Hyper-Variable Spike Protein of Omicron Corona Virus and Its Differences with Alpha and Delta Variants: Prospects of RT-PCR and New Vaccine

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Journal of Emerging Diseases and Virology

ISSN 2473-1846 |

Open Access

J Emerg Dis Virol | JEDV

RESEARCH ARTICLE

Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences

with Alpha and Delta Variants: Prospects of RT-PCR and New Vaccine

Asit Kumar Chakraborty*

Department of Biotechnology and Biochemistry, Oriental Instute of Science and Technology, Midnapore, West Bengal, India

Received: 24 Dec, 2021 | Accepted: 07 Feb, 2022 | Published: 24 Feb, 2022

Volume 7 - Issue 1

*Corresponding author: Asit Kumar Chakraborty, Rered, Department of Biotechnology and Biochemistry, Oriental Instute of Science and

Technology, Midnapore, West Bengal, India, Tel: +917679154141; E-mail: chakraak[email protected]

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta Variants:

Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

which permits unrestricted use, distribuon, and reproducon in any medium, provided the original author and source are credited.

Abstract

NCBI SARS-CoV-2 Database was analyzed between November-December, 2021 to decipher the spread of Delta corona virus variants in the USA

and compared with highly transmissible new omicron variant recently originated in South Africa. Presently, B.1.617.2 and AY.103 lineages Delta

variants with spike protein L452R, T478K, P681R mutaons and F157/R158 two amino acids deleons were predominant in the USA and superseded

the deadly outbreaks of B.1.1.7 Alpha variant with deleons of H69, V70 and Y145 amino acids as well as N501Y, and D614G highly transmissible

mutaons. Interesngly, omicron variant has six H69, V70, V143, Y144, Y145, L212 immune-escape deleons as well as 29 mutaons in the spike

protein including most deadly N501Y (Y498 in omicron) and D614G (G611 in omicron). This indicated that omicron variant was originated by

combinaon among B.1.1.7, AY.X and B.1.617.2 lineages. A unique three amino acids (EPE) inseron at 215 posion of spike protein was detected to

compensate six deleons suggesng further recombinaon events. Three Serine residues were mutated at amino acids 371 (S=L, L368 in omicron),

373 (S=P, P370 in Omicron), 375 (S=F, F372 in omicron) but compensated at 446 (G=S, S443 in omicron) and 496 (G=S, S493 in omicron) at the RBD

domain of omicron virus. The three amino acids (ERS) deleon at posion 30 in the N-protein acts as another signature of omicron virus. Omicron

variant has less mutaon in the 2/3 5’-end of the genome that codes for ORF1ab poly-protein but dominant P4715L mutaon in the RNA-dependent

RNA polymerase. However, overall amino acid composion, alipathic index, and instability index were found fairly constant although hydrophobic

plot gave some dierence between spike protein of Wuhan and omicron corona viruses. BLAST search detected 20nt and 19nt perfect match of

hyper-variable 22957-22977nt region comprising 488-493 amino acids (NH2-PLRSYS-CO2H) of the spike protein of omicron virus with the ch-2 of

Seladonia tumulorum or ch-16 of Steromphala cineraria respecvely. A primer set designed from the RBD domain of spike gene did not detected

the omicron genome by BLAST search but primers from the constant regions of the genome worked well. Such hyper-variaon in the spike protein

suggested that DNA vaccine or mRNA vaccine using spike gene of corona virus may not eciently protect omicron virus infecon and aenuated

whole corona virus vaccine will be safer vaccine.

Keywords: NCBI SARS CoV-2 Database; Signatures of omicron corona virus; Hyper-variable spike protein; RT-PCR diagnosis; Vaccine failure; Anbody

resistance

Introduction

Since the December 2019 Wuhan corona virus (severe acute

respiratory syndrome virus or SARS-CoV-2) has caused 0.58 million

deaths worldwide with thousand mutations creating many dominant

forms like alpha, delta, delta-plus and very recently omicron. Corona

virus is a large positive-sense RNA virus with a compact 29,980

nucleotides-long genome and COVID-19 is related to six dierent

corona viruses like CoV-229E, CoV-HKU1, CoV-OC43, CoV-NL63,

SARS-CoV and Middle East respiratory syndrome corona virus

(MERS-CoV) [1-5]. It has structural proteins (S, M, N, E) at the 3’-

end and 5’ two very large poly-proteins (2/3 of the genome) which

degraded into sixteen non-structural proteins (nsp1-16) including

RNA-dependent RNA polymerase (nsp12) [6], two proteases (nsp3

and nsp5) [7,8], RNA topoisomerase (nsp2) [9], RNA helicase

(nsp13) [10], nucleases (nsp15) [11] and methyl transferases (nsp16)

[12] (gure 1A). Spike protein (1273 aa) is a trimeric class 1 trans-

membrane glycoprotein and its RBD domain (335-515 aa) acts as

receptor binding domain to bind ACE-2 receptor of host cells for virus

entry [13] (gure 1B). S protein 1-13 AA acts as signal peptide and the

S1 subunit is 14-685 AA containing RBD domain and S2 subunit is

686 to 1273 amino acids with fusion contact peptide (788-806 AA)

as well as two hepta-peptide (HPPHCPC) repeats at 1163 and 1213

positions [14]. Among the other structural proteins N-protein (419

aa) binds to leader RNA of replicating corona virus and also regulates

host-pathogen interactions.

