P-ISSN 2587-2400 | E-ISSN 2587-196X
Ejmo Kapak
EJMO Volume : 5 Issue : 2 Year : 2021
EJMO. 2021; 5(2): 163-180 | DOI: 10.14744/ejmo.2021.52103

In Silico Characterisation of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) based on the Spike Protein Gene

Ashish Warghane1, Tejaswini Petkar2, Usha Preeyaa S3, Nishi Kumari4, Lavanya Ranjan5
1Department of Life Sciences, Mandsaur University, Mandsaur, Madhya Pradesh, India, 2Department of Agriculture and Food Sciences, Faculty of Agriculture, University of Mauritius, Reduit, Mauritius, 3Department of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India, 4Department of Molecular Biophysics, Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India, 5Department of Zoology, Maitreyi College, University of Delhi, New Delhi, India,

Objectives: The Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has been the current global pandemic concern. With a high transmission rate, especially through direct contact, this disease spreads from person to person, and this has in turn led to a huge number of infections on a global scale. Methods: In present study, comparative genomic analysis was performed using 151 gene sequences of the viral spike protein retrieved from NCBI and along with its translated nucleotide sequences using MEGAX software. Variation in the nucleotide and amino acid positions were identified. Results: Our analysis revealed that 22 nucleotide variations observed in positions 13, 141, 162, 233, 284, 328, 455, 459, 716, 773, 784, 882, 1686, 1715, 1749, 1841, 2031, 2076, 2383, 2520, 2533, 3300 and 17 amino acid variations observed in position 5, 54, 78, 90, 95, 152, 153, 239, 258, 262, 572, 583, 614, 684, 677, 795 and 845. Further, phylogenetic analysis was used to uncover the patterns of spread of the virus across the affected countries. Although, certain strains showed patterns of transmission within communities, a vast majority revealed an evident mosaic pattern. Conclusion: The data obtained provides a clear understanding of variations in the nucleotide and translated nucleotide sequences, which can be targeted towards drug designing and to study evolutionary analysis. Keywords: In silico, SARS-CoV-2, Spike protein gene, mutation, Multiple Sequence Alignment, Variation


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Warghane A, Petkar T, Preeyaa S U, Kumari N, Ranjan L. In Silico Characterisation of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) based on the Spike Protein Gene. EJMO. 2021; 5(2): 163-180

Corresponding Author: Ashish Warghane

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In Silico Characterisation of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) based on the Spike Protein Gene . Abstract : Objectives: The Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has been the current global pandemic concern. With a high transmission rate, especially through direct contact, this disease spreads from person to person, and this has in turn led to a huge number of infections on a global scale. Methods: In present study, comparative genomic analysis was performed using 151 gene sequences of the viral spike protein retrieved from NCBI and along with its translated nucleotide sequences using MEGAX software. Variation in the nucleotide and amino acid positions were identified. Results: Our analysis revealed that 22 nucleotide variations observed in positions 13, 141, 162, 233, 284, 328, 455, 459, 716, 773, 784, 882, 1686, 1715, 1749, 1841, 2031, 2076, 2383, 2520, 2533, 3300 and 17 amino acid variations observed in position 5, 54, 78, 90, 95, 152, 153, 239, 258, 262, 572, 583, 614, 684, 677, 795 and 845. Further, phylogenetic analysis was used to uncover the patterns of spread of the virus across the affected countries. Although, certain strains showed patterns of transmission within communities, a vast majority revealed an evident mosaic pattern. Conclusion: The data obtained provides a clear understanding of variations in the nucleotide and translated nucleotide sequences, which can be targeted towards drug designing and to study evolutionary analysis. Keywords: In silico, SARS-CoV-2, Spike protein gene, mutation, Multiple Sequence Alignment, Variation The SARS-CoV-2 (Severe Acute Respiratory SyndromeCoronavirus-2), the causative agent of COVID-19 is found to be similar to Middle East Respiratory SyndromeCoronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV). This virus has led to infecting over 223 countries worldwide with over 133 million (133,552,774) confirmed cases and 2 million (2,894,295) confirmed deaths as of World Health Organisation (WHO) reports on 10th April, 2021.[1] The mortality rate of SARSCoV-2 lies between 1-35% and is similar to SARS-CoV and MERS-CoV during the year 2003 and 2012 respectively.[2] With a higher infectivity rate than its mortality rate, COVID19 finds itself easily unfurling across six continents in the form of droplets, sneezing and cough from one individual to another.[3,4] The disease is primarily characterized by fever, sore throat, common cold, fatigue, lack of smell and taste. People having comorbidity such as heart disease, diabetes or chronic lung disease may further develop severe symptoms including pneumonia and acute respiratory distress syndrome. A few people also develop asymptomatic conditions of the disease.[5,6] The Coronavirus also designated as ‘Severe Acute Respiratory Syndrome Coronavirus 2’ (SARS-CoV-2) is a positivesense single stranded RNA virus belonging to Order Nidovirales, Family Coronaviridae and Subfamily Coronavirinae. [7] The subfamily is further divided as Alphacoronavirus and Betacoronavirus infecting the mammals; and Gammacoronavirus and Deltacoronavirus infecting the birds respectively.[8,9] These viruses have a genome size of 26-32kb encoding 4 structural proteins [spike glycoprotein (S), envelope glycoprotein (E), matrix protein (M), nucleocapsid protein (N)] and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14)] (Fig 1).[10] Their genome is protected through a layer of capsid proteins, or nucleocapsid, which forms a covering. This viral nucleocapsid possesses a helical symmetry, an atypical feature of positive-sense RNA virus which is further surrounded by an envelope glycoprotein layer.[11] Among the 4 structural proteins, spike glycoprotein is highly essential for the virus to enter the host via interaction with host cellular receptors like Angiotensin Converting Enzyme-2 (ACE-2).[12,13] S protein belongs to class I viral transmembrane protein and has 1160 to 1400 amino acids. It is a trimer found on the surface of virus which gives it a crown-like appearance. It has 2 ectodomains, S1 and S2. S1 enables host receptor binding and S2 helps in fusion process. S1 is further subdivided into N-terminal domain (NTD), central receptor binding domain (RBD) and a C-terminal domain (CTD). This S1 CTD has receptor binding motif (RBM) and S1 trimer stalks itself upon the trimeric S2 stalk. There are 27 amino acid substitutions seen in 1273 amino acid stretch sequence. Of these, 6 are from RBD and 4 from RBM at CTD of S domain. These rapid substitutions depict the rapid changes in virus evolution.