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 where 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 analysis on Fig. 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.
Even though 11 patients SARS CoV-2 genome samples from Ethiopia were grouped as in same genetic distance as it marked in red color of tree, it is reflected that sub family member of EPI ISL 1897884, EPI ISL 1899281 and EPI ISL 1899047 were clustered with same sub-group and genotype association to SARS CoV-2 Nigeria, Tunisia, Gabon and Reunion patients samples as of yellow color of a tree node as it is illustrated on Fig. 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 the next 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 3 samples were absolutely different and categorize as GR clad distributions which consist of EPI_ISL_1899281, EPI_ISL_1899047 and EPI_ISL_1897884 were confirmed as a new strains of VUI202012/01 GRY (B.1.1.7). Pango linage B.1.1.7 was first detected in UK and now circulating more than 134 countries including Ethiopia.
We had also performed spike glycoprotein mutational ancestral relationship against to Wuhan genome and SARS. 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 Fig. 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 Fig. 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 shown that how the spike proteins changed over the time as compared to the previous coronavirus and probable that will change rigorously.
SARS CoV 2 Spike dynamic mutational changes and its antigenicity
The sum total of 14 SARS CoV-2 genome 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. Especially, the changes on S gene or Spike proteins those will have effect on site of receptor binding and able to alter host receptors or antigenicity was considered for this analysis. Essentially 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 and none structural S13T(N-term) M1229I(C-term) as it has depicted on Fig. 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 mutational changes those happened in Ethiopia exclusively such as Spike T478X, Spike H69X, Spike, N440K and Spike D614X.
Therefore, the genetic mutation changes is expected vary depend open the immune response of the host cells and contumely fluctuations across each patients reaction. Though most mutational could be as nonthreatening, the spike glycoproteins mutational changes of Spike_D614G help strengthen the viral survival capability . N501Y mutation might be lead 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 to 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 give us sufficient of 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 that searching of conserved domain of spike protein is one of the technique 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 were 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 enable to attach on the receptor cells perfectly . 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 on Fig. 3. As a result of changes in the receptor domain affects its adhesiveness of affinity and the virus is more likely to be transmitted to humans easy as compare to the previous coronavirus. Annotated Spike protein (Ethiopia-QSM35588.1) structural modeling and ACE2 confirmation was done through modeler and taken its analogues crystallographic structure from 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 viral receptor binding domain and i-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 Fig. 3B & 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 .
Specifically, the mutational changes on PI_ISL_1898258 samples was detected as N440K amino acid changes and expected that which will determine the notarization of human antibody and vaccine escape mechanism because as it was illustrated on Fig. 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. This indicates how the viral infection could re-evasive the community through go through the existence antibodies or vaccines. Consequently, the total confirmed cases and number of death increase as per the national COVID − 19 dash board as it has shown the graph on Fig. 3 (F&G).