Severe COVID-19 is more common in adults aged ~70 years with

co-morbidities such as diabetes, cardiovascular disease and chronic

respiratory disease. A dierence in case fatality rates across countries

was observed, possibly due to a diverse demographic composition

and the type of control measures that have been taken in dierent

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

countries to stop viral spreading [15]. According to 2020 database,

three major Clades of SARS-CoV-2 can be identied and named as

Clade G (variant of the spike protein S-D614G), Clade V (variant

of the ORF3a coding protein NS3-G251V), Clade GR (S-D614G +

N-G204R) and Clade S (variant ORF8-L84S) [16,17]. SARS-CoV-2

variants emerged many fold in late 2020, and at least three variants

of concern (B.1.1.7, B.1.351, and P1) have been reported by WHO.

Alpha variant B1.1.1.7 had caused havoc calamity in India, UK

and USA between August 2020 and March 2021. Delta and Delta-

plus variants specically AY.103 and B.1.617.2 caused much fatality

between March-December 2021. Delta spike triggers faster fusion

with ACE-2 receptor of host lung cells relative to only D614G mutants

suggesting greater pathogenicity of delta variants than B.1.1.7 lineage.

Beta B.1.351, Gamma P.1, Epsilon B.1.427, Iota B.1.526, Mu B.1.621

and Zeta P.2 variants corona viruses were also indicated some

concern in dierent demography [18]. However, recently omicron

variant is highly spreading in South Africa and already has detected

in 90 countries including Europe, USA, Australia and India. It has

hyper-variable thirty spike mutations with important deletions and

insertions. us, more complete sequences needed to dene specic

geographic distributions of omicron virus variant. Most importantly,

clinical and political strategies at the local level must be augmented

because spike gene DNA and mRNA vaccines may not work well for

hyper-variable spike gene of omicron corona virus.

Presently, at least ten vaccine candidates vaccinated 70% world

population. Vaccine usually is a protein or synthetic peptides from

Coronavirus that can elicits humoral antibody (IgG) as well as T-cell

mediated ability to destroy virus. Attenuated or killed Corona virus

(Covaxin, Bharat Biotech, India) also used like Pox vaccination. As

genetic information in cells processed from DNA to RNA to protein,

scientists have exploited DNA vaccine as well as RNA vaccine for the

protection of Corona virus. Indian Serum Institute uses killed virus

where as Russia uses mRNA vaccine (Sputnik V) and England (Oxford

+ Astra-Zeneca) uses S gene DNA vaccine using adenovirus vector

(Ad5 or Ad26). USA (Moderna/ Pzer) and Germany’s BioNTech uses

S gene mRNA vaccine [19]. e most companies used spike protein

(S gene) which was the receptor protein of corona virus that bound to

ACE-2 receptor of lung cells of human and animal.

Mutation greatly aected increase modes of virus transmission

as in case of D614G and N501Y mutations [16]. Further, a decrease

vaccine utility (protection against virus) was reported with immune

escape (T cell immunity) as in case of 69, 70 and 145 amino acids

deletions in alpha corona virus (B.1.1.7) [20]. Further, mutations

like L452R, E484K, and other at the RBD of virus greatly lower the

neutralization ecacy of serum antibody from earlier corona patients

to mutant viruses [21]. Presently, deletion of F157 and R158 in AY.X

and B.1.1.617.2 Delta variants produced increased transmission in

presence of D614G mutation together further lowering the vaccine

ecacy. Very recently, distinct omicron virus new mutations found

lowering vaccine utility and increasing transmission rate but reports

of conrmed immunological data yet to come [22-24]. We will

molecularly study the spread of omicron virus specically in the USA

by analyzing NCBI Virus Database between 20

November to 25

December, 2021 using dierent free soware available in the net.

Methods

Database analysis

We used NCBI (www.ncbi.nlm.gov) SARS-CoV-2 database only as

it gave multi-alignment data for up to 500 sequences. Such alignment

detected most sequences were incomplete and separately analyzed. But

Figure 1A: Structure of human corona viruses (SARS CoV-2 or COVID-19).

Figure 1B: Primary amino acid sequence of the spike protein of

Omicron corona virus. Two deleon points were shown by Del-1

and Del-2 arrows, Inseron point was denoted by Ins-1 and stars.

