Abstract

Introduction: The emergence of a novel coronavirus, SARS-CoV-2, an etiologic agent of coronavirus disease (COVID-19), has led to a pandemic of global concern. Considering the huge number of morbidity and mortality worldwide, the World Health Organization declared, on 11th March 2020, the pandemic as an unprecedented public health crisis. The virus is a member of plus sense RNA viruses that can show a high rate of mutations. The ongoing multiple mutations in the structural proteins of coronavirus drive viral evolution, enabling them to evade the host immunity and rapidly acquire drug resistance. In the present study, we focused mainly on the prevalence of mutations in the four types of structural proteins- S (spike), E (envelope), M (membrane), and N (nucleocapsid)- that are required for the assembly of a complete virion particle. Further, we estimated the antigenicity and allergenicity of these structural proteins to design and develop a potentially good candidate vaccine against SARS-CoV-2.


Methods: In the present in silico study, envelope protein was found to be highly antigenic, followed by the nucleocapsid, membrane, and spike proteins of SARS-CoV-2.


Results: In this study, we detected 987 mutations from 729 sequences from Asia in October 2020, and compared them with China's first Wuhan isolate sequence as a reference. Spike protein showed the highest mutations with 807 point mutations among the four structural proteins, followed by nucleocapsid with 151 mutations, while envelope showed 19 mutations and membrane only 10 point mutations.


Conclusion: Taken together, our study revealed that variations occurring in the structural protein of SARS-CoV-2 might be altering the viral structure and function, and that the envelope protein appears to be a promising vaccine candidate to curb coronavirus infections.

Keywords: allergenicity antigenicity COVID-19, mutation SARS-CoV-2 structural proteins vaccine


Introduction

Human Coronavirus (SARS-CoV-2, Severe acute respiratory syndrome) is a positive-sense RNA virus. As an etiologic agent of coronavirus disease 2019 (COVID-19), the virus induces moderate to severe respiratory distress1. This pandemic originated from an animal market in Wuhan city of China2. The ripple effect of this contagious viral disease has created a humanitarian health crisis and has become an enormous challenge to the entire health systems across the globe.

SARS-CoV-2 is a member of the Coronaviridae family and Nidovirales order. The virus is considered the third zoonotic coronavirus (after SARS-CoV and MERS-CoV) and originated from bats. However, this novel coronavirus has been the only one having pandemic potential3, 4, 5, 6. SARS-CoV-2, a beta coronavirus, is an enveloped single-stranded, positive-sense, non-segmented and genetically diverse RNA virus with the largest genome size among known RNA viruses (29,891 ase pair, encodes for approximately 9860 amino acids)2, 7, 8. The genome of SARS-CoV-2 encodes both structural proteins like spike (S), envelope (E), membrane (M), and nucleocapsid (N), as well as non-structural proteins ranging from NSP1 to NSP16.

RNA viruses, generally, show a drastically high rate of mutation, substantially higher than those of DNA viruses. Due to this high rate of mutation shown by SARS-CoV-2 over a short period, it has been observed that viruses exhibit genomic variability which enables them to modulate virulence properties in the host and subsequently evade the host immunity9, 10.

In the present research work, we detected 987 mutations from 729 sequences derived from Asia in in the October. Altogether spike showed the highest mutations with 807 point mutations among the four structural proteins, followed by nucleocapsid with 151 mutations. Envelope showed 19 mutations and membrane showed only 10 point mutations. The results of our study suggest that mutational analysis of this virus might be considered as a new approach to help understand its genomic variability. Similarly, using the predictive tools of immunoinformatics approach, the antigenicity and allergenicity of the structural proteins of SARS-CoV-2 have been determined to develop efficacious antiviral therapeutics or vaccines against COVID-19.

Methods

Data mining

The full-length protein sequences of SARS-CoV-2 structural proteins, i.e., envelope protein, nucleocapsid phosphoprotein, surface glycoprotein and membrane glycoprotein, were retrieved from the NCBI virus database, as submitted from Asia in the month of October. There were 729 SARS-CoV-2 structural protein sequences submitted from Asia in the month of October, including sequences of 165 envelope proteins, 159 nucleocapsid phosphoproteins, 246 surface glycoproteins, and 159 membrane glycoproteins. A total of four reference sequences for envelope protein (YP_009724392), nucleocapsid phosphoprotein (YP_009724397), surface glycoprotein (YP_009724390), and membrane glycoprotein (YP_009724393) were also retrieved for mutational studies.

Multiple sequence alignment (MSA) and mutational identification

Multiple sequence alignment was performed using Clustal Omega online platform (http://www.clustal.org/) based on HMM profile seeded guide trees11. The envelope, nucleocapsid phosphoprotein, surface glycoprotein, and membrane glycoprotein were aligned with their respective reference sequences. The aligned files were viewed using Jalview (https://www.jalview.org/) to identify the point mutations occurring in different structural proteins with respect to the Wuhan type isolate.