[14-16] SARS-CoV-2 shares 89% similarity with SARS-CoV and MERS-CoV. SARS-CoV-2 consists of 12 functional open reading frames (ORFs) with the GC content of 38%.[17] The gene order: 5’cap-5’UTR-ORF1a-ORF1b-S-ORF3a-E-M-ORF6a-ORF7a-ORF7b-ORF8-N-ORF10-3’UTR-polyA tail.[18,19] Among these genes, ORF1ab contains the maximum number (21290 nucleotides) of nucleotides than the others. ORF1a-1b are arranged in the form replicase and helicase followed by 4 structural proteins.[20] The whole genome encodes a long polyprotein consisting of accessory proteins of 7096 residues long besides the structural proteins. These accessory proteins enable in viral replication, transcription, protein processing and response to antiviral actions.[21-23] The spike protein is a vital target molecule for the production of DNA and attenuated vaccines, being the SARSCoV-2 key protein responsible for infection, entry into host and pathogenesis. Here, we are performing a sequence analysis of the viral spike protein gene to enable clearer understanding of the regions easily variable both in nucleotide and translated nucleotide sequences. This data could be used to carefully modify those nucleotide or amino acid positions while preparing a remedy that could account for an enhanced protection from the SARS-CoV-2. Therefore, this present study which is carried out to identify the variation, mutations and conserved sequences present in the 151 nucleotide and amino acids of spike protein gene of SARS-CoV-2, is greatly important. Phylogenetic analysis of 151 sequences from different geographical regions worldwide was carried out to recognize the trend of nucleotide and amino acid variation and identify the different strains of SARS-CoV-2 present in studied sequences. Methods Retrieval of Sequences and Alignment A total of 151 complete sequences of spike protein gene of SARS-CoV-2 along with the reference sequence were retrieved from the NCBI Database (www.ncbi.nig.gov) (Table 1). The nucleotide sequences were downloaded in FASTA format ensuring that they were all 3822 bp in length (complete sequences). After retrieving, the sequences were then aligned using Multiple Sequence Alignment program CLUSTAL_W using MEGA X software.[24] The aligned sequence files were used for further analysis. MEGA Software Molecular Evolutionary Genetics Analysis (MEGA) software was initially (in the 1900s) programmed for gene sequence analysis. Its recent version, MEGA X, enables whole genome sequencing (both DNA and protein) via Pairwise or Multiple Sequence Alignment with CLUSTAL_W program. The Molecular Evolutionary Genetics Analysis (MEGA) software provides tools to conduct automatic and manual sequence alignment and includes a large repertoire of programs for Figure 1. Schematic representation of SARS-CoV-2 genome. This figure represents the gene order from 5’ to 3’ end along with the nucleotide length of each gene respectively. The red arrow indicates the three-dimensional view of spike glycoprotein.EJMO 165 assembling sequence alignments, estimating genetic distances and diversities, inferring evolutionary trees, computing time trees, inferring ancestral sequences and testing selection. In addition, this software has a bootstrap tree construction and model selection further enabling phylogenetic analysis. Nucleotide and Amino acid Variation In order to detect nucleotide variations in the spike protein gene sequences, we performed MSA of highly accurate and continuous assemblies of sequences. Variations were then curated manually from aligned sequences by individually hand-picking and using software display tools. The nucleotide variations of each of the 151 sequences are mentioned in Table 2. To overcome the problem of degeneracy of codon, variations in amino acid sequences were also determined and noted with respect to all positions in the alignment by robust analysis. Only those variations that were able to make a credible change in any amino acid residue within the protein sequences were counted and labelled as mutations. Multiple Sequence Alignment (MSA) and Phylogenetic Analysis To understand the homology and evolutionary relationship between these 151 retrieved sequences along with reference genome, we used MEGA X software (www.megasoftware.net)[24] to carry out MSA. This tool was preferred since it is one of the most cited tools used for evolutionary analysis in diverse biological fields. Multiple sequence alignment, following sequence retrieval, was performed using CLUSTAL W of MEGA X software with default parameters. This aligned sequence file was further analysed. Phylogeny was inferred using the Maximum Likelihood Method and the Tamura-Nei Model[25] – for Nucleotide Sequence Alignment, while Maximum Likelihood Method and a JTT matrix-based model[26] – was used for amino acid sequence alignment, both at 1000 bootstrap level in MEGA X. The Phylogenetic Trees thus created for both the alignments were then visualized in interactive Tree of Life (iTOL) (Fig. 2 and Fig. 3).[27] Results Complete 151 gene sequences of the SARS-CoV-2 spike gene along with the reference sequence were retrieved from NCBI Database randomly to avoid sample bias across the globe. Among the 150 sequences considered, partial sequences and incomplete sequences were manually excluded from the analysis to clearly understand the precise variations in nucleotide and translated nucleotide sequences. Of the 151 sequences, 91 sequences were from India, 27 sequences from United States of America (USA), 15 sequences from Japan, 11 sequences from Saudi Arabia, 2 sequences each from Serbia and Germany, 1 sequence each from China, Netherlands and Sri Lanka. While NCBI had over 4000 sequences deposited when we were conducting the study, we confined our analysis to 151 complete sequences since many sequences detected were incomplete and partial. Only the spike gene was retrieved as this was identified and confirmed for pathogenesis. MEGA X software is a user-friendly software enabling the alignment of both DNA and protein sequences. The nucleotide variations and amino acid variation positions selected sequences are mentioned in table 2 and table 3 respectively. The phylogenetic tree, also made using MEGA X software, aids us in understanding the common ancestor and how these sequences varied with time. This in turn permits the study of an evolutionary analysis as well. On performing MSA, we obtained 22 nucleotide variations in positions 13, 141, 162, 233, 284, 328, 455, 459, 716, 773, 784, 882, 1686, 1715, 1749, 1841, 2031, 2076, 2383, 2520, 2533, 3300 (Table 2). We also found 17 amino acid variations in position 5, 54, 78, 90, 95, 152, 153, 239, 258, 262, 572, 583, 614, 684, 677, 795 and 845 (Table 3). This observation reFigure 2. Evolutionary analysis of nucleotide sequence of Spike protein Gene by Maximum Likelihood method. The evolutionary history was inferred by using Maximum Likelihood method and Tamura-Nei model.