So far round 7000000 people have died between 2019–2023 due to deadly Delta, Alpha and to some extent Beta and Gamma lineages of SARS-CoV-2 (1, 2). However, Mutation, Deletion and Insertion in omicron coronaviruses have reduced the viral tire and disease severity (3). Still, NCBI Virus Database is depositing omicron COVID-19 sequences every day demonstrating that COVID-19 era has not been ended yet. Coronaviruses (family, Coronaviridae) are enveloped viruses with a largest positive sense, single-stranded RNA genome of 30kb. On genetic and antigenic criteria, CoVs have been organised into three groups: α-CoVs, β-CoVs, and γ-CoVs (4). Coronaviruses primarily infect birds, mammals and human, causing a variety of lethal respiratory diseases resembling the common cold, to lower respiratory tract infections such as bronchitis, pneumonia, and even severe acute respiratory syndrome (SARS) (5, 6). The Covid-19 (SARS-CoV-2) infects human only. The virus enters cells through ACE2 receptor-mediated endocytosis. The receptor ACE2, was abundant in lungs AT2 alveolar epithelial cell as well as cells in the kidney, nose, heart and blood vessels (7, 8).
SARS-CoV-2 had structural proteins (S, M, N, E) at the 3’- end and two polyproteins, ORF1ab, ORF1a (7096aa and 4405aa) were coded in same reading frame from 5’ of 2/3 of the genome. 7096aa) which were degraded into sixteen (nsp1-nsp16) non-structural proteins (9). The ORF1ab generates sixteen functional peptides proteolytically: Nsp1(1-180aa), Nsp2(181-818aa), Nsp3(819-2763aa), Nsp4(2764-3263aa), Nsp5(3264-3569aa), Nsp6(3570-3859aa), Nsp7(3860-3942aa), Nsp8(3943-4140aa), Nsp9(4141-4253aa), Nsp10(4265—4392aa), Nsp11(4393-4400aa), Nsp12(4401-5324aa), Nsp13(5325-5925aa), Nsp14(5926-6462aa), Nsp15(6453-6798aa) and Nsp16(6799-7096aa) (10). The functions of the most sixteen enzymes were reported: Trans-activator (nsp1), RNA topoisomerase (nsp2), two proteases (nsp3 and nsp5), RNA-dependent RNA polymerase (nsp12), RNA helicase and capping methyltransferase (nsp13), nucleases (nsp14 and nsp15) and methyl transferases (nsp16) and accessory proteins (nsp7, nsp8, nsp9 and nsp10) (11–18). The ORF1ab protein was reported as 7096 − 7092 AAs in different variants. The spike protein (1273 AAs) is a trimeric class 1 transmembrane glycoprotein and it’s RBD domain (335–515 aa) acts as receptor binding domain to bind ACE-2 receptor of host lung cells for virus entry (19). The spike protein 1–13 AA acts as signal peptide and the S1 subunit (14–685 AAs) containing RBD domain and S2 subunit (686 to 1273 AAs) are important due to furine cleavage. Spike protein also contains fusion contact peptide (788–806 AA) as well as two hepta-peptide (HPPHCPC) repeats at 1163 and 1213 positions (20). The ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF9 and ORF10 small proteins also coded from 3’ end of the SARS-CoV-2 genome and have roles in regulating cellular genes (21–26).
Spike protein in COVID-19 greatly varied in different lineages. Spike protein is 1273aa in Wuhan but it has changed in Alpha lineage to 1270 AA due to deletions of 69HV and 145Y positions; in Delta variant spike is 1271aa due to 156FR deletion only. Spike protein of omicron BA.1 variant is 1270aa due to 69HV, 143VYY and 212L deletions as well as 215EPE three amino acid insertion (27–30). The Spike protein of omicron BA.4 and BA.5 corona viruses is 1268 AA due to deletions of 24LPP and 69HV. Spike protein of omicron BA.2 has 1270 AAs due to 24LPP deletion but no 69HV and 143VYY deletions or 215EPE insertion (3, 10). The 69HV spike deletion found in B.1.1.7 first but also acquired in omicron BA.1, BA.4 and BA.5 variants but not in omicron BA.2 variant. Among the other structural proteins N-protein (419 AAs) binds to leader RNA of replicating corona virus and also regulates host-pathogen interactions. Three AAs deletion (31ERS) was found in N-protein (416 AAs) of all omicron corona viruses (BA.1/2/4/5) and was very useful for diagnostics. Three amino acid deletions (3675SGF) were found in ORF1ab protein (nsp6 protein region) of Alpha and Omicron BA.2 and BA.5 (ORF1ab = 7093) but at the same region 3674LSG deletion as well as extra 2083S deletion were found in omicron BA.1 corona virus (ORF1ab = 7092 AA). Interestingly, no ORF1ab deletions in notorious Delta variant (ORF1ab = 7096 AAs). Whereas, a three amino acids 141KSF deletion was found in omicron BA.4 variant only (ORF1ab = 7090 AAs) and such change was utilized to detect BA.4 omicron variant (31, 32). Further, D614G mutation was detected in all variants since March, 2021 ongoing and such mutation increased 80% higher transmission (33). The N501Y spike mutation was appeared first in Alpha (B.1.1.7) variant but also located in