A Method of Identification of SARS-CoV-2 Variant Using NCBI BLAST-2 100% Homology Search with Specific Oligonucleotides Selected at the Deletion Boundaries of S, N, ORF7a, ORF8 and ORF1ab Proteins

doi:10.21203/rs.3.rs-2082525/v1

Genomic sequencing of many SARS-CoV-2 variants with higher transmission and immune-escape were reported due to point mutations and deletions. Thus, whether a newly sequenced SARS-CoV-2 belongs to Alpha, Beta, Gamma, Delta, or Omicron (BA.1, BA.2, BA.4 and BA.5) variants must be known. We multi-aligned the different Spike, ORF1ab and Nucleocapsid proteins of those corona virus variants and detected different lineage specific deletions and point mutations. Different COVID-19 sequences were aligned with CLUSTAL Omega software and oligonucleotides from deletion boundary were selected. BLAST search using those oligonucleotides clearly predicted the specific variant type with 100% homology and was very useful for new corona virus sequence characterization. Selection of sub-variants were done by oligonucleotides selected at the specific point mutation boundaries leading to amino acid change. COVID-19 variant status was not reported in most published corona virus sequences and this method would be very useful application to understand the nature of expected prognosis of corona virus infected patients in less technology-equipped countries.

Virology

SARS-CoV-2

Variant identification

Deletion boundary

Oligonucleotide

BLAST-2 search

Since the December 2019 Wuhan corona virus (severe acute respiratory syndrome virus or SARS-CoV-2) has caused 6.4 million deaths worldwide. It caused many point mutations and deletions creating dominant forms like alpha, beta, delta and very recently omicron [1–3]. COVID-19 is a large positive-sense RNA virus with a compact 29,980 nucleotides-long genome. It is related to six different corona viruses like CoV-229E, CoV-HKU1, CoV-OC43, CoV-NL63, SARS-CoV and MERS-CoV. It had structural proteins (S, M, N, E) at the 3’- end and 5’ two (ORF1ab, ORF1a) very large poly-proteins (2/3 of the genome; in same reading frames) which were degraded into sixteen (nsp1-nsp16) non-structural proteins [4] including RNA topoisomerase (nsp2) [5], two proteases (nsp3 and nsp5) [6, 7], RNA-dependent RNA polymerase (nsp12) [8], RNA helicase (nsp13) [9], nucleases (nsp15) [10] and methyl transferases (nsp16) [11] (figure-1). ORF1ab protein was reported as 7096 − 7092 AA in different variants where Wuhan corona virus was 7096 AA. Spike protein (1273 AA) 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 [12]. S 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. S protein also contains fusion contact peptide (788–806 AA) as well as two hepta-peptide (HPPHCPC) repeats at 1163 and 1213 positions [13]. RNA sequencing clearly established many S-protein deletion variants with epidemic spread at different time points since December, 2019. In USA Wuhan-D614G first peak 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. 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/BA.5 variants are increasing worldwide. Spike protein in COVID-19 alpha is 1270 AA due to deletions of H⁶⁹V⁷⁰ and Y¹⁴⁵ positions but delta variant has E¹⁵⁶F¹⁵⁷ deletion only (S = 1271 aa). Spike protein of omicron BA.1 variant has H⁶⁹V⁷⁰, V¹⁴³Y¹⁴⁴Y¹⁴⁵ and L²¹² deletions as well as ²¹⁵EPE three amino acid insertion but no ²⁴LPP deletion (1270aa) [14]. Spike protein of omicron BA.4 and BA.5 corona viruses are 1268 AA due to deletions of L²⁴P²⁵P²⁶ and H⁶⁹V⁷⁰ but no L²¹² deletion or ²¹⁵EPE insertion. S protein of omicron BA.2 has 1270 AAs due to L²⁴P²⁵P²⁶ deletion but no H⁶⁹V⁷⁰ and V¹⁴³Y¹⁴⁴Y¹⁴⁵ deletions or E²¹⁵P²¹⁶E²¹⁷ insertion. H⁶⁹V⁷⁰ deletion found in B.1.1.7 also acquired in BA.1/4/5 but not in BA.2. Among the other structural proteins N-protein (419 AAs) binds to leader RNA of replicating corona virus and also regulates host-pathogen interactions. Three AA deletions (E³¹R³²S³³) were found in N-protein (416 AAs) of all omicron corona viruses (BA.1/2/4/5 and BE.3) and was very useful for diagnostics [14–16]. Three amino acid deletions (S³⁶⁷⁵G³⁶⁷⁶F³⁶⁷⁷) were found in ORF1ab protein (nsp6 protein region) of alpha and omicron BA.2/BA.5 (ORF1ab = 7093) but at the same region L³⁶⁷⁴S³⁶⁷⁵G³⁶⁷⁶ deletion as well as extra ²⁰⁸³S deletion were found in omicron BA.1 corona virus (ORF1ab = 7092 AA) but no such deletions in delta variant (ORF1ab = 7096 AA). Whereas, extra three amino acids (K¹⁴¹S¹⁴²F¹⁴³) deletions were found in omicron BA.4 variant only (ORF1ab = 7090 AAs) and such change was utilized to detect BA.4 omicron variant. Further, D⁶¹⁴G mutations were detected in all variants and such mutation increased 80% higher transmission. N⁵⁰¹Y mutation was appeared first in alpha variant but also located in omicron variants BA.1/BA.4/BA.5 but not in BA.2 and such mutation increased transmission by more than 20% as well as immune escape [17–20]. 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. Delta variant contained L⁴⁵²R immune-escape mutation which also gained by omicron BA.1 as well as BA.4 and BA.5 variants. BA.4 and BA.5 variants also gained important F486V mutation [21]. However, BA.2.12.1 lineage recently gained L⁴⁵²Q mutation facilitating immune-escape and high infectivity [22–24]. 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 are diminishing recently. P⁶⁸¹R mutation in delta variant was replaced by P⁶⁸¹H in all omicron variants (BA.1/2/4/5). Gamma (P.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. We will analyzed deeply the genomes of different lineages of corona virus (COVID-19) as discussed above to get lineage specific primer.