Red denotes mutant amino acids and with underlined means well

characterized mutaons that enhanced viral transmission (G611 and

Y498 here).

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

middle complete sequences were checked for comparable sequences

by looking red lines for mutations and mostly AY.x variant corona

viruses. Omicron viruses have many red lines between 21000-28000nt

for the hyper-variable spike protein and other structural proteins

(Figure 1C). us, we covered many sequences to few omicron virus

sequences helpful for analysis by freely available Multalin soware

and CLUSTAL-Omega soware. It took 2-3 minutes for spike protein

(1273 aa) alignment by Multalin soware but it took 30-40 minutes

for CLUSTAL Omega soware 30kb RNA genome alignment. As we

found most sequences were AY.X Delta type (~85%) and some Delta

B.1.1.617.2 type (~10%). We only documented data for omicron

sequences (0.5-5%) deposited between 23

November and 24

December, 2021. Date of sequence deposition in the NCBI Database,

Author’s name and Collection date of virus were used to analyze

sequence sets. Although rst we detected one omicron corona virus

sequence in such search. en, we BLAST analyzed the hyper-variable

regions (60 nt) to get more three omicron sequences but one US such

US originated sequence had incomplete spike protein and two complete

sequences each from Canada and Belgium. However, from December

6, more and more omicron viruses were detected in the database. Our

guess sequencing primers used for Wuhan, Alpha and Delta variants

were not worked well using standard kits available. But more and

more omicron virus complete sequences will be deposited aerwards.

We have no access to GISAIP database and the accession number for

the rst omicron virus genome (29684 bp) is EPI_ISL_6640916 with

collection date 11-11-2021 and submission date 23-11-2021. Parts of

multi-alignments were presented with dierent omicron signatures.

During review of the paper, we also analyzed the database and huge

omicron sequences were deposited in last week of December, 2021 and

rst week of January, 2022.

Results

We rst detected an omicron strain (B.1.1.529 or BA.1) of corona

virus on 7

December by analyzing the NCBI virus database deposited

on 6

December 2021 by Puehringer, et al., from Austria (accession

no. OL721912; Date of isolation 1

December, 2021). We had not

detected any omicron variants in US from data deposited by Bankers

L, et al., and mostly found delta B.1.617.2 and AY.X strains (Figure

2). e 7

December 2021 deposited sequences by Howard D, et al.,

Schmedes, et al., Bankers L, et al., Pachucki R, et al., Buck GA, et al.,

Pokharel A, et al., Blankenship HM, et al. and Lighthouse Lab, et

al., did not found any omicron as well. en, we planned to BLAST

search of 60nt (22894 5’- AAC TGA AAT CTA TCA GGC CGG TAA

CAA ACC TTG TAA TGG TGT TGC AGG TTT TAA TTG TTA-3’

22953) hyper-variable region of omicron corona virus genome. e

study resulted three hyper-variable spike protein of omicron B.1.1.529

corona virus with accession nos. OL698718 (USA, incomplete S),

OL677199 (Canada) and OL672836 (Belgium). We presented multi-

alignment data to show mutations and deletions as compared with

most deadly B.1.1.7, AY.103 and B.1.1617.2 variants (Figures 3A-3C).

It proved that omicron variants had six H69, V70, E144, F145, R146

and L212 immune-escape deletions as well as 29 mutations in the spike

protein including most deadly D614G (G611 in omicron virus) and

N501Y (Y498 in omicron virus). Other mutations were: A67V (V67),

T95I (I93), N211I (I206), L212V (V207), V215P (P210), R216E (E211),

G341D (D336), S373L (L368), S375P (P370), S377F (F372), K419N

(N414), N442K (K437), G448S (S443), S479N (N474), E486A (A481),

Q495R (R490), G498S (S493), Q500R (R495), Y507H (H502), T549K

(K544), H657Y (Y652), P683H (H678), N766K (K761), D798Y (Y793),

N858K (K853), Q956H (H951), N971K (K966), and L983F (F978) [in

sate values for omicron virus positions] (gure 1B and gures 3A-3C).

e spike protein in Wuhan virus had 1273 aa, in B.1.1.7 variant had

1270 aa, in B.1.1.617.2 and AY.X variants had 1271 aa and in omicron

variant had 1270 aa. e roles of those mutations were not clear yet

and more research needed! P681R mutation (P683H disclosed here)

was found in B.1.1.617.2 variant but no L452R mutation in omicron

virus. It appeared EPE sequences inserted at 215 position and then

substitutions would be happened. Receptor Binding Domain (RBD)

of Spike protein bound to its ACE-2 receptor of human lung cells

needed for virus entry. Analysis of ORF1ab protein (7096 aa long)

suggested that dominant P4715L mutation in the RNA-dependent

RNA polymerase and further K564N, K856R, L2084I, A2710T and

P3395H mutations were happened in ORF1ab protein of omicron

virus. P4715L, as well as L2084I mutations were also detected in Delta

variants. However, S2083 deletion in omicron could be critical but

no data available. In Delta variants other important mutations like

A1306S, P2046L, P2287S, T2836I, V2930L, G5063S, P5401, A6319V,

Figure 1C: Mul-alignment of 200-500 sequences at the NCBI SARS CoV-2 database showing gray gap (arrow) for incomplete sequence and heavy

red lines for mutaon in the spike protein between 21000-25000nt (blue box) that acted as signatures of omicron corona virus. Such signature

is important to search omicron in the database where 200-500 sequences were aligned. Otherwise it was very hard to nd omicron sequences.