[25] This analysis involves 151 nucleotide sequences. There was a total of 3822 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.[24]166 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 vealed that N-terminal region of the spike protein gene is more prone to mutations whereas C-terminus region of the spike protein gene was found to be conserved. Out of 17 variations occurred in the studied amino acid sequences, 14 variations were observed in the S1 subunit of spike protein gene. Phylogenetic analysis was carried out using nucleotide and amino acid sequences of spike protein gene of SARS-CoV-2. The phylogenetic tree was built using Maximum Likelihood method and Tamura-Nei model.[25] All the sequences retrieved from NCBI, belonging to different countries were sketched, and grouped into six and three clades based on their respective nucleotide and amino acid sequences (Fig. 2 and Fig. 3). In a closure view of nucleotide based phylogenetic tree, the studied isolates were grouped in 22 clades (Fig. 2). However, 17 clade groups were seen based on the amino acid sequences. As the origin of SARS-CoV-2 and its subsequent rapid transition from an epidemic to pandemic are still clouded in ambiguity, it was interesting to try to identify the pattern of viral spread across various geographical locations. We discovered that, in both the nucleotide and amino acid alignments, the reference sequence (isolated from Wuhan, China) clustered closely with strains from Gujarat and Puerto Rico, followed by several different strains from the USA. Discussion SARS-Coronavirus-2, the unexpectedly relentless virus, has been extending its tentacles all over the world uncaring of borders and confinements. Since the declaration of the disease as a pandemic by WHO, many countries have implemented complete lockdowns, sealing of international borders, and instructing people to step out only during emergencies. Since it is transmissible via contact and respiratory fluids,[30] it has emerged as one of biggest factors posing public health risk. In the first four months of 2021 many countries facing the second wave of virus and found to be more severe than the first one. Coronavirus is also seen to affect the nervous system of individuals.[29] The current speedy transmission and worldwide spread of SARSCoV-2 have raised life-threatening questions about the drastic evolution and its adaptation. The RNA genome of virus is prone to mutations, recombination’s deletions and they are attacking different hosts having diverse strength of the immune response, which is responsible for variation in the genome of the viruses. In the present study, comparative genomic analysis was used to identify the conserved sites and major hotspots. Vaccines designed by considering these variations, could cut losses in terms of time and expenditure that might incur during the vaccine production. Similar studies have also been found, conducted in Dengue virus,[32] Saint Louis encephalitis virus,[33] Rotavirus,[34] H1N1 Influenza A virus,[35] Zika virus[36] and Coronavirus.[37] The viral particle exhibits 76-78% similarity with its ancestor- SARS-CoV.[31] The envelope of SARS-CoV-2 consisting of trimeric spike protein in the S1 domain (14–685 amino acid residues), is responsible for the binding to the ACE2 receptor of the host (Fig. 4). We infer that the major 14 amino acid variations observed in our studied sequences may thus account for the strain variation in the domain (Walls et al., 2020., Yan R et al., 2020). The first step of viral infection is the binding of the virus particle to receptor present of the host cell. The virus has to therefore recognise specific receptor to enter into the host and this being a crucial step and found to be one of the key targets for drug designing. The amino acid variation identified in the present study is useful for drug design, diagnostics and vaccine development programs (Huang et al., 2020). In our study, variations from only the spike gene sequences were identified as the major gene involved pathogenesis and entry of virus in human host. We found 22 nucleotide variations in the positions: 13, 141, Figure 3. Evolutionary analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model.[26] The tree with the highest log likelihood (-3898.48) is shown. Bootstrap Values of more than 50% are represented on branches as grey dots with sizes corresponding to the respective Bootstrap Values. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 151 amino acid sequences (including the Reference Sequence). There were a total of 1273 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.[24]EJMO 167 162, 233, 284, 328, 455, 459, 716, 773, 784, 882, 1686, 1715, 1749, 1841, 2031, 2076, 2383, 2520, 2533, and 3300 (Table 3). To our surprise, many of these nucleotide variations contributed to synonymous variations in aligned amino acid sequences. We found 17 amino acid variations in the positions: 5, 54, 78, 90, 95, 152, 153, 239, 258, 262, 572, 583, 614, 684, 677, 795 and 845 (Table 3) when we translated the nucleotide sequence of spike protein gene into amino acid sequence. The code degeneracy restricts the all-nucleotide variation into the amino acid variations. C- terminal region of the spike protein was found to be conserved and very few variations were observed in this region. Further studies might be required to ensure a clearer understanding especially during vaccine preparation.[39-41] A similar kind of study conducted by the Syed et al., 2020 involved MSA of 320 whole sequences and spike protein sequences and they found 483 new variations in the whole genome in SARS-CoV-2. This included 25 synonymous mutations and 1 deletion in spike protein of SARS-CoV-2. Of these 26 variations, 12 were present in NTD and 6 variations in RBD of spike protein. They also found 22 amino acid variations when compared with SARS-CoV-2, whereas in our study we found 17 variations in the spike proteins gene. This observation revealed the need for screening a larger number spike protein genes of SARS-CoV-2, and might be helpful to understand in variation at both levels i.e., nucleotide and amino acid. The above-mentioned observation shows that this might affect the receptor recognition of virus during host viral interactions and the phylogeny analysis reveals that the present SARS-CoV-2 is closely similar to the bat corona virus. Phylogenetic analysis also reveals that some strains belonging to a particular geographical area (Ahmedabad, Vadodara, Surat and Palanpur from Gujarat, and Saitama, Chiba and Ishikawa from Japan) clustered closely with one another indicating viral spread due to transmission between people in nearby communities. However, similar patterns in other strains were rather rare as other strains from Gujarat and Japan notably clustered with those belonging to Serbia or Heinsberg. Our observations revealed that, a state or country contains more than one type of strains of SARS-CoV-2. Furthermore, most of the strains from a single state (such as Gujarat) or from a single country (USA or Japan), clustered distantly from one another, and instead showed relatedness with strains from other geographical locations. This was interesting to note as the absence of connection among strains from neighbouring regions or countries might suggest that each strain was brought in by the travel of infected individuals to different countries or regions. This in turn might have caused the development of a mosaic pattern of phylogenetic placements. Similar studies reporting mosaic pattern of phylogeographical distribution have also been conducted.