Multi-alignment of different COVID-19 proteins and genome sequences (www.ncbi.nlm.nih.gov/nucleotide or /protein) were done by Multalin software for protein analysis and CLUSTAL Omega software for protein and DNA analyses. Known COVID-19 lineages were selected from NCBI SARS-CoV-2 Database. Deletion boundaries were selected for specific variant and same position in the genome sequence was selected for oligonucleotides. NCBI BLAST was done with oligonucleotides selecting sequence parameter 10, 50, 100, 200 or 5000 to check the abundant of oligonucleotide specific COVID-19 sequence in the Database. Align with two sequence were done with BLAST Seq-2 sequence homology search between two sequences and parameter selected “highly similar sequence” or “somewhat similar sequence”. 100% homology defined a correct lineage of COVID-19 and mutation or deletion could be found with selecting “some what similar sequences” in the NCBI BLAST web portal (www.ncbi.nlm.nih.gov/blast). Because we found mutational variation within same lineages (omicron BA.4) due to error, we multi-aligned many same lineages and truthful major similar sequence class was assigned for further use as standard.

For oligonucleotide selection of sub-lineages, we seq-2 BlastN searched two sequences (BA.5.0 vs BA.5.1) putting accession numbers. The total mutation numbers were obtained and each matched 60nt sequence was BlastX searched to get amino acid sequence and mismatched coding amino acid (1, 2 or 3 reading frames). Minimum 51nt in same reading frame (17 AA) was required for BlastX search which would be 52nt for 2nd reading frame or 53nt if 3rd reading frame. Usually, 1–2 mutation(s) with amino acid change occurred (ATG = ATT; Methionine to Isoleucine) from 8–10 total mutations being most mutations were silent (GTT = GTG = Valine; TGC = TGT = Cysteine). After getting coding mutated amino acid, we have to select the amino acid position. We also found that BlastX search with few 60 nt from COVID-19 sequence did not produce any data possibly due to TGA like termination codon in the sequence. As for example, the sequence (position-3196nt) 5’-ACA AGA CGG CAG TGA GGA CAA TCA GAC AAC TAC TAT TCA AAC AAT TGT TGA GGT TCA ACC-3’ would not give amino acid sequence. Similar non-coding sequences were found at the 21533nt region (5’-AAC AAC TAA ACG AAC AAT GTT TGTT TTT CTT GTT TTA TTG CCA CTA GTC TCT AGT CAG TG-3’) and 195nt region (5’-ATC ATC AGC ACA TCT AGG TTT CGT CCG GGT GTG ACC GAA AGG TAA GAT GGA GAG CCT TG -3’ and also 25294nt region (5’-ACA CAT AAA CGA ACT TAT GGA TTT GTT TAT GAG AAG CTT CAC AAT TGG AAC TGT AAC TTT-3’).

For getting amino acid change, we looked Amino Acid Codon Table in 1,2,3 reading frames (as for example here, 3rd reading frame; AGG = ATG; Arginine to Methionine). We found during BlastX search that all ORF1ab AA sequences were more than 7096 AA (7097–7108 AA) due to error data (we found many xx notations instead AA single letter code and “GTG” AA insertion sequence and no “SGF” AA deletion in such protein sequences). In such case, sequence of an authentic lineage (as for example, BA.5.0 = ON658807) would be seen for ORF1ab AA mutated position as we knew that total AA of ORF1ab protein varied with different corona virus variants. However, we also changed the window parameter from 100 to 5000 to get many similar sequences and in that case, you should get a sequence with proper total AA of ORF1ab but it would take time for analysis to get correct AA position. We also checked the date of isolation of the virus as date also predicted type of variant in the database (as for example, for BA.5.x lineages should be isolated recently e.g. June-September, 2022 but Delta variant between June to September, 2021, while BA.2.x lineages were isolated little bit earlier e. g. March-July, 2022). For selection of omicron BA.5.0 oligonucleotide, BA.4.0 sequence aligned with BA.5.0 and to get oligonucleotides for other BA.5.x lineages, omicron BA.5.0 sequence was aligned individually with BA.5.1, BA.5.1.1, BA.5.2, BA.5.2.1 etc. Sometime during oligonucleotide selection seq-2 alignment was not proper as the codon did not specified the correct AA as selected by multi-alignment of protein. That is due to similar other sequence nearby and you have to adjust the proper adjustment of deleted nucleotide with AA codon to-and-fro of the sequence as happened for oligonucleotide DelS341-Beta. Such problem was also seen in Fig. 2 for ²¹²L deletion followed by ²¹⁵EPE insertion region of spike proteins multi-alignment (Multalin software). All oligonucleotides are in reading frame except DelORF8-Delta which had TAA termination codon at the end. Minimum 18 nucleotides (or higher) was required for NCBI BLAST search and you could reduce oligonucleotide length from both side of red-labelled base. During Blast-2 search with your sequence, always see the homology pattern by pressing alignments bottom to check if your oligo was not fragmented giving two fragments with 100% cover and 100% similarity (a problem of the Blast-2 search result in the sub-window).

