In-silico Analysis for the impact of SARS-CoV-2
Variants on the STANDARDTM Q COVID-19 Ag Test
Revision History
Revision

Date

Description of changes

00

2021. 06. 24

01

2021. 07. 08

022021. 08. 18

032021. 09. 10042021. 11. 1205

2021. 11. 24

06

2021. 11. 29

072021. 12. 08

08

2021. 12. 23

092022. 01. 06

Updated the B.1.640.1 and B.1.640.2

102022. 04. 19

Updated the XD, XE, XF

112022. 08. 24

Updated the BA.4.1, BA.5, BA.2.75

Established In-silico Analysis for the SARS-CoV-2 Variants on
STANDARDTM Q COVID-19 Ag Test
Updated the in-silico Analysis for the B.1.617.1 and B.1.617.2
Updated the in-silico Analysis for the Delta virus designated byWHO
Updated the in-silico Analysis for the Mu (SARS-CoV-2) designatedby WHO and C.1.2
Updated the product category and WHO label.
Revised some descriptions.
Corrected typos.
Updated the in-silico Analysis for the Omicron (B.1.1.529)
designated by WHO
Updated the in-silico Analysis for the Stealth Omicron (B.1.1.529.2)
Updated the changed labelling for B.1.1.529
Sub-lineages (BA.1, BA.2 and BA3) from B.1.1.529

Authorization
Reviewed byApproved by

Dong-Hyuk, Kim
Executive manager of
R&D/BioNote

Jeong-Ho, Kim
Executive Director of
R&D/BioNote

Written by

Chaeyeong, Lee
Assistant Manager of
R&D/BioNote

Se-Jung, Lee
Executive manager of
R&D/BioNote

1 / 16

TABLE OF CONTENTS
1. PURPOSE AND SCOPE

3

2. OVERVIEW

3

3. CRITERIA

7

4. RESULT

8

5. CONCLUSION

15

6. REFERENCES

16

2 / 16

1. PURPOSE AND SCOPE
1) Purpose
This document describes the in-silico analysis for the impact of SARS-CoV-2 variants on the
STANDARDTM COVID-19 Ag Test

2) GoalThis in-silico analysis aims to assess if a mutation in the nucleocapsid protein sequence of SARSCoV-2 variants could theoretically affect performance of the STANDARDTM COVID-19 Ag Test. For
this purpose, potential changes in reactivity of the antibodies to the antigen were predicted bycomparing the mutation site in the nucleocapsid (N) protein of each SARS-CoV-2 variant with the
epitope region of the antibodies used in the products.

Product Name

Used antibodies· STANDARDTM Q COVID-19 Ag Test (REF: Q-NCOV-01G)

Monoclonal antibody A
Monoclonal antibody B

3) Reference documents- Q COVID-19 Ag_Epitope mapping analysis for the antibodies_Rev.03- Q COVID-19 Ag_Wet-testing report_Rev.03

4) Scope
STANDARDTM Q COVID-19 Ag Test

2. OVERVIEW
1) Testing Condition
Investigating Date
Revised Date
Site
Testing
Representative
In-silico analyzer

2021. 06. 21 ~ 2022. 07. 29 (Regulately)2022. 08. 24
Department of R&D, BioNote, INC.
Preparer: Dong-Hyuk Kim, Bionote R&D
Operator: Jin-Soo Kim, Bionote R&D
Operator: Se-Jung Lee, Bionote R&D
Reviewer: Jung-Ho Kim, Bionote R&D