[28] Studies on phylogeny help us to comprehend pathogenesis and design potential inhibitors -therapeutic drugs, besides vaccines and anti-viral therapies.[38] The 2017 avian like H1N1 lineages was found to be similar to 2009 pandemic H1N1 lineages in parallel to SARS-CoV-2 being similar to MERS-CoV and SARS-CoV viruses. Such a phylogeny analysis could help to design antiFigure 4. Schematic representation of attachment of the Spike protein of SARS-CoV-2 and ACE-2 receptor on the human lung.168 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 SARS-CoV-2 antibodies with SARS-CoV-2 spike protein particularly targeting the spike protein gene to apprehend the trending variation and cross-reactivity. Consistent studies and constant monitoring of the SARS-CoV-2 spike protein gene is of immense importance for subsequent novel drug development, newer diagnostics and protection against this deadly COVID-19 crisis. Conclusion In the present study, we were able to identify both the nucleotide and amino acid variation in different 22 and 17 positions respectively from 151 SARS-CoV-2 spike protein sequences. Since, these positions occupy the active site of epitope-based vaccines and most drug targets, their inclusion, or careful modifications could be beneficial in many ways. The data extracted in this present study will be useful for the further drug designing and modification, development of serological and molecular based diagnostics tools, evolution and variation studies and could finally be implemented in vaccine development programs. Disclosures Acknowledgments: Authors acknowledge Virtual Internship with Science Leader platform and Dr. Felix Bast for providing us the opportunity to conduct this research work. Authors are also thankful to Faculty of Life Sciences, Mandsaur and Mandsaur University for its motivation and encouragement on our work on SARS-CoV-2. Peer-review: Externally peer-reviewed. Conflict of Interest: None declared. Authorship Contributions: Concept – A.W.; Design – A.W., U.P.S., N.K., L.R., T.P.; Supervision – A.W.; Materials – U.P.S., N.K., U.P.S.; Data collection &/or processing – A.W., U.P.S., N.K., L.R., T.P.; Analysis and/or interpretation – A.W., U.P.S., N.K., L.R., T.P.; Literature search – U.P.S., N.K., L.R., T.P.; Writing – A.W., U.P.S., N.K., L.R., T.P.; Critical review – A.W., U.P.S. References 1. WHO Coronavirus (COVID-19) Dashboard, WHO. Available from-https://www.who.int/emergencies/diseases/novelcoronavirus-2019 (Accessed on 10th April, 2021) 2. 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Lokman SM, Rasheduzzaman M, Salauddin A, Barua R, Tanzina AY, Rumi MH, Hossain MI, Siddiki AMAMZ, Mannan A, Hasan MM. Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach. Infect Genet Evol. 2020 Oct;84:104389. 39. Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020; 181:281–92 e286. 40. Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020; 367:1444–8. 41. Yuan Huang, Chan Yang, Xin-feng Xu, Wei Xu and Shu-wen Liu. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacologica Sinica. 2020. 41:1141–1149.170 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 Table 1. Table indicating the accession number, name of the isolate, name of the gene, location (country, state, district), and the published date of the 150 spike proteins along with the reference sequence at top as extracted from NCBI Database Sr. No Accession No. Isolate Name/number Name of gene Host Country: State: District Date 1. NC_045512.2 Wuhan-Hu-1 S Homo sapiens China 18-07-20 (Ref. Seq.) 2 MT419820 SARS-CoV2/human/USA/PR-CDC-S11/2020 S Homo sapiens Puerto Rico 01-05-20 3 MT419818 SARS-CoV2/human/USA/PR-CDC-S9/2020 S Homo sapiens Puerto Rico 01-05-20 4 MT419815 SARS-CoV2/human/USA/PR-CDC-S6/2020 S Homo sapiens Puerto Rico 01-05-20 5 MT419814 SARS-CoV2/human/USA/PR-CDC-S5/2020 S Homo sapiens Puerto Rico 01-05-20 6 MT419812 SARS-CoV2/human/USA/PR-CDC-S3/2020 S Homo sapiens Puerto Rico 01-05-20 7 MT396266 SARS-CoV2/mink/NLD/1/2020 S Mustela lutreola Netherlands: Milheeze 28-04-20 8 MT459979 SARS-CoV2/human/SRB/Novi Pazar-363/2020 S Homo sapiens Serbia: Novi Pazar 13-05-20 9 MT450872 SARS-CoV2/human/SRB/KV26/2020 S Homo sapiens Serbia 11-05-20 10 MT472624 SARS-CoV2/human/USA/FL-CDC-7619/2020 S Homo sapiens USA: FL 15-05-20 11 MT472626 SARS-CoV2/human/USA/IA-CDC-8200/2020 S Homo sapiens USA: IA 15-05-20 12 MT434799 SARS-CoV2/human/USA/NY-CDC-SURV0444NYC/2020 S Homo sapiens USA: NY 06-05-20 13 MT434785 SARS-CoV2/human/USA/NY-CDC-SURV039NYC/2020 S Homo sapiens USA: NY 06-05-20 14 MT472622 SARS-CoV2/human/USA/MD-CDC-0025/2020 S Homo sapiens USA:MD 15-05-20 15 MT434817 SARS-CoV2/human/USA/NY-CDC-SURV0985NYC/2020 S Homo sapiens USA: NY 06-05-20 16 MT434816 SARS-CoV2/human/USA/NY-CDC-SURV0592NYC/2020 S Homo sapiens USA: NY 06-05-20 17 MT434814 SARS-CoV2/human/USA/NY-CDC-SURV0168NYC/2020 S Homo sapiens USA: NY 06-05-20 18 MT434813 SARS-CoV2/human/USA/NY-CDC-SURV0144NYC/2020 S Homo sapiens USA: NY 06-05-20 19 MT434811 SARS-CoV2/human/USA/NY-CDC-SURV0996NYC/2020 S Homo sapiens USA: NY 06-05-20 20 MT434809 SARS-CoV2/human/USA/NY-CDC-SURV0983NYC/2020 S Homo sapiens USA: NY 06-05-20 21 MT434808 SARS-CoV2/human/USA/NY-CDC-SURV0982NYC/2020 S Homo sapiens USA: NY 06-05-20 22 MT434807 SARS-CoV2/human/USA/NY-CDC-SURV0874NYC/2020 S Homo sapiens USA: NY 06-05-20 23 MT434805 SARS-CoV2/human/USA/NY-CDC-SURV0862NYC/202 S Homo sapiens USA: NY 06-05-20 24 MT434804 SARS-CoV2/human/USA/NY-CDC-SURV0710NYC/2020 S Homo sapiens USA: NY 06-05-20 25 MT434803 SARS-CoV2/human/USA/NY-CDC-SURV0513NYC/2020 S Homo sapiens USA: NY 06-05-20 26 MT434801 SARS-CoV2/human/USA/NY-CDC-SURV S Homo sapiens USA: NY 06-05-20 27 MT370836 SARS-CoV-2/human/USA/NY-PV08436/2020 S Homo sapiens USA: NY 06-08-20 28 MT370831 SARS-CoV-2/human/USA/NY-PV08464/2020 S Homo sapiens USA: NY 06-08-20 29 MT371049 SARS-CoV-2/human/LKA/COV91/2020 S Homo sapiens Sri Lanka 23-04-20 30 MT325597 