Collection of specific variants of COVID-19 proteins (Spike, ORF1ab and Nucleocapsid) and multi-alignment

Important genomic sequences COVID-19 variants were obtained from NCBI SARS-CoV-2 Database and Spike (S), ORF1ab, Nucleocapsid (N), Envelope (E) as well as ORF3a and ORF8 protein amino acids sequences were collected and aligned separately (Table-1A). Figure-2 showed the alignment regions with deleted amino acids of different corona virus variants. We demonstrated the deletion of L²⁴P²⁵P²⁶ amino acid sequences in omicron BA.2, BA.4 and BA.5 corona viruses but not in omicron BA.1 variant as well as early Wuhan, Alpha, Beta, Gamma and Delta corona viruses. 2^nd H⁶⁹V⁷⁰ two amino acids deletion was demonstrated in B.1.1.7 as well as in omicron BA.1, BA.4 and BA.5 variants but not in omicron BA.2, Gamma and Delta variants. 3^rd V¹⁴³Y¹⁴⁴Y¹⁴⁵three amino acids deletion demonstrated only in Omicron BA.1 variant but one amino acid deletion (Y¹⁴⁵) was specific for B.1.1.7 variant (figure-2). 4^th major F¹⁵⁶R¹⁵⁷two amino acids deletion was found specific for Delta variants (AY.125; B.1.617.2). Next, we found ²¹²L single amino acid deletion followed by ²¹⁵EPE three amino acids insertion in Omicron BA.1 variant only but not in BA.2, BA.4 and BA.5 omicron corona viruses or in other early corona virus variants (Alpha, Beta, Gamma and Delta). We also detected Beta variant specific ³⁴¹LAL three amino acids spike protein deletion. Such multi-alignment helped to find genomic sequences of SARS-CoV-2 viruses across the deletion boundary useful for detection of specific type of corona virus after RNA sequencing from the sample collected from corona virus infected patients.

Figure-3 demonstrated the multi-alignment region of hypervariable region with 29 mutations in the RBD domain of omicron BA.1 variant useful for detection. In figure-4, we showed the dominant N⁵⁰¹Y and D⁶¹⁴G mutations regions found in most highly infectious variants of human corona viruses since March-June, 2020. Next, we reported the E³¹R³²S³³three amino acids deletion in N-protein of all omicron corona viruses (BA.1, BA.2, BA.4, BA.5) but not in Alpha, Beta, Gamma and Delta variants (figure-5). Multi-alignment of ORF1ab protein (~7096aa length) of different corona viruses showed K¹⁴¹S¹⁴²F¹⁴³ three amino acids deletion in nsp1 protein specific for omicron BA.4 variant only (figure-6). In figure-7, we demonstrated the region of P⁴⁷¹⁵L point mutation found in all highly infectious variants (Alpha, Beta, Gamma, Delta and Omicron) that was occurred since 2020. Figure-8 showed the Envelope protein mutation (P⁷¹L) in corona virus Beta variant and figure-9 showed the Q⁵⁷H mutation in ORF3a protein of the same variant.

TABLE-1A: Important accession numbers of different SRAS-CoV-2 lineages used in the study for multi-alignment and Blast-2 homology search
Variant type	Lineage type	Accession number	Date of isolation of virus	Homology	No of Base change	No of Spike AA change
Wuhan	B	NC_045512	00-12-2019	100%	0	0
Alpha	B.1.1.7	MZ821602	30-07-2021	99.8%	60	11
Beta	B.1.351	MZ433432	01-02-2021	99.85%	44	11
Gamma	P.1	MZ010005	01-04-2021	99.82%	54	14
Delta	B.1.617.2	OM542166	19-12-2021	99.82%	55	11
Zeta	P.2	MZ008795	01-04-2021	99.91%	28	3
Omicron	B.1.1.529	OM003685	27-11-2021	99.66%	102	40
Omicron	BA.1	OM542730	14-01-2022	99.66%	102	39
Omicron	BA.1.1	ON394519	15-02-2022	99.64%	108	40
Omicron	BA.1.1.18	ON386282	28-12-2021	99.65%	103	40
Omicron	BA.2	OM305333	28-12-2021	99.8%	60	12
Omicron	BA.2.3	ON386456	07-04-2022	99.7%	82	31
Omicron	BA.2.12.1	ON623660	07-05-2022	99.84%	32	33
Omicron	BA.2.48	ON957923	24-06-2022	99.58%	127	42
Omicron	BA.2.75	ON999338	03-07-2022	99.63%	110	37
Omicron	BA.4	ON907393	14-06-2022	99.62%	114	35
Omicron	BA.4.1	ON991461	05-07-2022	99.53%	141	35
Omicron	BA.4.2	OP257529	29-07-2022	99.62%	114	34
Omicron	BA.4.6	ON990652	02-07-2022	99.52%	144	36
Omicron	BA.5	ON658807	19-05-2022	99.66%	101	36
Omicron	BA.5.1	OP237923	15-06-2022	99.57%	129	34
Omicron	BA.5.2	ON999606	28-06-2022	99.64%	106	35
Omicron	BA.5.2.1	OP238223	22-06-2022	99.58%	130	34
Omicron	BA.5.5	OP237919	20-06-2022	99.58%	124	35
Omicron	BA.5.6	ON999542	27-06-2022	99.66%	102	34
Omicron	BA.5.8	OP327107	05-07-2022	99.56%	130	35
Omicron	BA.5.10	OP332021	13-08-2022	99.64%	107	35
Omicron	BF.1	OP335015	18-08-2022	99.63%	110	35
Omicron	BF.1.1	OP313354	05-07-2022	99.67%	98	21