1. Program: NCBI blastp (Needleman-Wunsch)
Onsite link: https://blast.ncbi.nlm.nih.gov/Blast.cgi

3 / 16

2) SARS-CoV-2 Variants
A total of 47 variants were analyzed and are listed in the table below.
For the N protein, the target protein of all SARS-CoV-2 antigen diagnostic products, the mutationsites found in variants with a frequency of >29% from 105-9475 sequences the public resource
GISAID for the respective variants were investigated. The number of sequences at the time ofanalysis was limited for some variants since not more sequences that are valid were available.
In Rev.06, New VOC (B.1.1.529) designated by WHO is added.
In Rev.07, Stealth Omicron (B.1.1.529.2= BA.2) is added.
In Rev.08, sub-lineages (BA.1, BA.2, BA.3) from B.1.1.529 were added, previous described
B.1.1.529 in rev.06 is sorted to BA.1. other B.1.1.529 is added. previous described B.1.1.529.2 in
rev.07 is revised to BA.2. B.1.1.529.2 and BA.2 are the synonym, but BA.2 has being usually used.
In Rev.09, two sub-lineages (B.1.640.1 and B.1.640.2) from B.1.640 were added. B.1.640 isdesigned “Variant Under Monitoring, VUM” from WHO. (06 January, 2022)
In Rev.10, three recombined variants (XD, XE and XF) from Omicron and Delta were added. XD, XE
and XF were given named by cov-lineages (the earliest date XD 03 January, 2022, XE 19 January,
2022 and XF 07 January, 2022, reference on site: https://github.com/cov-lineages).
In Rev.11, BA.4, BA.5 and BA.2.75 that are sub-lineages from Omicron were updated, BA.4.1 (hCoV19/South Africa/NCV1112/2022) was first designated as BA.4 on April 14, 2022, and re-designatedon May 22, 2022 (from pango-designation issue #548).

GISAID ACCESSION ID.

No.

