Phylogenetic Tree Analysis Related to Precursor
To study the genetic evolutionary relationship of the virus of that entered into Ethiopia, the evolutionary phylogenic tree analysis were conducted using MEGA using neighbor-joining statistical approaches and Poisson model along phylogeny test of 1000 bootstrap replication methods and found out that bat coronavirus RaTG13: MN996532.1 which were previously isolated from Yunnan province was considered as the nearest ancestor family to all Ethiopian SARS-CoV-2 followed by pangolin bat-SL-CoVZC45: MG772933.1 and bat-SL-CoVZXC21: MG772934.1 respectively. However, (MERS: KT806053.1 and MK564474.1) coronavirus was disclosed as a genetic distance as inferred on Figure 1A. This finding provisionally took a scientific evidence as per [10, 11], reported Rhinolophus affinis was consider a natural reservoir of the coronavirus and supposed to a source of future epidemic.
Nevertheless 11 patients SARS-CoV-2 genome samples from Ethiopia were grouped as in same genetic distance as it was marked in red color of the tree, it was reflected that sub family member of EPI ISL 1897884, EPI ISL 1899281 and EPI ISL 1899047 were clustered and genotype association to SARS-CoV-2 Nigeria, Tunisia, Gabon and Reunion patients samples as of yellow color of a tree node as it was illustrated on figure 1A phylogenetic tree.
Thus, it has theoretical to be the sample from 4 of them EPI_ISL_1169919, EPI_ISL_1170958, EPI_ISL_1170957 and EPI_ISL_1170956 categorized as clade of other (O) and it has not defined yet, whereas 7 of them were classified as GH clad distributions which include EPI_ISL_1899485, EPI_ISL_1899485, EPI_ISL_1898814, EPI_ISL_1898554, EPI_ISL_1898258, EPI_ISL_1897648, EPI_ISL_1897646 and EPI_ISL_1897644, but the rest 3 samples were absolutely different and categorize as GR clad distributions which including EPI_ISL_1899281, EPI_ISL_1899047 and EPI_ISL_1897884 were confirmed as a new varaints of VUI202012/01/GRY (B.1.1.7). Pango linage B.1.1.7 was first detected in the UK and is now circulating in more than 134 countries, including Ethiopia.
We performed spike glycoprotein mutational ancestral relationship against to wild Wuhan genome and SARS-like-CoV. It has proven that the gradual mutational changes on spike protein in related its ancestors. Accordingly the GASAID, 2020, the first 4 genome samples of (EPI_ISL_1169919; EPI_ISL_1170956; EPI_ISL_1170957 and EPI_ISL_1170958 there was at about 30 amino acid changes along 97.9% identity against to bat/Yunnan/RaTG13/2013 whereas 232 aa changed with 81.36% identity in contradiction of SARS-like/Bat/Nanjing/SL-CoVZXC21/2015 and 226 aa changed with 81.86% against to SARS-like/Bat/Nanjing/SL-CoVZC45/2017 respectively, but only 4 aa was altered with 99.6 % identity of hCoV-19/Wuhan/WIV04/2019 on Figure 1 (A1, B1, C1 and D1); whereas the second round 10 genome samples of EPI_ISL_1899485; EPI_ISL_1899281; EPI_ISL_1899047; EPI_ISL_1898814; EPI_ISL_1898554; EPI_ISL_1898258; EPI_ISL_1897884; EPI_ISL_1897648; EPI_ISL_1897646; EPI_ISL_1897644 had about 33 amino acid changes along 97.4% identity against to bat/Yunnan/RaTG13/2013 whereas 234 aa changed with 81.2% identity in contradiction of SARS-like/Bat/Nanjing/SL-CoVZXC21/2015 and 228 aa changed with 81.7% against to SARS-like/Bat/Nanjing/SL-CoVZC45/2017 respectively on Figure 1 (A2, B2, C2 and D2) this implies that the mutational changes on structural spike was before a decades and continue to at present SARS-CoV-2. This finding showed that how the spike proteins changed over time as compared to the previous coronavirus and is probable that they will change rigorously.