SARS-CoV-2/human/USA/NV-CDC-0052/2020 S Homo sapiens USA: NV 29-07-20 31 MT630432 SARS-CoV-2/human/SAU/85791C/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 32 MT630431 SARS-CoV-2/human/SAU/85790C/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 33 MT630430 SARS-CoV-2/human/SAU/832279/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 34 MT630429 SARS-CoV-2/human/SAU/86650/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 35 MT630428 SARS-CoV-2/human/SAU/86327/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 36 MT630427 SARS-CoV-2/human/SAU/86267/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 37 MT630425 SARS-CoV-2/human/SAU/85790/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 38 MT630424 SARS-CoV-2/human/SAU/85715/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 39 MT630423 SARS-CoV-2/human/SAU/85613/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 40 MT630422 SARS-CoV-2/human/SAU/42952/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 41 MT630421 SARS-CoV-2/human/SAU/4637/2020 S Homo sapiens Saudi Arabia: Jeddah 17-06-20 42 MT582498 SARS-CoV-2/human/DEU/NRW-02.1/2020 S Homo sapiens Germany: Heinsberg 09-06-20 43 MT607608 SARS-CoV-2/human/IND/GBRC182a/2020 S Homo sapiens India: Ahmedabad 15-06-20 44 MT582494 SARS-CoV-2/human/DEU/NRW-06/2020 S Homo sapiens Germany: Heinsberg 09-06-20 45 MT370960 SARS-CoV-2/human/USA/NY-PV09097/2020 S Homo sapiens USA: NY 23-04-20 46 MT370967 SARS-CoV-2/human/USA/NY-PV09303/2020 S Homo sapiens USA: NY 23-04-20 47 MT675954 SARS-CoV-2/human/IND/GBRC231a/2020 S Homo sapiens India: Surat 29-06-20 48 MT675952 SARS-CoV-2/human/IND/GBRC229b/2020 S Homo sapiens India: Palanpur 29-06-20EJMO 171 Table 1. CONT. Sr. No Accession No. Isolate Name/number Name of gene Host Country: State: District Date 49 MT675950 SARS-CoV-2/human/IND/GBRC229a/2020 S Homo sapiens India: Palanpur 29-06-20 50 MT675951 SARS-CoV-2/human/IND/GBRC230/2020 S Homo sapiens India: Surat 29-06-20 51 MT675945 SARS-CoV-2/human/IND/GBRC228b/2020 S Homo sapiens India: Ahmedabad 29-06-20 52 MT675944 SARS-CoV-2/human/IND/GBRC228a/2020 S Homo sapiens India: Ahmedabad 29-06-20 53 MT675943 SARS-CoV-2/human/IND/GBRC225a/2020 S Homo sapiens India: Savali 29-06-20 54 MT675942 SARS-CoV-2/human/IND/GBRC225b/2020 S Homo sapiens India: Savali 29-06-20 55 MT675940 SARS-CoV-2/human/IND/GBRC223b/2020 S Homo sapiens India: Vadodara 29-06-20 56 MT675941 SARS-CoV-2/human/IND/GBRC224b/2020 S Homo sapiens India: Vadodara 29-06-20 57 MT675939 SARS-CoV-2/human/IND/GBRC222b/2020 S Homo sapiens India: Vadodara 29-06-20 58 MT675938 SARS-CoV-2/human/IND/GBRC224a/2020 S Homo sapiens India: Vadodara 29-06-20 59 MT675937 SARS-CoV-2/human/IND/GBRC223a/2020 S Homo sapiens India: Vadodara 29-06-20 60 MT675933 SARS-CoV-2/human/IND/GBRC222a/2020 S Homo sapiens India: Vadodara 29-06-20 61 MT669322 SARS-CoV-2/human/IND/GBRC203b/2020 S Homo sapiens India: Palanpur 26-06-20 62 MT669321 SARS-CoV-2/human/IND/GBRC203a/2020 S Homo sapiens India: Palanpur 26-06-20 63 MT666042 SARS-CoV-2/human/IND/GBRC221/2020 S Homo sapiens India: Bhuj 25-06-20 64 MT665974 SARS-CoV-2/human/IND/GBRC220/2020 S Homo sapiens India: Mandvi 25-06-20 65 MT665972 SARS-CoV-2/human/IND/GBRC219b/2020 S Homo sapiens India: Kheda 25-06-20 66 MT665970 SARS-CoV-2/human/IND/GBRC219a/2020 S Homo sapiens India: Kheda 25-06-20 67 MT665028 SARS-CoV-2/human/IND/GBRC218b/2020 S Homo sapiens India: Vadodara 25-06-20 68 MT665006 SARS-CoV-2/human/IND/GBRC218a/2020 S Homo sapiens India: Vadodara 25-06-20 69 MT664990 SARS-CoV-2/human/IND/GBRC217b/2020 S Homo sapiens India: Vadodara 25-06-20 70 MT664986 SARS-CoV-2/human/IND/GBRC217a/2020 S Homo sapiens India: Vadodara 25-06-20 71 MT664822 SARS-CoV-2/human/IND/GBRC216b/2020 S Homo sapiens India: Vadodara 25-06-20 72 MT664808 SARS-CoV-2/human/IND/GBRC194b/2020 S Homo sapiens India: Savli 25-06-20 73 MT664807 SARS-CoV-2/human/IND/GBRC194a/2020 S Homo sapiens India: Savli 25-06-20 74 MT664796 SARS-CoV-2/human/IND/GBRC216a/2020 S Homo sapiens India: Vadodara 25-06-20 75 MT607608 SARS-CoV-2/human/IND/GBRC182a/2020 S Homo sapiens India: Ahmedabad 15-06-20 76 MT607611 SARS-CoV-2/human/IND/GBRC183a/2020 S Homo sapiens India: Ahmedabad 15-06-20 77 MT664774 SARS-CoV-2/human/IND/GBRC215/2020 S Homo sapiens India: Bharuch 25-06-20 78 MT664729 SARS-CoV-2/human/IND/GBRC214b/2020 S Homo sapiens India: Surat 25-06-20 79 MT664727 SARS-CoV-2/human/IND/GBRC214a/2020 S Homo sapiens India: Surat 25-06-20 80 MT664209 SARS-CoV-2/human/IND/GBRC210b/2020 S Homo sapiens India: Surat 25-06-20 81 MT664205 SARS-CoV-2/human/IND/GBRC210a/2020 S Homo sapiens India: Surat 25-06-20 82 MT664203 SARS-CoV-2/human/IND/GBRC209b/2020 S Homo sapiens India: Surat 25-06-20 83 MT664202 SARS-CoV-2/human/IND/GBRC209a/2020 S Homo sapiens India: Surat 25-06-20 84 MT664201 SARS-CoV-2/human/IND/GBRC208b/2020 S Homo sapiens India: Surat 25-06-20 85 MT664197 SARS-CoV-2/human/IND/GBRC208a/2020 S Homo sapiens India: Surat 25-06-20 86 MT664172 SARS-CoV-2/human/IND/GBRC207b/2020 S Homo sapiens India: Surat 25-06-20 87 MT664170 SARS-CoV-2/human/IND/GBRC207a/2020 S Homo sapiens India: Surat 25-06-20 88 MT664169 SARS-CoV-2/human/IND/GBRC206/2020 S Homo sapiens India: Surat 25-06-20 89 MT664161 SARS-CoV-2/human/IND/GBRC205b/2020 S Homo sapiens India: Surat 25-06-20 90 MT664143 SARS-CoV-2/human/IND/GBRC205a/2020 S Homo sapiens India: Surat 25-06-20 91 MT664118 SARS-CoV-2/human/IND/GBRC204b/2020 S Homo sapiens India: Palanpur 25-06-20 92 MT664117 SARS-CoV-2/human/IND/GBRC204a/2020 S Homo sapiens India: Palanpur 25-06-20 93 MT635858 SARS-CoV-2/human/IND/GBRC199/2020 S Homo sapiens India: Gujarat, Nadiad 18-06-20 94 MT635856 SARS-CoV-2/human/IND/GBRC202/2020 S Homo sapiens India: Gujarat, Dhanera 18-06-20 95 MT635857 SARS-CoV-2/human/IND/GBRC201b/2020 S Homo sapiensIndia: Gujarat, Mahemdavad18-06-20 96 MT635855 SARS-CoV-2/human/IND/GBRC201a/2020 S Homo sapiensIndia: Gujarat, Mahemdavad18-06-20 97 MT635410 SARS-CoV-2/human/IND/GBRC196/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 98 MT635409 SARS-CoV-2/human/IND/GBRC198/2020 S Homo sapiens India: Gujarat, Nadiad 18-06-20 99 MT635408 SARS-CoV-2/human/IND/GBRC190/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 100 MT635407 SARS-CoV-2/human/IND/GBRC195b/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20172 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 Table 1. CONT. Sr. No Accession No. Isolate Name/number Name of gene Host Country: State: District Date 101 MT635406 SARS-CoV-2/human/IND/GBRC191a/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 102 MT635404 SARS-CoV-2/human/IND/GBRC197/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 103 MT635405 SARS-CoV-2/human/IND/GBRC195a/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 104 MT635403 SARS-CoV-2/human/IND/GBRC188/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 105 MT635397 SARS-CoV-2/human/IND/GBRC187b/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 