TABLE-1B: Deletions in the spike (S), ORF1ab and N proteins of corona virus (SARS-CoV-2) variants
Variant	Length S	Deletion in the S	Insertion S	ORF1ab length	N length
Wuhan (B)	1273 AA	no	no	7096 AA	419 AA
Alpha (B.1.1.7)	1270 AA	⁶⁹HV ¹⁴⁵Y	no	7093 AA	419 AA
Beta (B.1.315)	1273 AA	²⁴¹LAL	no	7096 AA	419 AA
Delta (B.1.617.2)	1271 AA	¹⁵⁶FR	no	7096 AA	419 AA
Omicron BA.1	1270 AA	⁶⁹HV ¹⁴³VYY ²¹²L	²¹⁵EPE	7092 AA	416 AA
Omicron BA.2	1270 AA	²⁴LPP	no	7093 AA	416 AA
Omicron BA.4	1268 AA	²⁴LPP ⁶⁹HV	no	7090 AA	416 AA
Omicron BA.5	1268 AA	²⁴LPP ⁶⁹HV	no	7093 AA	416 AA

Note: Three amino acid deletions (³⁶⁷⁵SGF) were found in ORF1ab protein (nsp6 coding region) of alpha and omicron BA.2/4/5 (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) but no such deletions in delta variant (ORF1ab=7096 AA). Omicron BA.4 has extra ¹⁴¹KSF deletion in the nsp1 coding region. All omicron variants had ³¹ERS deletion in the N protein.

Selection of variant specific oligonucleotides at the deleted boundary

We aligned the genomic sequences of Wuhan, Alpha, Beta, Delta, Omicron (BA.1, BA.2, BA.4, BA.5) COVID-19 viruses and respective deleted boundaries were detected and selected for lineage specific oligonucleotides (figure-10, A-I; Table-1B).

DelNsp2-BA.4 oligonucleotide was selected at ~685nt region of the corona virus genome and was specific for omicron BA.4 lineage which was spreading very recently (figure-10A). The oligonucleotide detected only BA.4 variant of omicron corona virus as demonstrated by BLAST search (data not shown). In figure-10B, we showed the selection of DelS31-BA2/4/5 oligonucleotide (~21632nt region) which was specific for Omicron variants like BA.2, BA.4 and BA.5 which were spreading very fast recently. In figure -10C we showed the selection oligonucleotide at the H⁶⁹V⁷⁰deletion boundary (~21764nt region) specific for Alpha variant as well as omicron variants BA.1, BA.4, BA.5. Next, we selected DelS145-B.1.1.7 oligonucleotide (~21992nt region) specific for Alpha variant, DelS143-BA.1 oligonucleotide (~21986nt region) specific for omicron BA.1 variant and DelS157-Delta oligonucleotide (~22028nt region) specific for Delta (B.1.817.2 and AY.3) oligonucleotides as shown in figure-10D. Then, we selected DelS212-BA.1 oligonucleotide (~22193nt region) specific for omicron BA.1 variant and such oligonucleotide also contained high specific ²¹⁵EPS insertion sequence (figure-10E). Then, we selected from the genome alignment DelN-Omicron oligonucleotide (~28361nt region) specific for all omicron variants (figure-10F). We further selected few specific oligonucleotides based on dominant point mutations like N501Y and D614G variants which increased corona virus transmission to ~20% and ~80% as demonstrated in figure-11A and figure-11B. All oligonucleotide sequences were presented in Table-2A/2B/2C.

Then what could be the procedure when one got a new corona virus sequence? Presently, 4^th phase of omicron infection was going on in the USA and few AY.X and B.1.817.2 variants could be also detectable now [14]. In flow diagram figure-12A, we demonstrated the procedure of new corona virus variant identification and in figure-12B we presented data of such identification of an omicron BA.2 variant. In our opinion, the identification method is applicable for all laboratories performing COVID-19 sequencing. In figure-12C, we presented the procedure for selection the selection of BA.4.0 specific oligonucleotide. In Figure-12D we showed the genomic deletions of BA.4 variant as compared to Wuhan virus. In Figure-12E, we showed the multi-alignment of BA.4-lineages leading to oligonucleotide selection of BA.4.1 where as in Fig.12F we presented details flow-chart methodology of selection of BA.4.1 oligonucleotide. Similar procedure for the selection of oligonucleotides for recently isolated omicron BK.1 variant was given in figure-12G. We are unable to present the data for all oligonucleotides selection due to page limit.

TABLE-2A: Oligonucleotides selected at the deletion boundaries of COVID-19
No	Oligo name	Position	Sequence of the variant specific oligonuclotide
P1	DelNsp1-BA.4	671	5’-TACGGCGCCGATCTAGACTTAGGCGACGAG-3’
P2	DelORF1ab3675-Gamma	11273	5’-GTTGATACTAGTTTGAAGCTAAAAGACTGTGTT-3’
P3	DelS24-BA.2/4/5	21612	5’-ATCTTATAACCAGAACTCAATCATACACTAATTCTTTCA-3’
P4	DelS69-Alpha-BA.4/5	21745	5’-TGTTACTTGGTTCCATGCTATCTCTGGGACCAATGGTAC-3’
P5	DelS69-BA.1	21745	5’-TGTTACTTGGTTCCATGTTATCTCTGGGACCAATGGTACT-3’
P6	DelS143-BA.1	21969	5’-GTAATGATCCATTTTTGGACCACAAAAACAACAAAAGTTGGA-3’
P7	DelS145-Alpha	21973	5’-TGATCCATTTTTGGGTGTTTACCACAAAAACAACAAAA-3’
P8	DelS157-Delta	22008	5’-ACAAAAGTTGGATGGAAAGTGGAGTTTATTCTAGTG-3’
P9	DelS212-BA.1	22182	5’-ACACGCCTATTATAGTGCGTGAGCCAGAAGATCTCCCTCA-3’
P10	DelS241-Beta	22269	5’-AGGTTTCAAACTTTACATAGAAGTTAT-3’
P11	DelORF8-Delta	28227	5’-CATGACGTTCGTGTTGTTTTAATCTAAACGAACAAAC-3’
P12	DelN31-Omicron	28341	5’-CAACTGGCAGTAACCAGAATGGTGGGGCGCGATCAAAAC-3’
P13	DelORF1ab82-BA.6	495	5’-GAACTGCACCTCATGTGGTTGAGCTGGTAG-3’
P14	DelORF7a-BA.7	27553	5’-TTTGCACTGACTTGCTTGCTTTTGCTTGTC-3’