omicron variants BA.1, BA.4, and BA.5 followed by recent BF.1, BQ.1, XBB.1, JN.1, HV.1 and FL.1 lineages and sub subvariants (34). The N501Y mutation increased transmission by more than 20–50% as well as immune escape. The P4715L ORF1ab mutation at RdRP was found in all variants since March, 2020 similar to D614G mutation. As N501Y and D614G both mutations appeared in omicron BA.1, BA.4 and BA.5, such viruses gained more spread than alpha, delta as well as BA.2 corona viruses but gave mild pathogenicity due to 25 more mutations in the spike and 26nt 3’-LTR deletion (20, 26). The BA.2.12.1 lineage gained L452Q mutation facilitating immune-escape and high infectivity. However, BA.2.75.2 sub-lineage carrying additional R346T, F486S and D1199N mutations in the spike protein and gained more mutations and rearrangement to produce XBB variant. The BA.4.6 variant with R346T and N658S mutations was one of the dominating variants (26). Delta variant contained L452R immune-escape mutation which also gained by omicron BA.1 as well as BA.4 and BA.5 variants. The BA.4 and BA.5 variants also gained important F486V mutation to produce immune escape similar to L452R mutation. A single mutation (E484K) present in SARS-CoV-2 Beta (B.1.351) and Gamma (P.1) lineages may alter the neutralising activity of convalescent (infected with previous COVID-19 strains) and post-vaccination polyclonal serum. Delta (B.1.617.2) variant has no N501Y mutation and spread of such viruses were diminished since 2021. The P681R mutation in Delta variant was replaced by P681H in all omicron variants (BA.1/2/4/5). The Gamma (P.1 or B.1.1.28.1) variant had 3675LSG deletion in the ORF1ab protein (nsp6 region) and such information was used to make DelORF1ab3675-P1 oligonucleotide specific for P.1 variant (35).
Data analysis detected one amino acid deletion (140Y=TAT; 145Y in B.0) in spike in BA.4.6, BQ.1.5, BQ.1.8, BQ.1.14, BQ.1.1.5, XBB.1 as well as related AZ.3, BU.1, BW.1, CR.2, CP.1 and CQ.1 subvariants. But Y140 deletion was not detected in BA.2.75, BF.7, XBD, BQ.1, BQ.1.1, BQ.1.2, BQ.1.6, BQ.1.10, BQ.1.12, BQ.1.16, BQ.1.19, BQ.1.22, BQ.1.1.1, BQ.1.1.4, BQ.1.1.12 and related BK.1, BN.1, BM.1.1.1, BR.2, BU.1, CA.1, CD.2, CH.1.1 subvariants (36). Spike protein mutations were rampant in omicron coronaviruses. The 91% nucleotides changes in spike protein of BQ.1 variant was resulted in AA changes whereas only 52% nucleotides changes resulted in AAs changes in ORF1ab polyprotein (37). The spike N460K and K444T mutations in BQ.1 may be important driving force for immune-escape similar to F486S and N480K mutations in BA.2.75 subvariant and related XBB.1 subvariant. Further, the R346T mutation as found in BA.4.6 and BF.7 was regained in the spike of BQ.1.1 and BA.2.75.2 or related recent lineages CH.1, BM.1 and CA.1 to enhance immune escape and infectivity (> 80%). The L452R and F486V spike mutations were main drivers of Omicron BA.2 conversion to BA.4 and BA.5 in presence of 69HV deletion and 30nt deletion in 3’-UTR. Whereas 24LPP spike deletion and 3675SGF ORF1ab protein deletion were found in all Omicron viruses including BQ.1 and XBB.1.
In USA, Wuhan D614G first peak occurred between March-August, 2020, Alpha (B.1.1.7) 2nd peak between January-June, 2021 followed by 3rd peak of Delta (B.1.617.2, AY.X) between June to December, 2021 (27). Since last week of December, 2022 4th peak of Omicron BA.1 variant (B.1.1.519) spread was evident followed by BA.2 variant spread in April, 2022. From June-July, 2022, omicron BA.4 and BA.5 variants are increasing worldwide (28). From November 2022 we found the spread of BQ.1 subvariants and from March 2023, we find spread of XBB.1 lineage. In August 2023, we detected more than 448 249RWMD spike insertion sequences spread in the USA and Europe. But such lineage was not prominent at the end of 2023 (37). Where as in December 2023 new sub-subvariants like XBB.1.16, XBB.1.5, EG.5.1, HV.1, FL.1.5, JN.1 and JD.1.1 were the major omicron coronaviruses sequences were deposited in the NCBI virus database (29, 30). Similarly, we detected ORF7a protein deletions in BQ.1 variant and ORF8 termination codon mutations in XBB.1 lineage as well as 141KSF, 82GHV and 82GHVMV deletions in the nsp1 protein of omicron BA.4 variant (22, 24, 26). All these changes caused recent corona viruses weak and non-life threatening with very low viral titer as compared to Alpha and Delta variants (36, 37). In this communication, we detected a continuous deletion and insertion in the spike of new corona virus variants like JN.1 and XBB.1.5.103 (38, 39). We discussed the potential issues of such changes for its pathogenicity and worldwide transmission as also reported in many newspapers recently.