Interesngly, deposited many omicron sequences were incomplete in the RBD domain (313-393 aa/ 303-393 aa/ 425-439 aa) of the spike protein

due to mutaons.

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

Figure 2: Most variant of corona virus detected in the USA before November 30, 2021 was AY.X Delta variant that superseded UK alpha B.1.1.7

variant.

Figure 3A: Deleons and inserons in the spike protein of omicron corona virus as compared with alpha and delta variants. Protein IDs UFP04971,

UFO69279 and UFT26501 were omicron variant originated in Canada, Belgium and Austria respecvely. Protein ID UFT26468 was B.1.617.2 Delta

variant and highly acve worldwide.

and K6958R were noticed (see, accession no. OL721909) as compared

to Wuhan virus of December, 2019 (data not shown).

We identied few spike protein conserved regions rich in

hydrophobic amino acids (V, L, I, F) as indicated by green underline

in gure 3B and gure 3C (green underlined) to explain a view that

potentiality of Wuhan S gene vaccine for omicron virus neutralization

as reported recently but much reduced ecacy (8x) would be possible.

us, attenuated whole virus vaccine like Covaxin (India made) may

be more important to control omicron corona virus spread until new S

gene vaccine with omicron genome will be made.

We continued our analysis as more new omicron sequences were

deposited. Data deposited on 10-12-2021 by Lemieux JE, et al., and

Howard D, et al., did not produce any omicron but mostly AY.X and

some B.1.1.617.2 variants. However, data deposited on 11-12-2021

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

Figure 3B: Hyper-variable regions of RBD domain of spike protein of Omicron variant corona virus as compared with Alpha and Delta variants. Three

Serine residues at 373, 375, and 377 were changed in omicron variants and compensated by G448S and G498S mutaons. Conserved hydrophobic

region was shown by green underline.

Figure 3C: Universal D614G mutaon (100% now) of deadly corona viruses and new H657Y, N681K and P683H mutaons in omicron variant.

Conserved hydrophobic region was shown by green underline.

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

by Holland SL, et al., Howard D, et al., and Pinet K, et al., produced

fourteen omicron variants between November 24 to December 6, 2021

collection dates. e accession numbers were: OL815080/81 (USA,

AZ), OL815417 (USA, TX), OL815350/51 (USA, MA), OL815447/48

(USA, GA), OL815449/50 (USA, NY), OL8154/51/52/53 (USA,

CA), and OL815455/56 (USA, MA). Multi-alignment produced very

similar result as depicted in gure 4. Panel C data was not adjusted

where RE insertion appeared not EPE due to unability of Multalin

soware to detect deletion L212. We kept that data because CLUSTAL-

Omega protein multi-alignment also did not correct the data which we

conrmed by DNA alignment as shown in gure 5.

Data analysis of dated 13-12-2021 was resulted three omicron

isolates deposited by Lemieux JE, et al., but appeared all incomplete

sequences (accession nos. OL823147 and OL823148 (USA, MA)

and OL822906 (USA, NY). However, the same day deposited many

sequences by Howard, et al., and Blankenship HM, et al., had no

omicron virus but mostly AY.X and few B.1.1.617.2. Similarly data

deposited on 16-12-2021 had no omicron variant (Howard D, et al.,

and Diagnostics G, et al.,) and for dated 17-12-2021 only two omicron

selected (accession nos. OL890283 (Lemieux J, et al.) and OL901854

(Linares-Perdo DJ, et al.). Interestingly, twenty four omicron

viruses were deposited on 18-12-2021 by Lemieux JE, et al., USA.

Accession nos. were OL903123 (NH, 3-12-2021), OL880661(MA,

3-12-2021), OL902594 (CT, 4-12-2021), OL903509(MA,

5-12-2021), OL913145(RI, 6-12-2021), OL904534(MA, 6-12-

2021), (OL904790(MA, 6-12-2021) OL904791(MA, 6-12-

2021), OL903931(VT, 8-12-2021), OL904803(NH, 8-12-2021),

OL904422(MA, 8-12-2021), OL904499(MA, 9-12-2021) and seven

omicron data from Massachusetts with collection date 8-12-2021

and accession nos. OL903558, OL903553, OL903821, OL903690,

OL903698, Ol903660, OL903604 and three from New York (accession

nos. OL903848, OL903853, OL903850) and two from Rhodes Island

(accession nos. OL903865, Ol903866). On the same day (18-12-2021)

Nickerson DA, et al., (USA) was deposited four omicron variants with

accession no. OL903977 (collection date 9-12-2021) and accession nos.