TABLE-2B: Oligonucleotides selected for higher transmission and immune-escape
No	Oligo name	Position	Sequence of the oligonucleotides
P15	MutN501Y-Alpha	23048	5’-GGTTTCCAACCCACTTATGGTGTTGGTTACCAACC-3’
P16	MutN501Y-Omicron	23048	5’-GGTTTCCGACCCACTTATGGTGTTGGTCACCAACC-3
P17	MutD614G-all	23392	5’-TCTTTATCAGGGTGTTAACTGCACAGAAGTCC-3’
P18	MutHyper-Omicron	23034	5’-CCTTTACGATCATATAGTTTCCGACCCACTTATGG-3’
P19	MutS-Gamma	21552	5’-CAGTGTGTTAATTTTACAAACAGAACTCAATTACCCTCTGCATACAC-3’

TABLE-2C: Oligonucleotides selected st the variant specific point mutation boundary for sub and sub-sub variants
No	Oligo name	Position	Sequence of the oligonucleotides
P20	MutE71-Beta	26441	5’-ATTCTTCTAGAGTTCTTGATCTTCTGGTCT-3’
P21	MutORF3a57-Beta	25550	5’-TTGCTGTTTTTCATAGCGCTTCCAAAATCA-3’
P22	MutS701-BA.1.1-Beta	23650	5’-TCACTTGGTGTAGAAAATTCAGTT-3’
P23	MutORF1ab1887-BA.1.1	5912	5’-AAATTGGATGGTATTGTTTGTAC-3’
P24	MutORF1ab2554-BA.1.1.18	7910	5’-TGTGAAGAATCATCTGTAAAATCAGCGTCT-3’
P25	MutS446-BA.2	22886	5’-GATTCTAAGGTTAGTGGTAATTATAAT-3’
P26	MutORF1ab2910-BA.2.3	8978	5’-TACACTGACTTTGTAACATCAGCGTG-3’
P27	MutE12-BA.2.75	26263	5’-GAAGAGATAGGTGCGTTAATAGTTAAT-3’
P28	MutS245-BA.2.76	22289	5’-GCTTTACATAGAAGTAATTTGACTCCTGGTGAT-3’
P29	MutN151-BA.4.0	28712	5’-GGCACCCGCAATTCTGCTAACAATGCT-3’
P30	MutS3-BA.4.1	21538	5’- CGAACAATGTTTGGTTTTCTTGTTT-3’
P31	MutORF1ab1636-BA.4.2	5156	5’-GCTTTTGAGTACTACCAAACAACTGATCCTAGT-3’
P32	MutS653-BA.4.4	23526	5’-ATGTCAACAGCTCATATG-3’
P33	MutS341-BA.4.6	22583	5’-GTTTTTAACGCCACCACATTTGCATCTGT-3’
P34	MutS493-BA.5.0	22669	5’-CTTTCCTTTACGATCATATAGTTTCCGACCCAC-3’
P35	MutORF1ab3344-BA.5.1	10285	5’-TATTGGACATTCTATTCAAAATTGTGTACT-3’
P36	MutORF3a94-BA.5.1.1	25658	5’-CAGTTTACTCACACATTTTGCTCGTTGCTGC-3’
P37	MutORF1ab5448-BA.5.2	16601	5’-TAGCTAACACCTGTAATGAAAGACTCAAGCT-3’
P38	MutN30-BA.5.2.1	28345	5’-TGGCAGTAACCAGAATTTTGGGGCGCGATCAA-3’
P39	MutS71-BA.5.5	21776	5’-GGGACCAATGGTATTAAGAGGTTTGATA-3’
P40	MutORF1ab5710-BA.5.6	17386	5’-TTGAGTGTTGTCAATGTCAGATTATGTGCTAAG-3’
P41	MutORF1ab2096-BA.5.8	6539	5’-CACACAGATCTACTGGCTGCTTATGTA-3’
P42	MutORF1ab993-BA.5.10	3230	5’-GGTCAACAAGACTGCAGTGAGGACAA-3’
P43	MutORF1ab3919-BA.5.10	12014	5’-GTTTTGCTTTCCATGCGGGGTGCTGTAGAC-3’
P44	MutE50-BA.5.10	26380	5’-ATTGTTAACGTGGGTCTTGTAAAACCTT-3’
P45	MutS-22821-BF.1	22865	5’-GCTTGGAATTCTAATAAGCTTGATTCTAAG-3’
P46	MutORF8-52-BF.8	28035	5’-AGAGTAGGAGCTATAAAATCAGCACCT-3’
P47	MutN13-BF.10	28297	5’-TCAGCGAAATGCATTCCGCATTACGT-3’
P48	MutCys-BE.3	17191	5’-GAGAAGGCATTAAAATGTTTGCCTATAGATAAA-3’
	MutORF1ab3839-BK.1	11781	5’-AGCATAGATGCCTTCAGACTCAACATTAAATTG-3’

Method of detection of corona virus sub-variants and sub-sub-variants

After the detection of SARS-CoV-2 variant, it is good to check the sub-variant using specific point mutant oligonucleotide specific for sub-variant or sub-sub-variant. As for example, in case of omicron BA.4.0 subvariants are BA.4.1/2/4/6 and important sub-sub-variants are BA.4.1.6 and so on. In Table-2C we have presented such oligonucleotides. For analysis of MutORF3a-BA.4.1, we first made BA.4.0 vs BA.4.1 seq-2 BlastN homology alignment and collected the six distinct point mutations (figure-12C). Then 60nt sequence with mutation (position of mutation varied in individual BlastX due to difference in 5’ sequence and we presented six BA.4.1 accession numbers) from both sequences performed BlastX search to get mutant amino acid and codon preference at 1,2 or 3 starting base to get mutant amino acid codon. In most cases though, no amino acid change occurred and mutation was silent (data not shown here).