Virus sort

WHO label

PANGO Lineage

1

SARS-CoV-2

N/A

B

402125China

2

SARS-CoV-2

N/A

A.23.1925892

United kingdom

3

SARS-CoV-2

N/A

AT.1

2385327

Russia4

SARS-CoV-2

N/A

AT.1

1259283

Russia5

SARS-CoV-2

Alpha

B.1.1.7

835226

United kingdom

6

SARS-CoV-2

Beta

B.1.351

660190South Africa

7

SARS-CoV-2

EpsilonB.1.4271060793

USA

8

SARS-CoV-2

EpsilonB.1.429

1771435

USA

9

SARS-CoV-2

EpsilonB.1.4291194304

USA

10

SARS-CoV-2

Eta

B.1.5252432518

Nigeria11

SARS-CoV-2

N/A

B.1.526.1

2204920

USA

12

SARS-CoV-2

N/A

B.1.526.2

1080752

USA

13

SARS-CoV-2

B.1.526

B.1.526

1227165

USA

14

SARS-CoV-2

N/A

B.1.616

1239370

France15

SARS-CoV-2

Kappa

B.1.617.1

1360306

India

16

SARS-CoV-2

Delta

B.1.617.2

1508996

India

17

SARS-CoV-2

N/A

B.1.617.3

1704494

India

4 / 16

EPI_ISLOutbreak country18

SARS-CoV-2

Mu

B.1.621

1582980

Colombia

19

SARS-CoV-2

N/A

C.36

1936140

Egypt

20

SARS-CoV-2

LambdaC.37

1111296

Peru

21

SARS-CoV-2

Gamma

P.1

792680Japan/Brazil

22

SARS-CoV-2

LambdaP.2

1182578

Brazil23

SARS-CoV-2

Gamma

P.3

1213573

Philippines

24

SARS-CoV-2

Zeta

B.1.617.1

1789542

India

25

SARS-CoV-2

Theta

B.1.617.1

1620161

India

26

SARS-CoV-2

Kappa

B.1.617.1

1545312Angola / Luanda

27

SARS-CoV-2

Kappa

B.1.617.1

1823120

Jordan / Amman

28

SARS-CoV-2

Kappa

B.1.617.1

1904467

Australia / New South Wales

29

SARS-CoV-2

Kappa

B.1.617.1

1660436

Bahrain

30

SARS-CoV-2

Kappa

B.1.617.1

1913208

Canada / Victoria

31

SARS-CoV-2

Kappa

B.1.617.1

1969991

Pakistan

32

SARS-CoV-2

Delta

B.1.617.2

1970310

India / Delhi

33

SARS-CoV-2

Delta

B.1.617.2

1660458

Bahrain

34

SARS-CoV-2

Delta

B.1.617.2

1807318

Australia / Capital Territory

35

SARS-CoV-2

Delta

B.1.617.2

1913205

Canada / Victoria

36

SARS-CoV-2

Delta

AY.1

3244751

India

37

SARS-CoV-2

Delta

AY.2

3123565

Albania38

SARS-CoV-2

Delta

AY.3

3352221

USA

39

SARS-CoV-2

Delta

AY.3.12920875

USA

40

SARS-CoV-2

Mu

B.1.6213477571

South America / Colombia

41

SARS-CoV-2

N/A

C.1.2

2695610South Africa

42

SARS-CoV-2

OmicronB.1.1.529

6647959South Africa / Gauteng

43

SARS-CoV-2

OmicronBA.1

6640917

Africa/ Botswana

44

SARS-CoV-2

OmicronBA.2

7190366

Oceania / Australia

45

SARS-CoV-2

OmicronBA.3

7526186

United Kingdom / England

46

SARS-CoV-2

N/A

B.1.640.1

6700813

Republic of the Congo

47

SARS-CoV-2

N/A

B.1.640.2

7181977

Europe / United Kingdom

48

SARS-CoV-2

Omicron + Delta

9879437

France9177743

United Kingdom8894978

United Kingdom

XD*(Delta and BA.1)
XE*
49

SARS-CoV-2

Omicron(BA.1 and BA.2)
XF*

50

SARS-CoV-2

Omicron + Delta(Delta and BA.1)

51

SARS-CoV-2

Omicron

BA.4.1a)12043292

South Africa

52

SARS-CoV-2

OmicronBA.5

11903045

South Africa

53

SARS-CoV-2

OmicronBA.5

12307612 **South Africa

5 / 16

54

SARS-CoV-2

OmicronBA.2.75

13826295***

India

55

SARS-CoV-2

OmicronBA.2.75

13711333***

India* XD, XE, XF are characterized by combining with the other two lineages (Delta + Omicron) byconsidering all mutation site including both spike protein and nucleocapsid protein. However, if only
the sequence of nucleocapsid protein is considered, a single lineage can be characterized.
** Accession number of 12307612 is BA.5 sub-lineage with very small portion (7.86% by GISAID,
2022.06.16)*** E31, R32 and S33 amino acid deletions of BA.2.75 occur in 74.15%, 74.15% and 72.79%
respectively (Covspectrum, 2022.07.22).
a) BA.4.1 (hCoV-19/South Africa/NCV1112/2022) was first designated as BA.4 on April 14, 2022,
and re-designated on May 22, 2022 (from pango-designation issue #548).
3) Epitope of antibodies
Studies investigating the epitopes of the antibodies in the materials used in all SARS-CoV-2 antigen
diagnostic products were conducted previously. (Document: Q COVID-19 Ag_Epitope mapping
analysis for the antibodies_Rev.03) The epitopes of paired antibodies used (A and B) are located in
the CTD region spanning the amino acids (aa) 258-361 of the SARS-CoV-2 N protein.

6 / 16

3. CRITERIA
The most frequent mutation site(s) of each variant was (were) compared to the epitope region by in-silico
analysis. If the mutation site is located within the epitope region, a possible impact on test performance waspredicted. If the mutation site is not located within the epitope region, impact on test performance waspredicted to be unlikely.
The criteria below were used for classification.

Result of in-silico analysis

Classification

Mutation site is not located in the epitope regionN

Mutation site is located in the epitope region

P

Prediction
Achangeperformanceisunlikely
There is a possibility of a change
in performance

If the in-silico result is decision “P”, the variant should be tested by wet testing.

7 / 16in4. RESULT[Overall Variants list]

No.