OL903978/79/80 with collection date 12-12-2021. Most incomplete

sequences were found at the AAs 303-393, 313-393, and 425-439

indicating primers would not worked during initial sequencing due

to strong variations in the genome of omicron virus. Such incomplete

S proteins were analyzed by Multalin soware and the resulted with

unique deletions and insertions for omicron (data not shown).

Data analyzed for day 20-12-2021 deposit by Howard D, et al.,

Linares-Perdo J, et al., and Grimaldo V, et al., appeared mostly AY.X

and few B.1.617.2 . e data analysis for day 21-12-2021 deposit by

Parrott T, et al., gave four omicron complete sequences with accession

no OL960535/36/37/38 with collection date 8-12-2021. Same day,

Gran J, et al., deposited 28 omicron sequences from Minnosota,

USA with accession nos. OL964103, OL964105/06, OL964108/09,

OL964111/12/13/14/15, OL964117/18, OL964120/21/22/23,

OL964125, OL964127/28/29/30/31/32, and OL964134/35/36/37/38.

S proteins were partial and few only analyzed to conrm omicron

signatures (data not shown). However, sequences deposited by Howard

Figure 4: Database Analysis of dated 11-12-2021deposit of Omicron variant and compared with Delta and Alpha variants. Parts of the major

dierences were shown here to demonstrate major feature of Omicron having deleons at H69, V70, V143, Y144, Y145 amino acids but not in

F167, R168 amino acid posions as found in Delta variants. L212 deleon was not aligned here and RE inseron will be EPE based on corona virus

genome alignment (see, Figure) due to similarity of VR sequences (green circles) between omicron and Wuhan and Delta viruses. Corona virus

paent samples were Nasal swab, Oral swab, Saliva and Mucus origin between 24th November 2021 to 5th December 2021 from United States (CA,

MI, MA, GA, NY, AZ, AL, KS, CO, TX).

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

Figure 5: Mul-alignment of omicron corona virus genome (30kb) which conrmed a deleon of one amino acid (L212) following three amino acid

(EPE) inseron at 215 posion of spike protein as well as H69, V70, G143, Y144, Y145 deleons. Part of the genome was shown here.

Figure 6: Complete omicron corona virus spike protein sequences up to 24th December, 2021. Protein IDs and collecon dates in 2021 were given

and compared with Wuhan, Alpha and Delta corona virus spike proteins. Part of the alignment was given showing V143, Y144 and Y145 three amino

acids deleons.

D, et al., Blankenship HM, et al., Pokhard A, et al., and Ritter J, et al.,

produced mostly AY.x delta corona virus (data not shown).

Data analyzed on 22-12-2021 deposit by Gohl DM, et al., Banu

LA, et al., Irfan M, et al., Beukelman R, et al., Bankers L, et al., were

mostly incomplete and multi-alignment produced some false image

with no omicron sequence was interpreted. But Lemieux JE, et al.,

deposited six omicron sequences with accession nos. OL976589 (USA,

MA), OL976472 (USA, MA), OL976989 (USA, ME), OL976899 (USA,

MA), OL977025 (USA, VT), OL977069 (USA, VT)] in the same day

with collection dates 7-12-2021 to 11-12-2021. Also Gener A, et al.,

deposited few complete omicron sequences from California with

accession nos. OL977473 (4-12-2021), OL977502 (13-12-2021) as well

as some incomplete omicron sequences with accession nos. OL977503

(13-12-2021), OL977504 (13-12-2021) and OL977661 (14-12-2021)

(data not shown).

e omicron sequences were deposited by Lemieux JE, et al., from

USA with accession nos. OL976589, OL976472, OL976899 (USA,

MA) and OL977025, Ol977069 (USA, VT) and OL976989 (USA, ME)

on dated 23-12-2021. Kandal S, et al., deposited one omicron sequence

on 23-12-2021 with accession no. OL988626 (USA, AR; 10-12-2021).

Howard D, et al., deposited on the same day many omicron sequences

with accession numbers OL991113 (USA, OH), OL991168 (USA, TX),

OL991171 (USA, WV) with collection date 1-12-2021 and OL991968

(USA, MD) with collection date 7-12-2021 and a partial omicron

sequence with accession no. OL991968 (CA, 4-12-2021) (data not

shown).