We noticed that many COVID-19 sequences did not properly report the variant status. As for example, a BA.4.0 variant (accession no. OP051217) 100% aligned to BA.4.0, and BA.4.4 oligonucleotides but not with BA.4.1, BA.4. 2 and BA.4.6. So, it must be assigned as BA.4.4.X. It has double sub-variants character and sub-sub variant for new mutations T26I and R185M (figure-14A). We also found similar case in COVID-19 omicron BA.5.2.3 variant (accession no. OP257552) where we found the dominant BA.4.0 signature deletion in nsp1 protein of ORF1ab. It 100% matched to DelNsp1-BA.4.0 oligonucleotide (figure-14B). It also had no 100% match to BA.5.0, BA.5.1, BA.5.1.1, BA.5.2 and BA.5.2.1oligonucleotides. The new variant status would be BA.4.1.X. Further, a newly assigned BA.5.2.3 variant (accession no. OP436327 had no “KSF” nsp1 deletion (see, figure-12D) and had 99.91% low homology to OP257552 (BA.4.1.X) with 27 bases mutations. It indeed had 100% homology BA.5.2 oligonucleotide but not with BA.5.0, BA.5.1, BA.5.1.1, BA.5.2.1 and BA.5.6 specific oligonucleotides suggesting our method was correct (Figure-14C). Truly our analysis with a BA.4 variant (accession no. ON836740) was very much confusing. IT had omicron signature “ERS” deletion in N-protein including “LPP” spike deletion which also omicron signature (BA.2/BA.4/BA.5) but no ⁶⁹HV spike deletion which contradicted for BA.4 or BA.5 (Table-1B). It has no KSF nsp1 deletion but upstream of “KSF” another “GHV” deletion (5’-GGT CAT GTT-3’) in nsp1 protein including “FSTQ” deletion (5’-TTT AGC ACT CAA-3’) in ORF7a protein. However, such double deletions were only found in accession no. ON836740 whereas single “GHV” deletion ORF1ab mutants were abundant (>1000) and designated as BA.6 variant (accession numbers: OP435120, OX317539, OX324794, OM296404). The ”FSTQ” deletied (ORF7a protein) variant was less abundant (<400) and designated as BA.7 variant (accession numbers: OP423060, OX270666, ON944596, OK107068). We presented the two oligonucleotides (red) in table-2A.

We have clearly showed by analyses that oligonucleotides selected at the deletion boundaries of different COVID-19 proteins useful to detect unknown variant after sequencing (Table-1). In truth, submitted sequences have no data regarding specific variant status and we are unable to get such information after down loading the sequence (www.ncbi.nlm.nih.gov/nucleotide). However, the SARS-CoV-2 Database has such information (not always) to get the variant status. Moreover, many sequences deposited without giving expressed protein data (accession numbers starting OV and OX). Thus, we always need an easy method to know correct lineage of the corona virus. So far, US scientists (Howard D et al; Bankers et al.) are giving correct and enough sequencing data to predict type of corona virus infection is rampant [14]. By comparing the few sequences from other country, one can predict type of infection may arrive in the neighbouring countries [9]. Thus, analysis we have presented giving few oligonucleotides for BLAST-2 sequence comparison to get corona virus variant status is logistic and demanding (see, figures-2,3,4). We hope many remote sequencing facilities in the poor countries have no important software for sequence analysis. But our approach using free NCBI Database, free NCBI/BLAST system and free CLUSTAL-Omega software may be a safe haven for scientists and it will be very applicable to Africa, Latin America, Middle East, and South Asian countries.

We analysed the amino acid composition of spike proteins and found no gross changes in omicron amino acid composition as compared to Wuhan and Alpha strains (www.expasy.org/cgi-bin /portparam). Acidic amino acid (Asp + Glu) and basic amino acids (Arg + Lys) were found 110 and 103 in Wuhan virus, 109 and 103 respectively for B.1.1.7 alpha virus where as such amino acids in Omicron corona virus (BA.1) were found 111 for both acidic and basic amino acids [14]. Instability index for Wuhan, alpha and omicron viruses were found 33.01, 32.82 and 34.65 respectively [14]. Alipathic index for Wuhan virus was found 84.67 and for omicron virus 84.95 and for alpha virus 84.65. Hydrophobic plot indicated some differences as omicron virus is more hydrophobic (data not shown). Such study indicated that during evolution different variants were selected by deletion and point mutation but 3-D structures never drastically changed keeping the Spike-ACE-2 interaction intact as well as SARS-CoV-2 life cycle processes unchanged. In truth, increase of infection during D⁶¹⁴G and N⁵⁰¹Y mutations was important as well as antibody escape due to E⁶⁹V⁷⁰ deletion as well as E⁴⁵²R mutation critically enhanced recurrent infectivity. However, rampant other mutations in the RBD domain in omicron corona virus must have role in transmission and immunity. Perhaps, further development of highly transmissible omicron BA.2, BA.4 and BA.5 corona viruses due to mutation (N⁴³⁵K, E⁴⁸⁴A, P⁶⁸¹H, Q⁹⁴⁹H) needs to be evaluated in the future. In this aspect, BA.2.75 variant was declared variant of concern by WHO. When compared spike protein of BA.2.48 with BA.2.75, we detected 9 mutations (K¹⁴⁴E, W¹⁴⁹R, F¹⁵⁴L, S²⁴⁵Y, G²⁵⁴S, D³³⁶H, G⁴⁴³S, N⁴⁵⁷K, R⁴⁹⁰Q). We concluded W¹⁴⁹R and N⁴⁵⁷K could be specific for omicron BA.2.75 variant. Interestingly, BA.2.75 spike have 31 mutations as compared to Wuhan virus.