Virus sort

WHO label

PANGO Lineage

Country of first

GISAID ACCESSION

Most frequentID.

mutation sites

EPI_ISL(amio acid numbers)

outbreakEpitope region

Classification

1

SARS-CoV-2

N/A

B

China

402125

N/A (as standard)

N/A

2

SARS-CoV-2

N/A

A.23.1

United kingdom925892

202

N

3

SARS-CoV-2

N/A

AT.1

Russia238532767, 203, 204

N

4

SARS-CoV-2

N/A

AT.1

Russia1259283

203, 204

N

5

SARS-CoV-2

Alpha

B.1.1.7

United kingdom

8352263, 203, 204, 235

N

6

SARS-CoV-2

Beta

B.1.351

South Africa

660190205

N

7

SARS-CoV-2

EpsilonB.1.427

USA

1060793205

N

8

SARS-CoV-2

EpsilonB.1.429

USA

1771435

205, 234N

9

SARS-CoV-2

EpsilonB.1.429

USA

1194304

205

N

10

SARS-CoV-2

Eta

B.1.525

Nigeria

2432518

2, 12, 205

N

11

SARS-CoV-2

N/A

B.1.526.1

USA2204920

205, 234

12

SARS-CoV-2

N/A

B.1.526.2

USA1080752

13, 202

N

13

SARS-CoV-2

B.1.526

B.1.526

USA

1227165

199, 234

N

14

SARS-CoV-2

N/A

B.1.616

France1239370

325

P

15

SARS-CoV-2

Kappa

B.1.617.1

India1360306

203, 377

N

16

SARS-CoV-2

Delta

B.1.617.2

India1508996

63, 203, 215, 377

N

17

SARS-CoV-2

N/A

B.1.617.3

India1704494

67, 203, 377

N

18

SARS-CoV-2

Mu

B.1.621

Colombia

1582980

205

N

19

SARS-CoV-2

N/A

C.36

Egypt

1936140

203, 204, 212

N

20

SARS-CoV-2

LambdaC.37

Peru111129613, 203, 204, 214, 366

N

21

SARS-CoV-2

Gamma

P.1

Japan/Brazil

79268080, 203, 204N

8 / 16258~361N22

SARS-CoV-2

LambdaP.2

Brazil1182578

119, 203, 204, 234

N

23

SARS-CoV-2

Gamma

P.3

Pilippines

1213573

203, 204

N

24

SARS-CoV-2

Zeta

B.1.617.1

India

1789542

203, 377, 385

N

25

SARS-CoV-2

Theta

B.1.617.1

India

1620161

3, 203, 377

N

26

SARS-CoV-2

Kappa

B.1.617.1

Angola / Luanda

1545312203, 204

N

27

SARS-CoV-2

Kappa

B.1.617.1

Jordan / Amman

1823120

203, 236, 377

N

28

SARS-CoV-2

Kappa

B.1.617.1

Australia / New South Wales

1904467

3, 13, 203, 243, 377

N

29

SARS-CoV-2

Kappa

B.1.617.1

Bahrain

16604363, 63, 203, 377

N

30

SARS-CoV-2

Kappa

B.1.617.1

Canada / Victoria

191320830, 203, 377

N

31

SARS-CoV-2

Kappa

B.1.617.1

Pakistan

1969991

203, 310, 377

P

32

SARS-CoV-2

Delta

B.1.617.2

India / Delhi

197031063, 203, 377. 385

N

33

SARS-CoV-2

Delta

B.1.617.2

Bahrain

1660458

63, 203, 377

N

34

SARS-CoV-2

Delta

B.1.617.2

Australia / Capital Territory

180731863, 203, 204, 205, 206, 207,
N
208, 377, 385
35

SARS-CoV-2

Delta

B.1.617.2

Canada / Victoria

191320563, 203, 215, 377

N

36

SARS-CoV-2

Delta

AY.1

India

324475163, 203, 215, 377

N

37

SARS-CoV-2

Delta

AY.2

Albania

3123565

63,203,377

N

38

SARS-CoV-2

Delta

AY.3

USA

335222163, 203, 215, 377

N

39

SARS-CoV-2

Delta

AY.3.1

USA2920875

63, 203, 215, 377

N

40

SARS-CoV-2

Mu

B.1.621

South America / Colombia

3477571

205

N

41

SARS-CoV-2

N/A

C.1.2

South Africa

2695610204, 13, 384, 203

N

42

SARS-CoV-2

OmicronB.1.1.529

South Africa / Gauteng

6647959

13, 31del, 32del, 33del,N
203, 204BA.1
13, 31del, 32del, 33del,43

SARS-CoV-2

Omicron(previously

Africa/ Botswana

6640917

N
203, 204B.1.1.529)

