Genome sequence analysis of SARS-COV-2 isolated from a COVID-19 patient in Erbil, Iraq.

In the city of Wuhan, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first recognized among humans at the end of December 2019, and has since spread to every country around the world. The emergence of this new coronavirus has attracted global attention to work towards finding a treatment and developing an effective vaccine against the virus. In this study, we sequence a full genome of SARS-COV-2 isolated from a male patient in the city of Erbil, Iraq. The virus was sequenced using Sanger sequencer and 21 distinct mutations were found in our isolate compared to the full genome sequence of the SARS-COV-2 isolated from the city of Wuhan/China (Accession number: NC_045512.2). Sequence analysis showed that four of the mutations were located at the spike glycoprotein (S), and ten of them were in nonstructural proteins (nsp1, nsp3, nsp12, and orf3a), which had been shown to be related to structural changes at various sites. Moreover, phylogenetic analysis and transmission supported the conclusion that the cases in Iraq were of independent origins of infections and had a close relation to the isolates from Iran. This is the first report on the DNA sequence of the SARS-CoV-2 genome isolated from the Kurdistan region of Iraq. © King Abdulaziz City for Science and Technology 2022.

Introduction

The recent appearance of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has attracted global attention and prompted an international campaign to develop a treatment and vaccine. The outbreak began in Wuhan, China, in December 2019 (Zhu et al. 2020) and has since spread to every country and impacting virtually everyone in the world (Hussen et al. 2021a). Since April 19, 2021, there have been 140,886,773 confirmed cases of COVID-19, with 3,012,251 deaths reported.

The SARS-CoV-2 virus is an enveloped single-stranded positive-sense RNA (+ ssRNA), encoding around 9860 amino acids (Kim et al. 2020; Sabir et al. 2020). Typical SARS-CoV-2 has one of the largest genomes ∼ 30 kb, among all RNA virus families (Sidiq et al. 2020) (Fig. 1).

Fig. 1

Graphical representation of the mutations in the SARS-CoV-2 genome isolated from Erbil/Iraq. A SARS-CoV-2 virion structure. B Location of the non-synonymous mutation in the whole genome of SARS-COV2 isolated from Erbil/Iraq

Efforts to develop successful vaccines have been separately undertaken in response to the pandemic's urgency. However, like other pathogens, staying in a hospital or living in a crowded place makes it easier for viruses to spread (Saleh et al. 2021; Ali et al. 2020; Al-Sa'ady et al. 2020; Mostafaei et al. 2021; Al-Bdery et al. 2020) and as a response to this, self-quarantine has been shown to have a negative impact which disturbs the lifestyle (Hussen et al. 2021b).

Viral genome sequences from different countries can assist in determining the virus's mutational tendencies as it crosses diverse geographical environments and peoples of various cultures, with differing genetic profiles and immunological responses (Parlikar et al. 2020; Khailany et al. 2020). Therefore, whole viral genome sequencing is important for diagnosing viral infectious diseases and developing vaccines. In addition, genome sequencing could be used to study the virulence and pathogenicity of the virus, as well as the evolutionary linkages between hosts and viruses (Shaibu et al. 2021). SARS-CoV-2 is rapidly spreading through countries, and there has been considerable concern about whether the spread is due to mutations in the virus (Aldiabat et al. 2018; Alhayani and llhan 2021).

A significant feature of this virus is its high mutation rate, with recent studies showing that, the viral genome develops new mutations as it spreads through various environments (Pachetti et al. 2020; Junejo et al. 2020; Khailany et al. 2020). As an RNA virus, SARS-CoV-2 does not have a ‘proof reading’ function when it replicates, and so random mutations occur. These have become quite prolific and are the primary circulating forms of the virus in many areas (Alhayani et al. 2021; Hasan and Alhayani 2021). ORF1ab, ORF3a, ORF8, N, M, and S regions have all been found to have mutations, with nt8782 in ORF 1a and nt28144 ORF 8 showed mutation rate of 29.47% and 30.53%, respectively (Wang et al. 2020; Hasan and Alhayani 2021; Mohammed and Daham 2021). In addition, it is believed that nucleotide variations, one of the most important indicators of viral development, are also responsible for modifying the virus in all conditions (Mostafaei et al. 2021; Hui 2006; Domingo 2000; Taheri et al. 2021; Ibrahim et al. 2021; Khan et al. 2021).