Whether deletions of nsp1 K¹³¹S¹³²F¹³³ amino acid sequence as well as nsp6 S³⁶⁷⁵G³⁶⁷⁶F³⁶⁷⁷ amino acid sequence favours omicron BA.4 variant for transmission is not known (figure-10). But acquisition of both N⁵⁰¹Y and D⁶¹⁴G mutations in omicron corona viruses including other 27 mutations in RBD domain suggested higher transmission than SARS-CoV-2 Alpha and Delta variants. BA.2 is about 1.5 and 4.2 times as contagious than BA.1 and Delta respectively. Further, BA.2 is found to be able to alarmingly re-infect patients originally infected by omicron BA.1 [25]. BA.4 and BA.5 omicron viruses shows reduced neutralization by the serum from individuals vaccinated with triple doses of AstraZeneca or Pfizer vaccine compared with omicron BA.1 and BA.2 [26].

Two serine mutations to S³⁷⁰F and S³⁷⁴F in omicron BA.2/4/5 and a third serine mutation in BA.1 to S³⁷²L as well as in BA.2/4/5 to S³⁷²P were surely significant in determining enhanced transmission (figure-3). BA.4 and BA.5 also had F⁴⁹¹V mutation that differed from BA.1 and BA.2 and such variants were increasingly replacing BA.1.1 and BA.2.9. Spike protein of B.1.1.7, omicron BA.1 and BA.2 variants were 1270 AA although had differential deletions and insertions where as delta variant S protein was 1271 AA long and that of omicron BA.4 and BA.5 variants were 1268 AA. Thus, length of S protein also predicted a specific type of SARS-CoV-2 variant (figure-2; Table-1B).

However, we admit that many sequencing errors present in the database. As for example, several higher molecular weight ORF1ab proteins (7108 − 7099) were available in the database (UEP73521, UPH81172, UEE28211, UES61213, UFC36273) but might be sequencing error due to duplication or integration of unusual sequence in same reading frame. Further, 1/3 of the SARS-CoV-2 sequences deposited had no protein prediction. Such sequences start with OV and OX (OV316200, OV315626, OX197261, OX129695 etc) and UK origin. One multi-alignment using BA.2 variants predicted 21 mutant oligonucleotides but BLAST search found no such sequences in the specific variant (data available on request). Particularly, CvsT silent mutations in the 3rd codon noticed in most sequences. However, BA2.3-CvsT-8991 oligonucleotide (5’-aga gta cac tga ctt tgt aac atc agc gtg cag t-3’) appeared specific for omicron BA.2.3 variant. Thus, deletions found in proteins of COVID-19 variants were more authentic and such oligonucleotides gave good analyses and reproducible. Thus, our method is reliable to find specific variant type after new sequencing of SARS-CoV-2 (figures-12A/12B).

Question arises if such hyper-variable difference of omicron spike protein could be involved in the vaccine failure for the corona vaccine made from S gene of Wuhan corona virus! Study indicated that only 20% and 24% of BNT162b2 vaccine recipients had detectable neutralizing antibody against the omicron variant HKU691 and HKU344-R346K, respectively, while none of the Coronavac recipients had detectable neutralizing antibody titre against either omicron isolate. Omicron variant escapes neutralizing antibodies elicited by BNT162b2 or Coronavac [27, 28]. Further study suggested that E⁴⁸⁴K mutation evaded antibody neutralization elicited by infection or vaccination and further enhanced by K⁴¹⁷N and N⁵⁰¹Y mutations [29–32]. Another study indicated that neutralizing antibodies elicited by inactivated corona virus vaccine and RBD-subunit spike protein vaccine against B.1.617 and B.1.1.7 variants enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization [33–35]. L⁴⁵²R mutation may be important in BA.4 and BA.5 omicron variants for immunity to neutralization antibody but such mutation not present in BA.1 and BA.2 variants (figure-3). Thus, new omicron vaccine may be needed although in India third booster dose with Covishild vaccine is ongoing. BA.1/2/4/5 omicron corona viruses were found to be highly resistant to the antisera elicited by the mRNA-1273, ChAdOx1, and BNT162b2 vaccines [26, 33]. Surely, oligonucleotides from the hypervariable region could differentiate BA.1 to BA.2. When we aligned different BA.4 and BA.5 variants, we found S gene has 2–3 mutations but we suspect authenticity of such data. As for example, few BA.4.6 variant or BA.5.6 variant have mutation in S gene sequence but not in other designated as same variant! (data available on request). It was reported that one amino acid differences between the BA.4 and BA.5 lineages in OFR1a, ORF6, ORF7b, and Nucleocapsid (N) proteins but no amino acid differences in proteins such as ORF1b, Spike, Envelope (E), Membrane (M), ORF8, and ORF10. Furthermore, only one amino acid in ORF10 differs between the BA.5 and BA.5.1 sub-lineages. Similarly, OFR1a-DEL141/143, ORF7b-D⁶¹L, and N-P¹⁵¹S mutations are specific to BA.4, and ORF6-D³N mutation is specific to BA.5 and BA.5.1 lineages [35]. However, we found ORF7b-D61L will be ORF6-D61L because ORF7b is only 43 amino acids and such mutation is prominent in BA.2 lineages (accession nos. ON957923, ON999338). N-P151S mutation as described above was correct for BA.4 lineages but N-S413R was found not present in BA.1 lineage but BA.2/4/5 as well as BE.x and BF.x lineages. We also found N-P13L mutation for all omicron whereas in one BF-10 lineage (accession no. OP257537) N-P13F mutation was noticed. Moreover, BA.4 and BA.5 have been found to be 4–20 times more resistant to monoclonal antibodies such as cilgavimab and Evusheld than BA.2. We also found that Q⁴⁹³R spike mutation located in omicron BA.1 and BA.2 lineages but not detected in BA.4 and BA.5. Our analysis however detected many mutations in spike having contradictory to Desingu & Nagarajan (2022) in sub-variants of BA.4 as well as BA.5 indicating rapid spread of such variant replacing omicron BA.2 variant [35]. As for example, we found V³G, T³⁴¹R and S⁶⁵³N spike mutations in BA.4 lineages including one K¹⁴²E (protein id. OPO51195) and one R¹⁸⁵M mutation (protein id. OPO51217). Analysis also detected L⁵F, G¹⁷⁶E, S⁸⁷⁹F, Q¹¹⁹⁶L mutations in one BA.4.5 variant (accession no. OP257738) as well as one T¹²⁶⁸L mutation in BA.4.6 (accession no. OP258078). Similarly, BA.5 lineages have some T⁷¹I as well as Q⁹⁴⁹R/H mutation in many omicron BA.5.5 lineages, one S¹¹⁶⁵F mutation in BA.5.2 (protein id. UTN60872) and N⁴³⁵K mutation in many omicron BA.5.2.1lineages.