106 MT635393 SARS-CoV-2/human/IND/GBRC193a/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 107 MT635392 SARS-CoV-2/human/IND/GBRC193b/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 108 MT635391 SARS-CoV-2/human/IND/GBRC191b/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 109 MT635339 SARS-CoV-2/human/IND/GBRC192/2020 S Homo sapiens India: Gujarat, Vadodara 18-06-20 110 MT635328 SARS-CoV-2/human/IND/GBRC185a/2020 S Homo sapiens India: Gujarat, Ahmedabad18-06-20 111 MT635272 SARS-CoV-2/human/IND/GBRC186b/2020 S Homo sapiens India: Gujarat, Ahmedabad18-06-20 112 MT635271 SARS-CoV-2/human/IND/GBRC185b/2020 S Homo sapiens India: Gujarat, Ahmedabad18-06-20 113 MT635269 SARS-CoV-2/human/IND/GBRC186a/2020 S Homo sapiens India: Gujarat, Ahmedabad18-06-20 114 MT635270 SARS-CoV-2/human/IND/GBRC184/2020 S Homo sapiens India: Gujarat, Ahmedabad18-06-20 115 MT608648 SARS-CoV-2/human/IND/GBRC183b/2020 S Homo sapiens India: Ahmedabad 15-06-20 116 MT607618 SARS-CoV-2/human/IND/GBRC176/2020 S Homo sapiens India: Vadodara 15-06-20 117 MT607621 SARS-CoV-2/human/IND/GBRC178a/2020 S Homo sapiens India: Ahmedabad 15-06-20 118 MT607619 SARS-CoV-2/human/IND/GBRC182b/2020 S Homo sapiens India: Ahmedabad 15-06-20 119 MT607620 SARS-CoV-2/human/IND/GBRC180b/2020 S Homo sapiens India: Ahmedabad 15-06-20 120 MT607617 SARS-CoV-2/human/IND/GBRC181a/2020 S Homo sapiens India: Ahmedabad 15-06-20 121 MT607615 SARS-CoV-2/human/IND/GBRC175/2020 S Homo sapiens India: Vadodara 15-06-20 122 MT607616 SARS-CoV-2/human/IND/GBRC173b/2020 S Homo sapiens India: Kalol 15-06-20 123 MT607613 SARS-CoV-2/human/IND/GBRC173a/2020 S Homo sapiens India: Kalol 15-06-20 124 MT607614 SARS-CoV-2/human/IND/GBRC179a/2020 S Homo sapiens India: Ahmedabad 15-06-20 125 MT607612 SARS-CoV-2/human/IND/GBRC171/2020 S Homo sapiens India: Gandhinagar 15-06-20 126 MT607609 SARS-CoV-2/human/IND/GBRC177a/2020 S Homo sapiens India: Ahmedabad 15-06-20 127 LC547528 hCoV-19/Japan/P4-6/2020 S Homo sapiens Japan: Saitama 16-05-20 128 LC547532 hCoV-19/Japan/P5-2/2020 S Homo sapiens Japan: Chiba 16-05-20 129 LC547531 hCoV-19/Japan/P5-1/2020 S Homo sapiens Japan: Chiba 16-05-20 130 LC547530 hCoV-19/Japan/P4-8/2020 S Homo sapiens Japan: Saitama 16-05-20 131 LC547529 hCoV-19/Japan/P4-7/2020 S Homo sapiens Japan: Saitama 16-05-20 132 LC547527 hCoV-19/Japan/P4-5/2020 S Homo sapiens Japan: Saitama 16-05-20 133 LC547526 hCoV-19/Japan/P4-4/2020 S Homo sapiens Japan: Saitama 16-05-20 134 LC547525 hCoV-19/Japan/P4-3/2020 S Homo sapiens Japan: Saitama 16-05-20 135 LC547524 hCoV-19/Japan/P4-2/2020 S Homo sapiens Japan: Saitama 16-05-20 136 LC547523 hCoV-19/Japan/P4-1/2020 S Homo sapiens Japan: Saitama 16-05-20 137 LC547522 hCoV-19/Japan/P3-2/2020 S Homo sapiens Japan: Ishikawa 16-05-20 138 LC547521 hCoV-19/Japan/P3-1/2020 S Homo sapiens Japan: Ishikawa 16-05-20 139 LC547520 hCoV-19/Japan/P2-2/2020 S Homo sapiens Japan: Chiba 16-05-20 140 LC547519 hCoV-19/Japan/P2-1/2020 S Homo sapiens Japan:Chiba 16-05-20 141 LC547518 hCoV-19/Japan/P1/2020 S Homo sapiens Japan: Kochi 16-05-20 142 MT416726 SARS-CoV-2/human/IND/8004/2020 S Homo sapiens India: Pune 01-06-20 143 MT416725 SARS-CoV-2/human/IND/8003/2020 S Homo sapiens India: Pune 01-06-20 144 MT435082 SARS-CoV-2/human/IND/GBRC5/2020 S Homo sapiens India: Ahmedabad 06-05-20 145 MT435086 SARS-CoV-2/human/IND/GBRC9/2020 S Homo sapiens India: Mansa 06-05-20 146 MT435085 SARS-CoV-2/human/IND/GBRC8/2020 S Homo sapiens India: Gandhinagar 06-05-20 147 MT435084 SARS-CoV-2/human/IND/GBRC7/2020 S Homo sapiens India: Ahmedabad 06-05-20 148 MT435083 SARS-CoV-2/human/IND/GBRC6/2020 S Homo sapiens India: Ahmedabad 06-05-20 149 MT435081 SARS-CoV-2/human/IND/GBRC4/2020 S Homo sapiens India: Ahmedabad 06-05-20 150 MT435080 SARS-CoV-2/human/IND/GBRC3/2020 S Homo sapiens India: Ahmedabad 06-05-20 151 MT483560 SARS-CoV-2/human/IND/GBRC81b/2020 S Homo sapiens India: Modasa 19-05-20EJMO 173 Table 2. Table indicating nucleotide variations in the selected 150 viral spike gene sequences along with the reference SARS-COV-2 (Wuhan Hu 1) sequence. Sr. No Isolate name Accession number Variations in nucleotide position of spike gene. 1 Wuhan-Hu-1 NC_045512 882 1841 2 SARS-CoV2/human/USA/PR-CDC-S11/2020 MT419820 882 3 SARS-CoV2/human/USA/PR-CDC-S9/2020 MT419818 882 716 4 SARS-CoV2/human/USA/PR-CDC-S6/2020 MT419815 882 5 SARS-CoV2/human/USA/PR-CDC-S5/2020 MT419814 882 6 SARS-CoV2/human/USA/PR-CDC-S3/2020 MT419812 882 1841 7 SARS-CoV2/mink/NLD/1/2020 MT396266 882 3300 8 SARS-CoV2/human/SRB/Novi Pazar-363/2020 MT459979 882 9 SARS-CoV2/human/SRB/KV26/2020 MT450872 882 10 SARS-CoV2/human/USA/FL-CDC-7619/2020 MT472624 882 11 SARS-CoV2/human/USA/IA-CDC-8200/2020 MT472626 882 773 12 SARS-CoV2/human/USA/NY-CDC SURV0444NYC/2020 MT434799 882 284 13 SARS-CoV2/human/USA/NY-CDC-SURV039NYC/2020 MT434785 882 1841 2533 14 SARS-CoV2/human/USA/MD-CDC-0025/2020 MT472622 882 15 SARS-CoV2/human/USA/NY-CDC-SURV0985NYC/2020 MT434817 882 16 SARS-CoV2/human/USA/NY-CDC-SURV0592NYC/2020 MT434816 882 17 SARS-CoV2/human/USA/NY-CDC-SURV0168NYC/2020 MT434814 882 1841 18 SARS-CoV2/human/USA/NY-CDC-SURV0144NYC/2020 MT434813 882 1841 19 SARS-CoV2/human/USA/NY-CDC-SURV0996NYC/2020 MT434811 882 20 SARS-CoV2/human/USA/NY-CDC-SURV0983NYC/2020 MT434809 882 21 SARS-CoV2/human/USA/NY-CDC-SURV0982NYC/2020 MT434808 882 22 SARS-CoV2/human/USA/NY-CDC-SURV0874NYC/2020 MT434807 882 1841 23 SARS-CoV2/human/USA/NY-CDC-SURV0862NYC/202 MT434805 882 24 SARS-CoV2/human/USA/NY-CDC-SURV0710NYC/2020 MT434804 882 25 SARS-CoV2/human/USA/NY-CDC-SURV0513NYC/2020 MT434803 882 26 SARS-CoV2/human/USA/NY-CDC-SURV0475NYC/2020 MT434801 882 27 hCoV-19/Japan/P4-6/2020 LC547528 882 1686 28 hCoV-19/Japan/P5-2/2020 LC547532 882 29 hCoV-19/Japan/P5-1/2020 LC547531 882 30 hCoV-19/Japan/P4-8/2020 LC547530 882 1686 31 hCoV-19/Japan/P4-7/2020 LC547529 882 1686 32 hCoV-19/Japan/P4-5/2020 LC547527 882 33 hCoV-19/Japan/P4-4/2020 LC547526 882 34 hCoV-19/Japan/P4-3/2020 LC547525 882 35 hCoV-19/Japan/P4-2/2020 LC547524 882 36 hCoV-19/Japan/P4-1/2020 LC547523 882 37 hCoV-19/Japan/P3-2/2020 LC547522 882 38 hCoV-19/Japan/P3-1/2020 LC547521 882 39 hCoV-19/Japan/P2-2/2020 LC547520 882 40 hCoV-19/Japan/P2-1/2020 LC547519 882 41 hCoV-19/Japan/P1/2020 LC547518 882 42 SARS-CoV-2/human/IND/8004/2020 MT416726 882 2031 43 SARS-CoV-2/human/IND/8003/2020 MT416725 882 2031 44 SARS-CoV-2/human/IND/GBRC5/2020 MT435082 882 45 SARS-CoV-2/human/IND/GBRC9/2020 MT435086 882 46 SARS-CoV-2/human/IND/GBRC8/2020 MT435085 882 47 SARS-CoV-2/human/IND/GBRC7/2020 MT435084 882 48 SARS-CoV-2/human/IND/GBRC6/2020 MT435083 882 2076174 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 Table 2. CONT. Sr. No Isolate name Accession number Variations in nucleotide position of spike gene. 49 SARS-CoV-2/human/IND/GBRC4/2020 MT435081 882 50 SARS-CoV-2/human/IND/GBRC3/2020 MT435080 882 51 SARS-CoV-2/human/IND/GBRC81b/2020 MT483560 882 233 52 