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

e last day of our analysis was dated 24-12-2021 deposited

sequences. We found by multi-alignment that Nickerson, et al.,

deposited many omicron sequences originated in Washington with

accession numbers OM003743 (19-12-2021), OM003730 (15-12-

20210, OMOM003721 (17-12-2021), OM003729/27/28/32 (15-12-

2021), OM003707/14/26/34 (13-12-2021), OM003719/35/37 (17-

12-2021), OM003711/17/23 (13-12-2021), OM003716 (15-12-2021)

and OM003744/41/42 (19-12-2021). We also found Ryan KA of

United Kingdom deposited one omicron sequence with accession

number OM003685 (27-11-2021). On 24-12-2021, Howard D, et

al., also deposited many omicron sequences originated in the New

York with accession numbers OM005692, OM005638, OM005669,

OM007728, OM007698, OM007702, OM007701 and OM007731

(collection dates 7-8

December, 2021). He also deposited many

omicron sequences originated in the dierent US States with

accession numbers OM007718, OM007685 (New Jersey, 8-12-2021),

OM007637 (Maryland, 7-12-2021), OM007625 (Pennsylvania, 7-12-

2021), OM007696 (District of Columbia, 8-12-2021) and OM007970

(Hawaii, 13-12-2021) (data not shown).

We got all the 141 suspected 1270 aa length complete + incomplete

omicron sequences and found 21 complete sequences. Multi-

alignment produced three mutations (D212Y in UHO53537, R343K

in UHO53468/91 and UHO53648 and A698V in UHO53131,

UGO96815 and UGO96803) in the omicron sequences. So, up to 24

December 2021, the Database (December 6 to December 24, 2021

total sequences deposited were 3,71,307) penetration of complete

+ incomplete omicron sequences were 0.0379% and for complete

sequences it further very reduced to 0.0056%. is indicated it was

very hard to get omicron virus sequences which was complete and

authentic (Figure 6).

Interestingly, on the Christmas day (25-12-2021) Howard D, et al.,

deposited 216 omicron virus sequences to the NCBI Virus database

indicating huge omicron virus transmission that likely competed the

huge transmission of the delta corona virus. To conrm the omicron

virus, we had selected few sequences from the dierent part of the

NCBI multi-alignment graph and analyzed (Figure 7).

We want to learn more signatures for omicron corona virus. We

found no changes in the furine cleavage site of S protein (Figure 8)

(Homann, et al., 2020). When we analyzed the N-protein sequences

from omicron viruses, we detected unique three amino acid deletion

(ERS) at position 30 and multi-alignment data presented in gure

9. Interestingly, N-protein had point mutations like P13L, R203K,

G204R and in some D343G. Similarly, two extra new mutation in the

M-protein were detected (D3G, A63T) (Figure 10) but no mutation in

the ORF3a protein (data not shown). Small structural E protein (75 aa)

in omicron virus has one mutation (T9I; data not shown). Never the

less omicron virus transmission is rapidly increasing in 90 countries

and in some US states it is about 10% where as in South Africa was

90% now. us, the rate of omicron transmission has increased in

UK and Germany where as about 400 patients were detected in

India. Death already was reported in England and USA although the

omicron disease appeared to be less virulent where oxygen support

and hospitalization were unnecessary.

Surprisingly, on 25-12-2021 huge omicron data deposited in the

NCBI SARS-CoV-2 Database as we reported higher than 216 omicron

sequences. But dated 27-12-2021 analysis we did not nd the pattern

of multi-alignment for omicron virus (Figure 1C). Ultimately we

discovered the heavy read lines were found in Delta variants (reversed)

due to huge deposition of omicron sequences from 25-12-2021

onwards. We then BLAST searched the 60 nt hyper-variable region of

spike protein (22894 5’- AAC TGA AAT CTA TCA GGC CGG TAA

CAA ACC TTG TAA TGG TGT TGC AGG TTT TAA TTG TTA-

3’ 22953) and we found 3815 possible omicron sequences instead of

only four (4) found on dated 08-12-2021 BLAST search. Such data was

astonishing and a huge spread of corona virus was evident in the USA

from the second week of December, 2021. Dated 29-12-2021 and 31-

12-2021 analysis, we discovered a mixed trend of alignment suggesting

our method still work well. Likely omicron penetration increased to

1.2% during end of December, 2021.

Next we analyzed the important of S gene mutations on corona

virus diagnostics. Many RT-PCR kits utilized the S gene primers

where some kits appeared unable to give RT-PCR data from the S gene

region. We determined the sequence variation in omicron virus as

compared to Wuhan 2019 strain. Data presented in gure 11 where

two or more regions in the genome were presented. We made 10

primers set using NCBI Primer Design soware and one was located

in the S gene. Analysis found by BLAST that forward primer (F3=

5’-23518GAC TAA GTC TCA TCG GCG GG23537-3’ would not

hybridize to omicron genome but reverse primer 5’-24130CCC ACA

TGA GGG ACA AGG AC24111-3’ did well. Old primers designed

for Wuhan strain were hardly identify S gene of omicron variants

pinpointing new primers design were necessary to track omicron

transmission using S gene primers. is is an example to be aware for

RT-PCR using old primers for the detection of omicron virus spread.