SARS-CoV-2 Spike Dynamic Mutational Changes and its Antigenicity
The sum total of 14 SARS-CoV-2 genomes from Ethiopia was sequenced and deposited in GISAID datasets as of April/2021 [12, 13]. And a number of structural and none structural mutational changes were evaluated. In particular, the changes on Spike proteins that will have an effect on site of receptor binding and able to alter host receptors or antigenicity were considered for this analysis. Essentially, a 21 number of notable mutational variations were recorded that will have probability antigenicity effects and found on structural termini including G142A;Y144del(143); E180Q; E309Q; P330L; N440K; N501Y; A520S; A570D; D614G; P681H(674); T716I; A771S; S939F; S982A; T1006I; D1118H; K1073N; A1078S while none structural S13T(N-term); M1229I(C-term) as it had depicted on Figure 2. However, Spike-D614G, N501Y, Spike_M1229I, NSP12_P323L mutation were reflected as high prevalence of SARS-CoV-2 and circulating in Ethiopia. Some of the mutational changes that happened in Ethiopia exclusively, such as Spike-T478X, Spike-H69X, Spike-N440K and Spike-D614X.
Therefore, the genetic mutation changes are expected to vary depending on the immune response of the host cells and contumely fluctuations across each patient's reaction. Though most mutations could be nonthreatening, the spike glycoprotein mutational changes of Spike_D614G help strengthen the viral survival capability [14]. The N501Y mutation might lead to the viral phenotypic character and its fusion or degradability of the receptor cells, whereas NSP5-S284G Non-structural protein-5 plays in the viral genome replication into the host for the formation of viral factories.
RBD of Spike Conserved Region and mAb Neutralizations Against Spike
It is important to investigate the function of the virus Receptor Binding Domain (RBD) and its motif and the targeted receptor host cells because the integration of molecular amino acid peptides and gene machinery gives us sufficient information about the mechanism of entry of virus and evasion mode of action into receptor cells and to know the mechanism of inhibitor drugs or to develop antibody neutralization and its vaccinations. So, searching for conserved domain of spike protein is one of the techniques to reveal the molecular evolution of genes and organisms. It has confirmed that the N-terminal domain (NTD) and C- terminal domain (CTD) of the S1 & S2 subunit of the Spike (S) proteins as reference QSM35600.1 accession number the sample was taken from one of Ethiopian patients were conserved from beta coronaviruses in the sarbecovirus subgenera (B lineage), and use the C-domain to bind their receptors with higher binding affinity than that of the previous SARS and MERS coronavirus and binding polypeptide sites of RBD and RBM has also noted. It enables itself to attach on the receptor cells perfectly [15]. Having to the conserved domain finding, S spike of SARS-CoV-2 of our sample (Ethiopia QSM35600.1) of fusion peptide site is highly conserved to pangolin coronavirus (QIQ54048.1), bat-SL-CoVZXC21, (AVP78042.1), HKU3 (Q3LZX1.1), SARS CoV-2 (PDB: 6VSB_A and 6ACC_A, 2021). However, exclusively SARS-CoV-2 has a functional polybasic (furin) cleavage site (R-arginine/S-threonine), which is absent in SARS coronavirus in figure 3. As a result, changes in the receptor domain affect its adhesiveness of affinity and the virus is more likely to be transmitted to humans easily as compared to the previous coronavirus. Annotated Spike protein (Ethiopia-QSM35588.1) structural modeling and ACE2 confirmation was done through modeler and taken its analogue crystallographic structure from the protein data bank (PDB: 6acc, EM 3.6 Angstrom) with RBD and Spike glycoprotein (PDB: 6acj, EM 4.2 Angstrom) in complex with host cell receptor ACE2. The interaction of the viral receptor binding domain and ACE-2 was revealed using molecular docking simulation through pyMol software. It has confirmed that the virus has four binding residues of Spike receptor binding sites, which are THR-486, TYR-436, ASN-473 and TYR-475 on figure 3 B & D. The interaction between Spike and ACE2 may be altered because of residual changes on the receptor binding sites and it could govern the responsibility of viral attachment and fusion activities [16].
Specifically, the mutational changes on PI_ISL_1898258 sample was detected as N440K amino acid changes and expected which will determine the neutralization of human antibody and vaccine escape mechanism because, as it was illustrated in figure 3 (B1 & B2) 7k8t PDB pyMol analysis ASN-440 glycoproteins of SARS-CoV 2 interact with SER-52 antibody might change the mode of action entirely and have antigenic-drift effects. This indicates how the viral infection could re-evasive the community through go through the existence of antibodies or vaccines. Consequently, the total confirmed cases and number of deaths increased as per the national COVID-19 dash board as it has shown the graph in figure 3 (F & G).