SARS-CoV-2/human/USA/NY-PV08436/2020 MT370836 882 53 SARS-CoV-2/human/LKA/COV91/2020 MT371049 882 54 SARS-CoV-2/human/USA/NV-CDC-0052/2020 MT325597 882 1841 13 55 SARS-CoV-2/human/USA/NY-PV08464/2020 MT370831 882 268 906 56 SARS-CoV-2/human/SAU/85791C/2020 MT630432 882 57 SARS-CoV-2/human/SAU/85790C/2020 MT630431 882 58 SARS-CoV-2/human/SAU/832279/2020 MT630430 882 59 SARS-CoV-2/human/SAU/86650/2020 MT630429 882 60 SARS-CoV-2/human/SAU/86327/2020 MT630428 882 61 SARS-CoV-2/human/SAU/86267/2020 MT630427 2051 62 SARS-CoV-2/human/SAU/85790/2020 MT630425 882 63 SARS-CoV-2/human/SAU/85715/2020 MT630424 882 64 SARS-CoV-2/human/SAU/85613/2020 MT630423 882 906 1841 65 SARS-CoV-2/human/SAU/42952/2020 MT630422 882 66 SARS-CoV-2/human/SAU/4637/2020 MT630421 882 67 SARS-CoV-2/human/DEU/NRW-02.1/2020 MT582498 882 1841 68 SARS-CoV-2/human/IND/GBRC182a/2020 MT607608 882 69 SARS-CoV-2/human/DEU/NRW-06/2020 MT582494 882 1841 70 SARS-CoV-2/human/USA/NY-PV09097/2020 MT370960 882 71 SARS-CoV-2/human/USA/NY-PV09303/2020 MT370967 882 72 SARS-CoV-2/human/IND/GBRC231a/2020 MT675954 882 162 73 SARS-CoV-2/human/IND/GBRC229b/2020 MT675952 882 162 74 SARS-CoV-2/human/IND/GBRC229a/2020 MT675950 882 141 75 SARS-CoV-2/human/IND/GBRC230/2020 MT675951 882 76 SARS-CoV-2/human/IND/GBRC228b/2020 MT675945 882 77 SARS-CoV-2/human/IND/GBRC228a/2020 MT675944 882 78 SARS-CoV-2/human/IND/GBRC225a/2020 MT675943 882 162 79 SARS-CoV-2/human/IND/GBRC225b/2020 MT675942 882 162 80 SARS-CoV-2/human/IND/GBRC223b/2020 MT675940 882 162 81 SARS-CoV-2/human/IND/GBRC224b/2020 MT675941 882 162 82 SARS-CoV-2/human/IND/GBRC222b/2020 MT675939 882 162 83 SARS-CoV-2/human/IND/GBRC224a/2020 MT675938 882 162 84 SARS-CoV-2/human/IND/GBRC223a/2020 MT675937 882 162 85 SARS-CoV-2/human/IND/GBRC222a/2020 MT675933 882 162 86 SARS-CoV-2/human/IND/GBRC203b/2020 MT669322 882 162 87 SARS-CoV-2/human/IND/GBRC203a/2020 MT669321 882 88 SARS-CoV-2/human/IND/GBRC221/2020 MT666042 882 328 2383 89 SARS-CoV-2/human/IND/GBRC220/2020 MT665974 882 90 SARS-CoV-2/human/IND/GBRC219b/2020 MT665972 882 162 91 SARS-CoV-2/human/IND/GBRC219a/2020 MT665970 882 162 92 SARS-CoV-2/human/IND/GBRC218b/2020 MT665028 882 162 93 SARS-CoV-2/human/IND/GBRC218a/2020 MT665006 882 162 94 SARS-CoV-2/human/IND/GBRC217b/2020 MT664990 882 162 95 SARS-CoV-2/human/IND/GBRC217a/2020 MT664986 882 162 96 SARS-CoV-2/human/IND/GBRC216b/2020 MT664822 882 162 97 SARS-CoV-2/human/IND/GBRC194b/2020 MT664808 882 162EJMO 175 Table 2. CONT. Sr. No Isolate name Accession number Variations in nucleotide position of spike gene. 98 SARS-CoV-2/human/IND/GBRC194a/2020 MT664807 882 162 99 SARS-CoV-2/human/IND/GBRC216a/2020 MT664796 882 162 100 SARS-CoV-2/human/IND/GBRC182a/2020 MT607608 882 101 SARS-CoV-2/human/IND/GBRC183a/2020 MT607611 882 102 SARS-CoV-2/human/IND/GBRC215/2020 MT664774 882 103 SARS-CoV-2/human/IND/GBRC214b/2020 MT664729 882 162 104 SARS-CoV-2/human/IND/GBRC214a/2020 MT664727 882 784 105 SARS-CoV-2/human/IND/GBRC210b/2020 MT664209 882 106 SARS-CoV-2/human/IND/GBRC210a/2020 MT664205 882 162 107 SARS-CoV-2/human/IND/GBRC209b/2020 MT664203 882 108 SARS-CoV-2/human/IND/GBRC209a/2020 MT664202 882 162 109 SARS-CoV-2/human/IND/GBRC208b/2020 MT664201 882 1841 110 SARS-CoV-2/human/IND/GBRC208a/2020 MT664197 882 906 111 SARS-CoV-2/human/IND/GBRC207b/2020 MT664172 882 162 112 SARS-CoV-2/human/IND/GBRC207a/2020 MT664170 882 162 113 SARS-CoV-2/human/IND/GBRC206/2020 MT664169 882 114 SARS-CoV-2/human/IND/GBRC205b/2020 MT664161 882 162 115 SARS-CoV-2/human/IND/GBRC205a/2020 MT664143 882 162 116 SARS-CoV-2/human/IND/GBRC204b/2020 MT664118 882 162 117 SARS-CoV-2/human/IND/GBRC204a/2020 MT664117 882 118 SARS-CoV-2/human/IND/GBRC199/2020 MT635858 882 455 119 SARS-CoV-2/human/IND/GBRC202/2020 MT635856 882 120 SARS-CoV-2/human/IND/GBRC201b/2020 MT635857 882 162 121 SARS-CoV-2/human/IND/GBRC201a/2020 MT635855 882 455 122 SARS-CoV-2/human/IND/GBRC196/2020 MT635410 882 123 SARS-CoV-2/human/IND/GBRC198/2020 MT635409 882 906 1841 124 SARS-CoV-2/human/IND/GBRC190/2020 MT635408 882 125 SARS-CoV-2/human/IND/GBRC195b/2020 MT635407 882 126 SARS-CoV-2/human/IND/GBRC191a/2020 MT635406 882 127 SARS-CoV-2/human/IND/GBRC197/2020 MT635404 882 128 SARS-CoV-2/human/IND/GBRC195a/2020 MT635405 882 2520 129 SARS-CoV-2/human/IND/GBRC188/2020 MT635403 882 130 SARS-CoV-2/human/IND/GBRC187b/2020 MT635397 882 131 SARS-CoV-2/human/IND/GBRC193a/2020 MT635393 882 132 SARS-CoV-2/human/IND/GBRC193b/2020 MT635392 882 162 133 SARS-CoV-2/human/IND/GBRC191b/2020 MT635391 882 134 SARS-CoV-2/human/IND/GBRC192/2020 MT635339 882 135 SARS-CoV-2/human/IND/GBRC185a/2020 MT635328 882 136 SARS-CoV-2/human/IND/GBRC186b/2020 MT635272 882 1749 137 SARS-CoV-2/human/IND/GBRC185b/2020 MT635271 882 138 SARS-CoV-2/human/IND/GBRC186a/2020 MT635269 882 139 SARS-CoV-2/human/IND/GBRC184/2020 MT635270 882 140 SARS-CoV-2/human/IND/GBRC183b/2020 MT608648 882 141 SARS-CoV-2/human/IND/GBRC176/2020 MT607618 882 142 SARS-CoV-2/human/IND/GBRC178a/2020 MT607621 882 143 SARS-CoV-2/human/IND/GBRC182b/2020 MT607619 882 144 SARS-CoV-2/human/IND/GBRC180b/2020 MT607620 882 145 SARS-CoV-2/human/IND/GBRC181a/2020 MT607617 882 1749 146 SARS-CoV-2/human/IND/GBRC175/2020 MT607615 882 176 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 Table 2. CONT. Sr. No Isolate name Accession number Variations in nucleotide position of spike gene. 147 SARS-CoV-2/human/IND/GBRC173b/2020 MT607616 882 148 SARS-CoV-2/human/IND/GBRC173a/2020 MT607613 882 149 SARS-CoV-2/human/IND/GBRC179a/2020 MT607614 882 1715 150 SARS-CoV-2/human/IND/GBRC171/2020 MT607612 882 459 151 SARS-CoV-2/human/IND/GBRC177a/2020 MT607609 882 1715EJMO 177 Table 3. Table depicting the positions of the corresponding amino acid variations from the selected 150 spike gene sequences along with the reference (Wuhan-Hu-1) sequence at the top Sr. No Isolate Name Accession Number A.A. Variations 1 Wuhan-Hu-1 NC_045512 614 2 SARS-CoV2/human/USA/PR-CDC- S11/2020 MT419820 3 SARS-CoV2/human/USA/PR-CDC- S9/2020 MT419818 239 4 SARS-CoV2/human/USA/PR-CDC- S6/2020 MT419815 5 SARS-CoV2/human/USA/PR-CDC- S5/2020 MT419814 6 SARS-CoV2/human/USA/PR-CDC- S3/2020 MT419812 614 7 SARS-CoV2/mink/NLD/1/2020 MT396266 8 SARS-CoV2/human/SRB/Novi Pazar- 363/2020 MT459979 9 SARS-CoV2/human/SRB/KV26/2020 MT450872 10 SARS-CoV2/human/USA/FL-CDC- 7619/2020 MT472624 11 SARS-CoV2/human/USA/IA-CDC- 8200/2020 MT472626 258 12 SARS-CoV2/human/USA/NY-CDC SURV0444NYC/2020 MT434799 95 13 SARS-CoV2/human/USA/NY-CDC- SURV039NYC/2020 MT434785 614, 845 14 SARS-CoV2/human/USA/MD-CDC-0025/2020 MT472622 15 SARS-CoV2/human/USA/NY-CDC- SURV0985NYC/2020 MT434817 16 SARS-CoV2/human/USA/NY-CDC- SURV0592NYC/2020 MT434816 17 SARS-CoV2/human/USA/NY-CDC- SURV0168NYC/2020 MT434814 614 18 SARS-CoV2/human/USA/NY-CDC- SURV0144NYC/2020 MT434813 614 19 SARS-CoV2/human/USA/NY-CDC- SURV0996NYC/2020 MT434811 20 SARS-CoV2/human/USA/NY-CDC-SURV0983NYC/2020 MT434809 21 SARS-CoV2/human/USA/NY-CDC- SURV0982NYC/2020 MT434808 22 