the obtained results are shown in Table 1.

The average and the Min/Max depth of coverage for the six described isolates was as follows, Cov0010: 12545 (0/35320), Cov0880: 9339 (0/41049), Cov1339: 10456 (0/

<figure><img src="image_3.png" /></figure>
<table><caption>**Table 1** Single nt polymorphisms (SNPs) deduced by comparison of six Tunisian whole genome SARS-CoV-2 sequences with reference SARS-CoV-2 NC045512 (Continued)</caption><thead><tr><th></th><th>SNPs</th><th>G&gt;T</th><th>C&gt;T</th><th>C&gt;T</th></tr></thead><tbody><tr><td>Cov1663</td><td>mutations</td><td></td><td></td><td></td></tr><tr><td></td><td>Proteins</td><td>V162L</td><td>R138C</td><td>T29I</td></tr><tr><td>Cov0425</td><td>mutations</td><td></td><td></td><td></td></tr><tr><td></td><td>Proteins</td><td></td><td></td><td></td></tr><tr><td></td><td>mutations</td><td></td><td></td><td></td></tr><tr><td>Cov1482</td><td>SNPs</td><td></td><td></td><td></td></tr><tr><td></td><td>mutations</td><td></td><td></td><td></td></tr><tr><td></td><td>Proteins</td><td></td><td></td><td></td></tr><tr><td></td><td>mutations</td><td></td><td></td><td></td></tr></tbody></table><table><thead><tr><th></th><th>A&gt;G</th><th></th><th></th><th></th></tr></thead><tbody><tr><td></td><td>D614G</td><td></td><td></td><td></td></tr><tr><td></td><td>A&gt;G</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>G&gt;T</td><td></td><td></td></tr><tr><td></td><td>D614G</td><td></td><td></td><td></td></tr><tr><td></td><td>A&gt;G</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>G&gt;T</td><td></td><td></td></tr><tr><td></td><td>D614G</td><td></td><td></td><td></td></tr><tr><td></td><td>A&gt;G</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>G&gt;T</td><td></td><td></td></tr><tr><td></td><td>D614G</td><td></td><td></td><td></td></tr></tbody></table>

(In bold and underlined: confirmed mutations with more than 2500 reads. In italics: validated mutations with less than 2500 reads)
35132), Cov1663: 13504 (0/31108), Cov0425: 12982 (0/
32281) and Cov1482: 12360 (0/30130). To properly
present the mutants described in Table 1, confirmed
mutations with more than 2500 reads are written in bold
and underlined, and validated mutations with less than
2500 reads are written in italics.

The six Tunisian SARS-CoV-2 sequences were non-identical with 0.1% nucleotide divergence between each other. Compared to the Wuhan (NC045512) reference, the reported Tunisian SARS-CoV-2 sequences showed several synonymous and non-synonymous mutations, distributed over the entire genome. A total of 43 SNP mutations were identified with 27 and 16 SNPs in the non-structural and structural region respectively. Each sequence showed a different SNPs profile; the number of detected SNPs in each sequence ranged between 6 and 13. Most of them were located in the non-structural region affecting 08 out of the 16 non-structural genes (Table 1). In the structural region, most of SNPs were detected in the S gene encoding for the spike protein.

Discussion

In this work, the full genomes sequences of six SARS-CoV-2 strains were obtained from RT-PCR positive samples collected in Tunisian patients between March and April 2020. Three cases had a recent travel history, one coming from Vietnam, one from Turkey and one from France; while the three remaining cases were infected locally through horizontal transmission. The six sequences were non-identical with 0.1% nucleotide divergence between each other and had several nucleotide changes compared to the first published COVID-19 sequence from Wuhan, China (NC045512). Phylogenetic analysis including representative sequences from 61 countries split the six sequences from Tunisia into 6 different clusters, together with sequences from various other countries. These results indicate multiple introductions of the SARS-CoV-2 virus in Tunisia.

According to recently published results by Yin in
2020, four major genotypes were suggested depending
on the positions of SNPs [8]. The SNPs here reported in
the positions 3037, 8782, 11,083, 14,408, 23,403 and 28,
144 allowed us to classify the Tunisian sequences into 3
distinct genotypes among the four major genotypes de-
scribed: Genotype I (COV0880), genotype III
(COV0010) and Genotype IV (COV0425, COV1339,
COV1482 and COV1663). Indeed, these SNPs were
identified as the most frequent mutations described
since of the beginning of the pandemic in comparison
with the Wuhan reference SARS-CoV-2 genome
NC045512.2 [8, 9]. The highly frequent SNP mutations
was previously described in the S protein, RNA polymer-
ase, RNA primase, and nucleoprotein, which are critical
proteins for diagnosis and vaccine development [8, 10].

Further complementary studies including clinical sta-
tus of COVID-19 cases will be of a great interest to bet-
ter understand the impacts of these SNP mutations on
the transmissibility and pathogenicity of SARS-CoV-2.

Conclusions

Analysis of SARS-CoV-2 sequences from the maximum of affected countries and in different phases of the COVID-19 outbreak is crucial to understand the virus transmission and its genetic evolution through time. This preliminary study reports the first SARS-CoV-2 whole genome sequences from Tunisia, North Africa. Further sequences from different clinical and epidemiological settings are still needed to monitor the molecular epidemiology of SARS-CoV-2 in the country and worldwide.

Methods

Detection of SARS-CoV-2 infection was performed on
nasopharyngeal swabs samples by specific real time re-
verse transcription polymerase chain reaction (RT-PCR)
according to the WHO approved protocol published by
Corman et al. and detecting specific sequences in the E,
N and RdRp genes [11]. A total of 6 SARS-CoV-2 highly
positive samples were assessed for whole genome se-
quencing using the NGS approach. The respective
COVID-19 cases were detected between March and
April 2020. They originated from six different districts
of Tunisia. Three cases had recent travel history and
three were infected locally.