9 / 16

13, 31del, 32del, 33del,44

SARS-CoV-2

OmicronBA.2

7190366

Oceania / AustraliaN
203, 204, 413
13, 31del, 32del, 33del,45

SARS-CoV-2

OmicronBA.3

United Kingdom / EnglandN

7526186
203, 204, 413

46

SARS-CoV-2

N/A

B.1.640.1

Republic of the Congo

670081363, 205, 378

N

47

SARS-CoV-2

N/A

B.1.640.2

Europe / United Kingdom

7181977

22, 205

N

48

SARS-CoV-2

Omicron + Delta

France9879437

63, 203, 215, 377

N

United Kingdom

9177743

XD*(Delta and BA.1)
XE*
49

SARS-CoV-2

13, 31del, 32del, 33del,Omicron(BA.1 and BA.2)
XF*

50

SARS-CoV-2

N
203, 204, 413
13, 31del, 32del, 33del,Omicron+ Delta

United Kingdom8894978(Delta and BA.1)

N
203, 204
P13L, E31del, R32del,51

SARS-CoV-2

Omicrona)BA.4.1

South Africa

12043292

S33del, P151S, R203K,

N

G204R, S413R
P13L, E31del, R32del,52

SARS-CoV-2

Omicron

BA.5

South Africa

11903045

S33del, R203K, G204R,

N

S413R
P13L, E31del, R32del,53

SARS-CoV-2

Omicron

BA.5

South Africa

12307612**

S33del, E136D, R203K,

N

G204R, S413R
P13L, E31del, R32del54

SARS-CoV-2

OmicronBA.2.75

India

13826295***

S33del, R203K, G204R,

N

S413R
P13L, G204R, R203K,
55

SARS-CoV-2

OmicronBA.2.75

India

13711333***

N
S413R

10 / 16

[Rev.10, XD, XE and XF nucleocapsid protein sequence analysis]
New investigating variant

Previous investigated variant

XD

B.1.617.2 (Delta)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_9879437)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_1913205)[D63G, R203M, G215C, D377Y][D63G, R203M, G215C, D377Y]

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEG

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKELKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKGLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGAN

DGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRSRNSTPGSSMGTSPARMAGNGCDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKNSSRNSTPGSSMGTSPARMAGNGCDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEAKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRISKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKAYETQALPQRQRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKAYETQALPQR

KKQQTVTLLPAADLDDFSKQLQQSMSSADSTQAQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA% of homology

100%

BA.2 (Omicron)

XE
Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_9177743)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_7190366)[P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R][P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R]

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRI

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA
BA.1 (Omicron)XF

11 / 16

100%

100%

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_8894978)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_6640917)[P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R][P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R]

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRI

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

KKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA

KKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA[Rev.11, BA.4.1, BA.5 and BA.2.75 nucleocapsid protein sequence analysis]

BA.4.1a)BA.2 (Omicron)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_12043292)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_7190366)[P13L, E31(Del*), R32(Del*), S33[Del*], P151S, R203K, G204R, S413R][P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R]

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNSANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRI

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

99%a) BA.4.1 (hCoV-19/South Africa/NCV1112/2022) was first designated as BA.4 on April 14, 2022, and re-designated on May 22, 2022 (from pango-designation issue #548).