The main aim of this study was to carry out whole-genome sequencing (WGS) of the local SARS-CoV2 variant to gain insights into the mutational variation of the virus compared to the first reported genome sequence of the SARS-COVID-2 genome isolated from Wuhan/China. In addition, this sequencing enabled a precise understanding of the novel variations, which are key indicators for viral development and vaccine response.

Materials and methods

This study was approved by the Medical Research Centre (MRC) ethical committee (Application number: IRB-20-357) at Hawler Medical University, Erbil, Iraq.

Clinical sample and processing

A nasopharyngeal swab collected from a patient (28-year-old male with respiratory tract infection in Erbil) received at the Immunogene Center for COVID-19 testing in December 2020 was used in this study. Viral nucleic acid extraction was performed using a QIamp RNA extraction kit (Qiagen, Maryland, USA) as per manufacturer recommendations. Viral presence was detected by using a Luna universal probe one-stage RT-qPCR kit (NEB, MA, USA) using primers and probes targeting nucleocapsid viral gene (Corman et al. 2019).

Genomic sequencing

Complementary DNA (cDNA) was synthesized from extracted RNA from a clinical sample that was positive for SARS-CoV-2. cDNA was then PCR amplified using a set of primers to obtain overlapping segments of the whole viral genome. High-performance sequencing was performed using a normal protocol Sanger sequencing (Applied Biosystems 3130xl genetic analyzer) according to the manufacturer's standards with a 150 bp read length. The assembly of the genome took place using MEGAHIT v1.2.9 (Li et al. 2015). Genome assembly was shown using MEGAHIT v1.2.9 (Sidiq et al. 2020), and the genome size was 29,803 bp and being 99% identical to the Wuhan sequence (NC_045512.2).

Genome assembly, alignment, and phylogenetic analysis

The software ClustalW was used to analyze the uniqueness of the sequenced genomes and to align full sequences of the genome to the SARS-CoV-2 reference genome (NC 045512.2) A phylogenetic analysis was done using Molecular Evolutionary Genetics Analysis version 7.0 (MEGA 7.0) to examine similarities and differences between the Erbil genome sequence and 27 SARS-CoV-2 genome sequence downloaded from GenBank and the GISAID database (Elbe and Buckland-Merrett 2017) (Fig. 2).

Fig. 2

Phylogenetic analysis based on nearly full gene sequence of SARS-COV-2 isolated from different geographical locations. The sequence was downloaded from GenBank and the GISAID database websites. The neighbor-joining method, bootstrapped with 1,000 replicate runs, was used to sequence analysis of the viruses by MEGA 7 tree-building program. The newly sequenced local isolate was highlighted orange on the tree. The accession number and EPI accession number are written next to the name of the country which the virus was isolated. The bar indicates substitutions per nucleotide

Structural characterization

The computation model structure of the SARS-COV-2 genome was created using the online service of the SIB Swiss Institute of Bioinformatics (Expasy) database (https://swissmodel.expasy.org/interactive). The 3S structure of SARS-COV-2 served as a template and was downloaded from Protein Data Bank (Pdb: 6vsb). UCSF Chimera 1.11.2 software was used throughout the investigation of the model structure (Pettersen et al. 2004).

Results and discussion

The ongoing SARS-CoV-2 epidemic is causing concern around the world due to its extreme contagiousness. Coronaviruses, contain the largest known RNA viruses (26.4 to 31.7 kb) (Woo et al. 2009; Mihindukulasuriya et al. 2008). A larger genome size offers greater possibilities for genetic modification (Woo et al. 2009; Mihindukulasuriya et al. 2008). RNA viruses often have extremely high mutation rates, making them very virulent and resulting in the development of new species and changes in the mortality rate and symptoms of host (Duffy 2018; Angeletti et al. 2020). This study uncovered several important facts that fill knowledge gaps in relation to the novel COVID-2019 virus (Barzinjy et al. 2021).

For genome analysis, our isolated genome sequence of the virus strain was uploaded to the NCBI databases accession number MW633517 on February 25, 2021. SARS-CoV-2 isolate sequences were aligned to a reference sequence (NC 045512.2) from Wuhan to identify mutations and the location of the mutations was predicted using the program ClustalW.

Based on mutation analysis, our isolate from Erbil-Iraq contained 21 various silent and missense mutations (Table 1). Nine mutations were highlighted as unique in the viral SARS-COV-2 isolate compared to the Wuhan reference sequence (Fig. 1). Of these nine mutations, three of them were identified in the spike glycoprotein (S), one in the Nucleocapsid protein (N), and five in nonstructural proteins (nsp1, nsp3, nsp12, and orf3a), which have been shown to be related to structural changes at various sites. Three of these (L22917R, E23311D, D23403G) lead to alterations in the structure of the spike glycoprotein (S), while the others change the structure of nsp2 (Table1).