However, the primer pairs F8-5’- GGC AAA CCA CGC GAA CAA

AT-3’ and R8 5’- GAG GGT CAA GTG CAC AGT CT-3’ (1145 bp;

Tm=60oC) worked well for all Wuhan (accession no. NC_045512.2),

alpha B.1.1.7 (accession no. OD984292), delta B.1.617.2 (accession no.

OV104747) and omicron B.1.1.529 (accession no. OL672836) corona

viruses. Similarly to sequence the hyper-variable point mutated region

Figure 7: Data analysis of omicron sequences deposited on 25-12-2021 by Howard D, et al., USA. Only three sequences (OM01136, OM010507,

OM011026) appeared complete and rest had ambiguity about three amino acids (EPE) inserons in the spike protein. Thus, although ~216 omicron

virus sequences were deposited by Howard D, et al., on dated 25th December 2021, authenc omicron corona virus spike protein sequence was

sll hard to detect.

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

Figure 8: Mul-alignment showing no mutaon in the spike protein furine cleavage site of Wuhan, Alpha, Delta and Omicron variants of COVID-19.

Figure 9: Three amino acids deleon of N-protein (416 aa) is an indicator of omicron variant corona virus. Data dated 18-12-2021 was analyzed and

compared with other old variants. P13L, R203K, G204R point mutaons were also detected.

(300-550 AA) we devised a forward primer from F8 reverse primer

region F9-5’-CTG TGC ACT TGA CCC TCT CTC-3’ and a reverse

primer downstream (R9-5’-CAC GGA CAG CAT CAG TAG TGT-3’)

giving a 863 bp DNA product (Tm=60oC) for all Wuhan, alpha, delta

and omicron corona viruses.

Interestingly, BLAST search of another part of hyper-variable

region (22954 5’- CTT TCC TTT ACG ATC ATA TAG TTT CCG

ACC CAC TTA TGG TGT TGG TCA CCA ACC ATA CAG-3’ 23013)

resulted same three omicron sequences with 100% similarity but also

we identied a 20nt exact match with chromosome-2 (nt. 13807869 to

13807888) of Seladonia tumulonum (bee) and 19 nt exact match with

chromosome 16 (nt. 1796578 to 1796596) of Stemomphata cineraria

(sea snail). It codes amino acids 488-493 (NH2-PLRSYS-CO2H)

region of spike protein of omicron corona virus (Figure 12). We do

not know why such similarity of viral sequence to lower eukaryotes

genome like y or mollusca! However, such information could have

some interest to some evolutionary biologist.

en, we wanted know how six amino acids deletions and three

amino acids insertions with many mutations could protect the

omicron virus S protein functional and stable giving the capacity for

higher transmission than alpha and delta variants? When we analyzed

the amino acid composition of spike proteins, we found no gross

changes in omicron as compared to Wuhan and Alpha strains. Data

presented in gure 13 (www.expasy.org/cgi-bin /portparam). Very

minor changes were noticed and boxed for Arginine, Aspergine,

Glutamine, Phenyl Alanine and Serine (Figure 11). Acidic amino acids

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

Figure 10: Mul-alignment of M-protein of dierent variant of corona virus. D3G and A63T mutaons in Omicron variant were detected.

Figure 11: Seq-2 BLAST similarity analysis between December 2019 Wuhan corona virus (accession no. NC_045512.2) and November 2021

omicron corona virus variant (accession no. OL721912). Only mulple nt. dierence posions of spike gene due to mutaon and deleon were

presented here.

(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 were found 111

for both acidic and basic amino acids. Alipathic index for Wuhan

virus was found 84.67 and for omicron virus 84.95 and for alpha virus

84.65. Instability index for Wuhan, alpha and omicron viruses were

found 33.01, 32.82 and 34.65 respectively explaining omicron corona

virus spike protein was very stable. at may be the one of the cause of

higher transmission of omicron corona virus due to stable interaction

with ACE-2 receptor and S protein. However, the interaction of other

>15 mutations in the RBD domain of omicron corona virus were

unknown. Hydrophobic plot of Wuhan and omicron corona viruses

were presented in gure 14. ere are some dierences as shown by

green box.

Discussion and Conclusion

Omicron variant of corona viruses are rapidly spreading in the world

including USA, UK, Australia, and India. We analyzed here the NCBI

database to conclude that the omicron virus was rapidly spreading

in the United States of America and spike protein of omicron corona

virus was very stable although it had more deletions and mutations

than alpha and delta variants. Recently, a paper was published on

African omicron corona virus phylogenetics using GISAIP database

and the accession no of the rst omicron virus genome (29684 bp) was

EPI_ISL_6640916 with collection date 11-11-2021 and submission

date 23-11-2021 [25]. However, I have no access to such database. e

molecular mechanism of the new 26 unknown spike protein mutations

were yet to know but such changes occurred with higher transmission

and combined 6 deletions of amino acid surely increased immune

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

Figure 12: Detecon of rst perfect sequence homology of S gene with bee ch-2 and snail ch-16.