SARS-CoV2/human/USA/NY-CDC- SURV0874NYC/2020 MT434807 614 23 SARS-CoV2/human/USA/NY-CDC- SURV0862NYC/202 MT434805 24 SARS-CoV2/human/USA/NY-CDC- SURV0710NYC/2020 MT434804 25 SARS-CoV2/human/USA/NY-CDC- SURV0513NYC/2020 MT434803 26 SARS-CoV2/human/USA/NY-CDC- SURV MT434801 27 hCoV-19/Japan/P4-6/2020 LC547528 28 hCoV-19/Japan/P5-2/2020 LC547532 29 hCoV-19/Japan/P5-1/2020 LC547531 30 hCoV-19/Japan/P4-8/2020 LC547530 31 hCoV-19/Japan/P4-7/2020 LC547529 32 hCoV-19/Japan/P4-5/2020 LC547527 33 hCoV-19/Japan/P4-4/2020 LC547526 34 hCoV-19/Japan/P4-3/2020 LC547525 35 hCoV-19/Japan/P4-2/2020 LC547524 36 hCoV-19/Japan/P4-1/2020 LC547523 37 hCoV-19/Japan/P3-2/2020 LC547522 38 hCoV-19/Japan/P3-1/2020 LC547521 39 hCoV-19/Japan/P2-2/2020 LC547520 40 hCoV-19/Japan/P2-1/2020 LC547519 32 hCoV-19/Japan/P1/2020 LC547518 33 hCoV-19/Japan/P4-4/2020 LC547526 34 hCoV-19/Japan/P4-3/2020 LC547525 35 hCoV-19/Japan/P4-2/2020 LC547524 36 hCoV-19/Japan/P4-1/2020 LC547523 37 hCoV-19/Japan/P3-2/2020 LC547522 38 hCoV-19/Japan/P3-1/2020 LC547521 39 hCoV-19/Japan/P2-2/2020 LC547520 40 hCoV-19/Japan/P2-1/2020 LC547519178 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 Table 3. CONT. Sr. No Isolate Name Accession Number A.A. Variations 41 hCoV-19/Japan/P1/2020 LC547518 42 SARS-CoV-2/human/IND/8004/2020 MT416726 677 43 SARS-CoV-2/human/IND/8003/2020 MT416725 677 44 SARS-CoV-2/human/IND/GBRC5/2020 MT435082 45 SARS-CoV-2/human/IND/GBRC9/2020 MT435086 46 SARS-CoV-2/human/IND/GBRC8/2020 MT435085 47 SARS-CoV-2/human/IND/GBRC7/2020 MT435084 48 SARS-CoV-2/human/IND/GBRC6/2020 MT435083 49 SARS-CoV-2/human/IND/GBRC4/2020 MT435081 50 SARS-CoV-2/human/IND/GBRC3/2020 MT435080 51 SARS-CoV-2/human/IND/GBRC81b/2020 MT483560 78 52 SARS-CoV-2/human/USA/NY- PV08436/2020 MT370836 53 SARS-CoV-2/human/LKA/COV91/2020 MT371049 54 SARS-CoV-2/human/USA/NV-CDC- 0052/2020 MT325597 5, 614 56 SARS-CoV-2/human/USA/NY- PV08464/2020 MT370831 90 57 SARS-CoV- 2/human/SAU/85790C/2020 MT630431 58 SARS-CoV- 2/human/SAU/832279/2020 MT630430 59 SARS-CoV2/human/SAU/86650/2020 MT630429 60 SARS-CoV- 2/human/SAU/86327/2020 MT630428 61 SARS-CoV- 2/human/SAU/86267/2020 MT630427 684 62 SARS-CoV- 2/human/SAU/85790/2020 MT630425 63 SARS-CoV- 2/human/SAU/85715/2020 MT630424 64 SARS-CoV- 2/human/SAU/85613/2020 MT630423 614 65 SARS-CoV- 2/human/SAU/42952/2020 MT630422 66 SARS-CoV-2/human/SAU/637/2020 MT630421 67 SARS-CoV-2/human/DEU/NRW- 02.1/2020 MT582498 614 68 SARS-CoV- 2/human/IND/GBRC182a/2020 MT607608 69 SARS-CoV-2/human/DEU/NRW- 06/2020 MT582494 614 70 SARS-CoV-2/human/USA/NY- PV09097/2020 MT370960 71 SARS-CoV-2/human/USA/NY- PV09303/2020 MT370967 72 SARS-CoV- 2/human/IND/GBRC231a/2020 MT675954 54 73 SARS-CoV-2/human/IND/GBRC229b/2020 MT675952 54 74 SARS-CoV-2/human/IND/GBRC229a/2020 MT675950 75 SARS-CoV-2/human/IND/GBRC230/2020 MT675951 76 SARS-CoV-2/human/IND/GBRC228b/2020 MT675945 77 SARS-CoV- 2/human/IND/GBRC228a/2020 MT675944 78 SARS-CoV- 2/human/IND/GBRC225a/2020 MT675943 54 79 SARS-CoV-2/human/IND/GBRC225b/2020 MT675942 54 80 SARS-CoV- 2/human/IND/GBRC223b/2020 MT675940 54 81 SARS-CoV-2/human/IND/GBRC224b/2020 MT675941 54 82 SARS-CoV-2/human/IND/GBRC222b/2020 MT675939 54 83 SARS-CoV-2/human/IND/GBRC224a/2020 MT675938 54 84 SARS-CoV-2/human/IND/GBRC223a/2020 MT675937 54 85 SARS-CoV-2/human/IND/GBRC222a/2020 MT675933 54 86 SARS-CoV-2/human/IND/GBRC203b/2020 MT669322 54 87 SARS-CoV-2/human/IND/GBRC203a/2020 MT669321 88 SARS-CoV-2/human/IND/GBRC221/2020 MT666042 795 89 SARS-CoV-2/human/IND/GBRC220/2020 MT665974 90 SARS-CoV-2/human/IND/GBRC219b/2020 MT665972 54 91 SARS-CoV- 2/human/IND/GBRC219a/2020 MT665970 54EJMO 179 Table 3. CONT. Sr. No Isolate Name Accession Number A.A. Variations 92 SARS-CoV-2/human/IND/GBRC218b/2020 MT665028 54 93 SARS-CoV-2/human/IND/GBRC218a/2020 MT665006 54 94 SARS-CoV-2/human/IND/GBRC217b/2020 MT664990 54 95 SARS-CoV-2/human/IND/BRC217a/2020 MT664986 54 96 SARS-CoV-2/human/IND/GBRC216b/2020 MT664822 54 97 SARS-CoV-2/human/IND/GBRC194b/2020 MT664808 54 98 SARS-CoV-2/human/IND/GBRC194a/2020 MT664807 54 99 SARS-CoV-2/human/IND/GBRC216a/2020 MT664796 54 100 SARS-CoV-2/human/IND/GBRC182a/2020 MT607608 101 SARS-CoV-2/human/IND/GBRC183a/2020 MT607611 102 SARS-CoV-2/human/IND/GBRC215/2020 MT664774 103 SARS-CoV-2/human/IND/GBRC214b/2020 MT664729 54 104 SARS-CoV-2/human/IND/GBRC214a/2020 MT664727 262 105 SARS-CoV-2/human/IND/GBRC210b/2020 MT664209 106 SARS-CoV-2/human/IND/GBRC210a/2020 MT664205 54 107 SARS-CoV-2/human/IND/GBRC209b/2020 MT664203 108 SARS-CoV-2/human/IND/GBRC209a/2020 MT664202 54 109 SARS-CoV-2/human/IND/GBRC208b/2020 MT664201 614 110 SARS-CoV-2/human/IND/GBRC208a/2020 MT664197 111 SARS-CoV-2/human/IND/GBRC207b/2020 MT664172 54 112 SARS-CoV-2/human/IND/GBRC207a/2020 MT664170 54 113 SARS-CoV-2/human/IND/GBRC206/2020 MT664169 114 SARS-CoV-2/human/IND/GBRC205b/2020 MT664161 54 115 SARS-CoV-2/human/IND/GBRC205a/2020 MT664143 54 116 SARS-CoV-2/human/IND/GBRC204b/2020 MT664118 54 117 SARS-CoV-2/human/IND/GBRC204a/2020 MT664117 118 SARS-CoV-2/human/IND/GBRC199/2020 MT635858 152 119 SARS-CoV-2/human/IND/GBRC202/2020 MT635856 120 SARS-CoV-2/human/IND/GBRC201b/2020 MT635857 54 121 SARS-CoV-2/human/IND/GBRC201a/2020 MT635855 152 122 SARS-CoV-2/human/IND/GBRC196/2020 MT635410 123 SARS-CoV-2/human/IND/GBRC198/2020 MT635409 614 124 SARS-CoV-2/human/IND/GBRC190/2020 MT635408 125 SARS-CoV-2/human/IND/GBRC195b/2020 MT635407 126 SARS-CoV-2/human/IND/GBRC191a/2020 MT635406 127 SARS-CoV-2/human/IND/GBRC197/2020 MT635404 128 SARS-CoV-2/human/IND/GBRC195a/2020 MT635405 129 SARS-CoV-2/human/IND/GBRC188/2020 MT635403 130 SARS-CoV-2/human/IND/GBRC187b/2020 MT635397 131 SARS-CoV-2/human/IND/GBRC193a/2020 MT635393 132 SARS-CoV-2/human/IND/GBRC193b/2020 MT635392 54 133 SARS-CoV-2/human/IND/GBRC191b/2020 MT635391 134 SARS-CoV-2/human/IND/GBRC192/2020 MT635339 135 SARS-CoV-2/human/IND/GBRC185a/2020 MT635328 136 SARS-CoV-2/human/IND/GBRC186b/2020 MT635272 583 137 SARS-CoV-2/human/IND/GBRC185b/2020 MT635271 138 SARS-CoV-2/human/IND/GBRC186a/2020 MT635269 139 SARS-CoV-2/human/IND/GBRC184/2020 MT635270 140 SARS-CoV-2/human/IND/GBRC183b/2020 MT608648 129 SARS-CoV-2/human/IND/GBRC176/2020 MT607618180 Warghane et al., In Silico Characterisation of SARS-CoV-2 based on the Spike Protein Gene / doi: 10.14744/ejmo.2021.52103 Table 3. CONT. Sr. No Isolate Name Accession Number A.A. Variations 130 SARS-CoV-2/human/IND/GBRC187b/2020 MT635397 141 SARS-CoV-2/human/IND/GBRC193a/2020 MT635393 142 SARS-CoV-2/human/IND/GBRC178a/2020 MT607621 143 SARS-CoV-2/human/IND/GBRC182b/2020 MT607619 144 SARS-CoV-2/human/IND/GBRC180b/2020 MT607620 145 SARS-CoV-2/human/IND/GBRC181a/2020 MT607617 583 146 SARS-CoV-2/human/IND/GBRC175/2020 MT607615 147 SARS-CoV-2/human/IND/GBRC173b/2020 MT607616 148 SARS-CoV-2/human/IND/GBRC173a/2020 MT607613 149 SARS-CoV-2/human/IND/GBRC179a/2020 MT607614 572 150 SARS-CoV-2/human/IND/GBRC171/2020 MT607612 153 151 SARS-CoV-2/human/IND/GBRC177a/2020 MT607609 57

EJMO & EJMO