Table 1

Total mutations, their locations, and their effects on the changes of the amino acid sequence within the SARS-COV-2 genome isolated from Erbil/Iraq (Accession number: MW633517)

Mutation	Location	Amino acid	Code
ORF1ab (216..21505)	10 mutations
	269 nsp1	Glutamic acid > lysine	GAG > AAG E2L
	3037nsp3	Phenylalanine > phenylephrine	TTC > TTT F924F
	5832nsp3	Serine > serine	TCC > TCT S1856S
	6293nsp3	Isoleucine > valine	ATA > GTA I2010V
	7675nsp3	Alanine > alanine	GCG > GCT A2470A
	11152nsp6	Valine > valine	GTC > GTT V3629V
	12142nsp8	Threonine > threonine	ACT > ACA T3959T
	14408nsp12	Proline > leucine	CCT > CTT P4715L
	16524nsp13	Threonine > threonine	ACA > ACG T5420T
	18877nsp14	Leucine > leucine	CTA > TTA L6205L
S gene 21513..25334	4 mutations
	22444	Aspartic acid > aspartic acid	GAC > GAT D294D
	22917	Leucine > arginine	CTG > CGG L452R
	23311	Glutamic acid > aspartic acid	GAG > GAT E583D
	23403	Aspartic acid > glycine	GAT > GGT D614G
ORF3a (25343..26170)	2mutations
	25440	Lysine > asparagine	AAG > AAT K16N
	25563	Glutamic acid > histidine	CAG > CAT Q57H
M gene (26,473..27141)	1 mutation
	26735	Tyrosine > tyrosine	TAC > TAT Y71Y
Gene N 28,224..29483	2 mutations
	28854	Serine > leucine	TCA > TTA S194L
	29167	Tyrosine > tyrosine	TAC > TAT Y298Y

Synonymous and non-synonymous mutations were observed in ORF1ab, spike glycoprotein (S), ORF3a, Membrane glycoprotein (M), and Nucleocapsid protein (N), but none were observed in ORF6, ORF7a, ORF7b, ORF8, or ORF10 gene. These co-occurring mutations were recently identified as a feature of one of the major SARS-CoV-2 variants found in Europe.

In the ORF1ab region, we found ten variations, which include both unique and previously described mutations at position 269 (G > A), 3037 (C > T), 5832 (C > T), 6293 (A > G), 7675 (GG > CT), 11152 (C > T), 12142 (T > A), 14408 (C > T), 16524 (A > G) and 18877 (C > T).

ORF1ab has multiple functions, influencing helicase activity, RNA-dependent RNA polymerase activity, methyltransferase activity, Zn– binding, and Fe–S cluster binding activity (Graham et al. 2008). The functional consequences of such mutations are yet unknown and future studies on functional alterations produced by such mutations may improve better understanding of viral pathogenesis.

Based on existing knowledge, mutations in nonstructural protein 1 (nsp1) at position 269 G > A (E2L) nsp1 and I2010V at position 6293nsp3 are unique and not previously described as non-synonymous mutations in SARS-CoV-2 nsp1 which exhibits a much higher capacity to block the host proteins synthesis, this may enhance the pathogenicity of SARS-CoV-2 (Shen et al. 2021).

The 14408 C > T (P4715L) and 3037 C > T variations in ORF1ab were reported to be common in neighboring countries (Rehman et al. 2020) and are thought to be related to and to have a causative role in, mutations in the RNA-dependent RNA polymerase (RdRP/nsp12) and nsp3 genes, respectively. Because RdRP/nsp12 is a core part of the replication or transcription process, the leucine mutation at position 14408 of RdRP/nsp12 may alter its function, ultimately increasing the mutation rate of the virus (Eskier et al. 2020).

Furthermore, the proline-to-leucine mutation has been identified as a prevalent mutation in earlier studies in Turkey (Rehman et al. 2020), Europe (51.6%) (Pachetti et al. 2020) and North America (58.1%) (Banerjee et al. 2021).

In addition, C3037T variation has been linked to a synonymous mutation in the region coding for nsp3 (nonstructural protein) and was observed in (71%) of Turkey. In humans, nsp2 and nsp3 are known to play important roles in SARS-CoV-2 pathogenesis (Angeletti et al. 2020).