BA.5

BA.2 (Omicron)

12 / 16100%

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_11903045)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_7190366)[P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R][P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R]

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRI

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

BA.5

BA.2 (Omicron)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_12307612***)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_7190366)[P13L, E31(Del*), R32(Del*), S33[Del*], E136D, R203K, G204R, S413R][P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R]

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATDGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRI

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

BA.2.75

99%

BA.2 (Omicron)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_13826295)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_7190366)[P13L E31(Del*), R32(Del*), S33(Del*), R203K, G204R, S413R][P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R]

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

100%

13 / 16

LKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRI

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

GMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

BA.2.75

BA.2 (Omicron)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_13711333)

Nucleocapsid Protein Sequence (accession no. GISAID ACCESSION ID. EPI_ISL_7190366)[P13L, G204R, R203K, S413R][P13L, E31(Del*), R32(Del*), S33[Del*], R203K, G204R, S413R]

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED

MSDNGPQNQRNALRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQR

KKQQTVTLLPAADLDDFSKQLQQSMSRADSTQAQKKQQTVTLLPAADLDDFSKQLQQSMSRADSTQA

14 / 1699%

5. CONCLUSION
The mutation sites of the most frequent mutations determined for 55 variants using the GISAID database in
the SARS-CoV-2 nucleocapsid protein compared to the Wuhan-Hu-1 sequence were compared to theepitope region of the paired antibodies used in the STANDARDTM COVID-19 Ag (aa 258-361). Two variants(B.1.616, listed as no.14 and B.1.617.1, listed as no.31) show a mutation within the epitope region (no.14 is
at aa 325, no.31 is at aa 310). B.1.616 variant was detected in France in the spring of 2021 and remained
very locally restricted. Per 18 November 2021, it is not currently classified as a VoC or Variants of Interest(VoI) by the WHO, nor is it classified as a Variant Being Monitored (VBM), a VOI, a VOC or a Variant of High
Consequence (VOHC) by the US CDC.
All other 45 variants showed mutations outside of the epitope region and would not affect assay performance.
Nonetheless, B.1.616 and B.1.617.1 (GISAID Accession ID EPI_ISL 1969991) should be wet-tested forpotential changes in assay performance.
The B.1.1.529 variant was designated by WHO at 2021.11.26 as the VoC.
New emerged variant B.1.1.529 (no.42, Omicron) and its sub-lineages (no.43-45, BA.1, BA.2, BA.3) haveespecially feature that the deleted sites (31del, 32del, 33del) of N protein. all mutation sites of B.1.1.529 andsub-lineages are not located in epitope region.
Nonetheless, wet-testing of the new emerged variants can help with confidence of performance along with
the in-silico analysis.
New emerging variants B.1.640.1 and B.1.640.2 are sub-lineage from B.1.640.
B.1.640.1 and B.1.640.2 would not affect assay performance, since the mutation sites on N protein of both
B.1.640.1 and B.1.640.2 are not located in epitope region.

New recombined variant (XD, XE and XF) were from Delta and Omicron. Nucleocapsid protein of
XD is same to the delta (B.1.617.2) variant, XE is same the omicron (BA.2) variant and XF is sameto the omicron (BA.1) We previously investigated the B.1.617.2, BA.2 and BA.2, all do not affectthe performance of STANDARDTM COVID-19 Ag Test.
To be conclusion, XD, XE and XF also do not affect the performance of STANDARDTM COVID-19
Ag and they are unnecessary to study with wet-testing.
BA.4 is very similar to BA.2, with only one additional mutation site and the mutation sites are not
located in epitope region.
BA.5 (GISAID ACCESSION ID. EPI_ISL_12307612) has especially other mutation site on the N
protein sequence comparing to BA.2. It appeared in the very small portion (7.86%) and thismutation site is also not located in epitope region.
New emerging variants B.2.75 are sub-lineages from BA.2. Two types of BA.2.75 have beenreported so far, and BA.2.75 (accession no. 13826295) has the same NP sequence as BA.2. The
other one (accession no. 13711333) shows 99% homology with BA.2 because the deletion site is

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missing. In both variants, mutation sites are not located in the epitope region. BA.2 wet-testing waspreviously performed, and there are no performance changes.

6. REFERENCES(1) GISAID: https://www.epicov.org(2) Nextstrain: https://nextstrain.org(3) PANGO lineages: https://cov-lineages.org(4) European Centre for Disease Prevention and Control: https://www.ecdc.europa.eu/en/covid-19/variants-concern(5) CDC: https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html#anchor_1632158885160(6) WHO tracking of variants: https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/

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