In recent omicron variant BA.2.75 we found new spike mutations K¹⁴⁴E, W¹⁴⁹R/H, G²⁵⁴S and N⁴⁵⁸K mutations but roles of such mutations remain elusive (figure-2; figure-13). As for example, N⁴³⁵K mutation in spike was found very much prominent in recent omicron isolates (BA.1.1, BA.2.75, BA.5.5 and BA.5.2.1). Thus, the oligonucleotide > MutS435-omicron (5’-gga att cta aca agc ttg att cta agg-3’) would be important to track such omicron transmission whereas N⁴³⁵K mutation was not present in Alpha and Delta variants. Further, oligonucleotide MutS341-BA.4.6 (5’-gtt ttt aac gcc acc aca ttt gca tct gt-3’) appeared very specific for omicron BA.4.6 which was spreading now. Thus, mutation might be there but specificity was not found when we analysed BlastN seq-2 between oligonucleotide and genomic sequences (Accession number) of BA.1, BA.2, BA.4, BA.5 omicron viruses as well as Alpha, Beta, Delta, Zeta, Gamma and Wuhan variant. Finally, our method was based on amino specific acid change which we had done by BlastX search of 60nt mutant oligonucleotide. Definitely we searched a lot but the major emphasis was given to omicron viruses which lately transmitted in huge population. Omicron viruses now combined with Alpha and Delta virusesSARS-CoV-2 producing many chimera human corona viruses. As for example, we detected a deletion in ORF7a in some variants (accession numbers: ON836740/ON944596/OP257615). Analysis of ON944596 produced ³¹LPP and ¹⁵⁷FR deletions in spike protein as well as FSTQ deletion (5’-TTT AGC ACT AAT-3’) in ORF7a protein near 27506nt suggesting it would be a Delta-BA.2 chimera. It had 65 mutations when aligned with BA.4.0 variant (accession no. ON907393) but had only 30 mutations with Delta variant (accession no. OM542166). Similarly, another FSTQ deleted SARS-CoV-2 had no KSF deletion at nsp1 but ³¹LPP deletion in spike and likely a BA.5-like (99.82% similarity; 54 mutations) human corona virus although no ⁶⁹HV deletion was found. Truly, the sequence 100% homology to MutORF8-64-BA.5.0 oligonucleotide confirming BA.5-like variant.

A distinct type of human corona virus variant was only reported in the SARS-CoV-2 database but was not located in the data of individual sequence. So, when you got a sequence with accession number search, you could not find the mention of variant status. Then, no way you could get again the data on variant type as Database was constantly loading with new sequences every day and now (15-09-2022) 6,263,795 COVID-19 sequences were deposited in the NCBI SARS-CoV-2 database. Thus, the described method for new COVID-19 variant detection is demanding and logistic. It is very simple and cost-effective using free computer-aided bioinformatics software and will solve the unresolved variant nomenclature problems located in the SARS-CoV-2 database. Definitely omicron variants like BE, BF, BG and BK are very similar (99.87%) to other important omicron variants and very hard to find a specific oligonucleotide for Blast-2 homology search.

Acknowledgement

We thank US NCBI Database and BLAST Search System for free service worldwide. CLUSTAL-Omega free software is also acknowledged. I am a retired professor.

Competent interests

The author declared no competent interest.

Ethical issues

The author declared no ethical issues as it was bioinformatics analyses and computer-generated data.

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A Method of Identification of SARS-CoV-2 Variant Using NCBI BLAST-2 100% Homology Search with Specific Oligonucleotides Selected at the Deletion Boundaries of S, N, ORF7a, ORF8 and ORF1ab Proteins

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Abstract

Figures

Introduction

Material And Methods

Results

Discussion

Conclusion

Declarations

References

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