Other mutations found in this study includes at one position 25440G > T (K16N) and another (Q57H) at position 25,563G > T within the region of ORF3a, which was seen in Turkish isolates and, one variation at position 26735 (C > T) in the M protein also was seen.

Similarly, one study (Omotoso et al. 2021) previously described a non-synonymous mutation at position 28854C > T (S194L), and in the current study such a mutation was observed in the N protein, which altered the original sequence of the open reading frame for the nucleocapsid (N) protein which is recognized to be important in virion assembly and structure (Weber et al. 2021). Apart from frequent mutations, four variants were found in the spike glycoprotein (S); while triple mutations observed (L452R, E583D, D614G) were non-synonymous and were reported previously in strains from other countries (Harvey et al. 2021) (Fig. 2).

The L452R mutation, which changes the amino acid Leucine (L) to Arginine (R) at position 22917, is the main change at receptor binding domain (RBD) that inhibit the Spike glycoprotein’s reactivity, so that it become resistant to neutralizing antibodies in the sera of patients (Li et al. 2020; Greaney et al. 2021). Additionally, the RBD binding site alterations enhance the free energy of the direct interaction with ACE2 to form RBD-ACE2 binding complex that makes viral attachment to cells more probable and, thus, increases viral infectivity (Chen et al. 2020; Tchesnokova et al. 2021).

The second mutation detected in the spike glycoprotein (S) is E583D, which is located in the S1 domain and was previously detected in Asia (Das et al. 2021). Signal peptide mutations might affect signaling processes within the intracellular secretory route, but the meaning behind these changes is yet to be defined.

The mutation is D614G, which modifies the amino acid aspartate (D) with a polar negative charged side chain to the amino acid glycine (G) with a non-polar side chain, is one of the most common mutations of SARS-CoV-2, and it was first found in China on January 24, 2020 (Wang et al. 2021).

The amino acids surrounding position 614 are quite conservative, showing that the D614G mutation will have a significant effect on the S protein functions of SARS-CoV-2.

The D614G mutation has been identified as the primary mutation responsible for the increased infectiousness of SARS-CoV-2 on a global scale (Korber et al. 2020; Yurkovetskiy et al. 2020).

Finally, the genome sequence of the located sequenced SARS-COV-2 was compared to genome sequences from Saudi Arabia, Turkey, Iran, America, China, and some European countries (Fig. 3).

Fig. 3

Location of non-synonymous mutation in the SARS-COV-2 Spike protein. A Trimer model structure of the SARS-COV-2 created using Expasy proteomic database based on the 3D structure of SARS-COV-2 (Pdb: 6vsb). B Visualizing the ribbon diagram of SARS-COV-2 S-protein using UCSF Chimera software. C Location of the three mutations R542, D538, and G164 on the model structure of Spike S protein are colored red

The phylogenetic tree was built based on the neighbor-joining method in MEGA 7 software (Kumar et al. 2016). The result shows how closely related our isolate is to previously sequenced SARS-COV-2 genome, such as from a male patient from the city of Shiraz/Iran (EPI Accession number: EPI_ISL_2455558). Considering, the close proximity of Iran and Iraq, perhaps it is not surprising that the variant of SARS-COV-2 found in Erbil originated from Iran. Notably, the collection date of the reported sample was in January, 25th, 2021.

Conclusion

In this study, we sequenced one SARS-CoV-2 genome from a clinical sample collected from Erbil, Iraq. Additional mutations and previously known mutations (synonymous and non-synonymous) were found in genome assembly and annotation research. We identified position-specific mutations in the nucleotide sequences, such as single-nucleotide variants. Additional analysis is performed on amino acid substitutions to determine the functional stability of the proteins. Our findings showed that SARS-CoV-2 proteins have 21 different variant locations in their coding regions. ORF1ab is the protein with the most mutations, followed by three structural proteins (S, ORF3a, N). We found fewer mutations in ORF3a and N proteins than in ORF1ab and S proteins. A higher quantity of single point mutation, C > T, is observed both in the second and 3rd positions in the codons.

Through analyzing the viral genome's phylogeny, it was discovered that they share many similarities with isolates that were found in Turkey, Saudi Arabia, and the United States, indicating that several introductions to the state may have occurred. To gain a better understanding of the possible function in terms of virulence, infectivity, and virus release, a complete investigation would be useful.