Figure 13: Dierence in amino acids composion among Wuhan, Alpha and Omicron variants of corona virus (COVID-19).

Figure 14: Hydrophobicity plot showing minor dierences (Boxed) between S protein of Wuhan and Omicron corona viruses.

escape [26]. We analyzed enormous data from NCBI SARS-CoV-2

database from November-December, 2021 and detected about ~141

omicron sequences (upto 24-12-2021 deposit). But such sequences

were mostly incomplete and we nally got about 21 authentic omicron

spike protein sequences (Figure 6). On Christmas day Howard D, et

al., deposited about 216 omicron sequences but mostly incomplete due

to ambiguity of insertion sequences but there were enough complete

sequences for spike protein to analyze. I faced tremendous problem to

get an authentic omicron spike protein sequence during last week of

November, 2021 when omicron reports were mounting in the media.

In January, 2022, the number of Omicron sequences were more than

Delta variants!

Sci Forschen

O p e n HUB for Sc i e n t i f i c R e s e a r c h

Citaon: Chakraborty AK (2022) Hyper-Variable Spike Protein of Omicron Corona Virus and Its Dierences with Alpha and Delta

Variants: Prospects of RT-PCR and New Vaccine. J Emerg Dis Virol 7(1): dx.doi.org/10.16966/2473-1846.166

Journal of Emerging Diseases and Virology

Open Access Journal

Question arises such hyper-variable dierence of omicron spike

protein could be involved in the vaccine failure for the corona vaccine

made from S gene! Analysis suggested that some hydrophobic regions

had similarity and surely partial protection possible! 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]. Using animal model Starr TN, et

al., [28] showed that the antibodies, S2H97 and S2E12 bound with

high anity across all sarbecovirus clades to a cryptic epitope and

prophylactically protects hamsters from viral challenge [28]. Further

study suggested that E484K mutation evaded antibody neutralization

elicited by infection or vaccination and further enhanced by K417N

and N501Y mutations [29]. A very similar conclusion was conrmed

by antibodies raised against deletion mutants of RBD domain of spike

protein [30]. Wang R, et al., [20,21] and others (2021) showed that

the South African variant B.1.351 was the most resistant to current

monoclonal antibodies and convalescent plasma from Wuhan virus

infected individuals, followed by United Kingdom alpha variant

(B.1.1.7) and the Brazilian gamma variant (P.1) with Y144del and

242-244del mutations and important D614G and N501Y mutations as

well as K417N/T, E484K/Q mutations in the RBD domain of the spike

protein of SARS-CoV-2 [27,31,32] (Wand, et al., 2021). 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]. us, all

natural mutations have allosteric eects that drive either interspecies

transmission or escape from antibody neutralization [36]. Omicron

virus was already transmitted in 90 countries and likely will be threat

to humanity [37]. Omicron may be ten times more contagious than

the original virus and twice more infectious than delta variant. We

have shown that omicron viruses are greatly aected many US States

including CA, NY, CO, MN and NJ. Further, omicron may be twice

more likely to escape current vaccines than the delta variant [38].

Wang R, et al., identied fast-growing RBD mutations like N439K,

S477N, S477R, and N501T that enhanced the RBD and ACE2 binding.

L452R mutation in the spike reduces its interaction with Wuhan

corona virus antibodies. Similarly, mutations E484K and K417N

found in South Africa and L452R and E484Q found in India variants

could be responsible for such reduced antibody interaction [21].

Miller NL, et al., preprint disclosed that the omicron variant increased

antibody escape due to mutations in class 3 and 4 antibody epitopes in

the spike protein as well as enhanced transmissibility via disruption of

ligand-receptor interface [26]. Finally, molecular biology of omicron

virus has just started to dene its functions of genetic changes [25].

Although mild symptoms of fever, cold and pneumonia reported,

delmicron (Delta + Omicron) has created a havoc calamity in the

world [39]. Remdesivir drug however has some benet to control

corona virus spread targeting RNA dependent RNA polymerase as

well as some immune drug were discovered [40]. We have pinpointed

the dierences in the spike protein of omicron but omicron spike

protein appeared very stable to interact with ACE-2 receptor. But

genomic mutations may aect RT-PCR (Figure 11) and thus new RT-

PCR primers were presented from conserved regions. Interestingly,

now more sequences for omicron virus will be available in the NCBI

SARS-CoV-2 Database.

Acknowledgement

e work was not funded by any agency. e author thanks CDC

and NCBI for the data provided. AKC is a retired professor.

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