A total of 140 µl of each nasopharyngeal sample was used for RNA extraction via the Qiamp viral RNA mini kit (Qiagen, Hilden, Germany). Reverse transcription of the whole genome was performed by The ProtoScript II First Strand cDNA synthesis kit (New England Biolabs, USA) using random hexamers. For the multiplex PCR approach, the general method described in (https://artic. network/nCoV-2019) was applied using Version 3 of amplicon set. After 40 amplification cycles, the PCR products were cleaned-up with AMPure XP magnetic beads (Beckman Coulter, USA) and quantified with the Qubit 2.0 fluorometer (ThermoFisher Scientific, USA).

The Nextera DNA Flex Prep kit (Illumina, USA) was
used for library preparation and thereafter qualified on
an Agilent Technologies 2100 Bioanalyser using a high-
sensitivity DNA chip following the manufacturer's in-
structions. Generated libraries were sequenced using the
MiSeq System (Illumina, USA) providing 2 × 250 bp read
length data.

To remove low quality reads, trim off low-quality and
contaminant residues and filter out duplicated reads,
fqCleaner v.0.5.0 was used, with Phred quality score of
28. Filtered reads were mapped against SARS-CoV-2 ref-
erence (NC045512) using 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. Whole genome sequences of the six SARS-CoV-2 Tunisian strains were submitted to NCBI database under accession number MT499215 to MT499220.

Multiple sequences alignment was performed using Multiple Sequence Comparison by Log-Expectation (MUSCLE) software implemented in Molecular Evolutionary Genetics Analysis software (MEGA) version 7 [12]. Phylogenetic analysis was performed using the neighbor-joining analysis method and the Kimura 2-parameter model with 1000 bootstrap replications. Genetic distances were calculated using the p-distance method.

**Abbreviations**

COVID: Corona Virus Disease; GISAID: Global Initiative on Sharing Avian Influenza Data; MEGA: Molecular Evolutionary Genetics Analysis software; MUSCLE: Multiple Sequence Comparison by Log-Expectation; NGS: New generation Sequencing; RNA: Ribo-Nucleic-Acid; RT-PCR: Reverse transcription polymerase chain reaction; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; SNPs: Single Nucleotides Polymorphism; SRA: Sequence read Archive

**Acknowledgments**

The authors thank the field staff from the Ministry of Health for their efforts in specimen collection and transportation to the laboratory. The authors thank also all the IPT volunteers who worked very hard to manage the huge number of samples from the reception to the result reporting.

**Authors' contributions**

WF Conceptualization and writing the original draft. AC contributing in writing the original draft. KG Formal Analysis and writing the original draft. MG Reviewing and editing. DR: Investigation process. S H-B: Investigation process. IBD Investigation process. HT Investigation process, WH Investigation process. ZM Investigation process. AS: Investigation process. NH: Investigation process. IA: Investigation process. AK: Formal analysis. VH: Formal Analysis. GM: Writing, reviewing and editing. VC Formal Analysis. J-CM Writing, reviewing and editing. NBA Reviewing, editing and supervising. HT Writing, reviewing, editing and supervising. All authors read and approved the final manuscript.

**Funding**

This work was supported by the Tunisian Ministry of High Education and Research and the MediLabSecure Project, founded by the European Commission (DEVCO: IFS/2018/402-247).

**Availability of data and materials**

The six SARS-CoV-2 Tunisian strains sequences, obtained in the current study, were submitted to NCBI database and the accession number with the corresponding strains is as follow MT499215, MT499216, MT499217, MT499218, MT499219 and MT499220 for strains COV0010, COV0880, COV1339, COV1663, COV0425 and COV1482, respectively (https://www.ncbi.nlm.nih.gov/nuccore/MT499215, MT499216, MT499217, MT499218, MT499219, MT499220). The raw reads datasets generated and analyzed during the current study were submitted to the NCBI Sequence Read Archive (SRA) repository and freely available under following accession numbers SRX11188766 to SRX11188771 for the corresponding whole genome sequence MT499215 to MT499220 respectively. The SARS-CoV-2 isolate Wuhan-Hu-1 sequence used us a reference strain is available on the https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.

**Declarations**

**Ethics approval and consent to participate**

Our work was a retrospective analysis of already available samples collected as part of the COVID-19 diagnostic effort, and all samples used for analysis were anonymized. This work was approved by the ethical institutional review board "Comité d'Ethique Bio-Médicale (CEBM)" from the Institut Pasteur de Tunis (ref.2020/14/I/LR16IPT/V1). Consequently, our institutional Review Board (IRB) deemed it unnecessary to obtain ethics approval for this work. The need for consent was also deemed unnecessary by the IRB.

**Consent for publication**

Not applicable.

**Competing interests**

The author's declare no conflict of interest.

**Author details**

¹Laboratory of Clinical Virology, Institut Pasteur, University Tunis-El Manar, Tunis, Tunisia. ²Laboratory of Bioinformatics, Biomathematics and Biostatistics (BIMS), Institut Pasteur Tunis, Tunis, Tunisia. ³Laboratory for Urgent Response to Biological Threats (CIBU), Environment and Infectious Risks (ERI) research and expertise unit, Institut Pasteur, Paris, France. ⁴National Observatory for New and Emerging Diseases, Ministry of Health, Tunis, Tunisia. ⁵Faculty of Medicine, University Tunis-El Manar, Tunis, Tunisia.

Received: 24 June 2020 Accepted: 5 July 2021

Published online: 14 July 2021

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