Antigenicity and allergenicity evaluation

Vaxijen v2.0 server was used for the estimation of antigenicity of all the four structural proteins to study the capability of structural proteins to be used in vaccine production. This online server predicts antigens as per the auto cross-covariance (called ACC transformation) of the peptide sequences submitted to it12. A good vaccine needs to be non-allergenic to the host, hence the rationale for evaluating the allergenicity of these structural proteins, AllerTOP server was used, which predicts allergenicity based on size, flexibility, and other parameters13.

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Figure 1 . Showing the total number of mutations occurring in the structural proteins. a . Surface glycoprotein, b . Envelope protein, c . Membrane glycoprotein and, d . Nucleocapsid phosphoprotein.

Table 1.

Mutational location after Multiple Sequence Alignment of SARS-CoV-2 envelope protein sequence with position and sequence

Serial No. Accession Mutated sequence and position
1. BCM16104 S68F
2. BCM16116 S68F
3. BCM16128 S68F
4. BCM16176 S68F
5. BCM16188 S68F
6. BCM16200 S68F
7. BCM16212 S68F
8. BCM16140 S68F
9. QOP57282 V75F
10. QOP57300 V75F
11. QOP57289 V75F
12. QOP57280 V75F
13. QOP57294 V75F
14. QOS50800 V75F
15. QOS50895 I46V
16. QOS50728 V75F
17. QOS50501 V75F
18. QOU99241 I46V
19. QOU99253 I46V

Table 2.

Mutational location after Multiple Sequence Alignment of SARS-CoV-2 nucleocapsid phosphoprotein sequence with position and sequence

Serial No. Accession Mutated sequence and position
1. QJF74875 R203K
2. QJF74875 G204R
3. QKM75385 R203K
4. QKM75385 G204R
5. QKM75397 R203K
6. QKM75397 G204R
7. QKM75409 R203K
8. QKM75409 G204R
9. QKM75421 R203K
10. QKM75421 G204R
11. QKM75433 R203K
12. QKM75433 G204R
13. QKM75445 P207L
14. QKM75445 M210I
15. QKM75505 R203K
16. QKM75505 G204R
17. QKM75505 D377G
18. QKM75517 R203K
19. QKM75517 G204R
20. QKM75517 D377G
21. QKM75529 R203K
22. QKM75529 G204R
23. QKM75529 D377G
24. QKM75541 G204R
25. QKM75541 D377G
26. QKM75541 R203K
27. QKM75552 R203K
28. QKM75552 G204R
29. QKM75552 D377G
30. QKM75563 R203K
31. QKM75563 G204R
32. QKM75563 D377G
33. QKM75575 R203K
34. QKM75575 G204R
35. QKM75587 R203K
36. QKM75587 G204R
37. QKM75599 R203K
38. QKM75599 G204R
39. QKM75647 R203K
40. QKM75647 G204R
41. QKM75659 R203K
42. QKM75659 R204R
43. QKM75683 R203K
44. QKM75683 G204R
45. QKM75695 R203K
46. QKM75695 G204R
47. QKQ30536 R203K
48. QKQ30536 G204R
49. QKQ30548 R40C
50. QKQ30560 R203K
51. QKQ30560 G204R
52. QKQ30572 R203K
53. QKQ30572 G204R
54. QKQ30584 R203K
55. QKQ30584 G204R
56. QLA10246 R203K
57. QLA10246 G204R
58. QLA10270 R203K
59. QLA10270 G204RR
60. QLA10282 R203K
61. QLA10282 G204R
62. QLA10294 P383L
63. QLA10294 R203K
64. QLA10294 G204R
65. QLA10306 R203K
66. QLA10306 G204R
67. QLA10318 G204R
68. QLA10318 R203K
69. QLA10330 G204R
70. QLA10330 R203K
71. QLA10342 G204R
72. QLA10342 R203K
73. QLA10354 R203K
74. QLA10354 G204R
75. QOI53600 P13L
76. QOQ57020 S194L
77. QOQ57032 S194L
78. QOQ57044 S194L
79. QOQ57056 S194L
80. QOQ57068 S194L
81. QOQ57092 M234I
82. QOQ57104 S194L
83. QOQ57116 S194L
84. QOQ57129 S194L
85. QOQ72552 S194L
86. QOQ72564 S194L
87. QOQ72576 S194L
88. QOQ84803 S194L
89. QOQ84834 S194L
90. QOR63442 T205I
91. QOR63454 S194L
92. QOR63466 T205I
93. QOR63514 A119S
94. QOR63514 S194L
95. QOR64241 S194L
96. QOR64253 S194L
97. QOS50459 P13L
98. QOS50495 T91I
99. QOS50507 P13L
100. QOS50519 P13L
101. QOS50531 P13L
102. QOS50590 P13L
103. QOS50650 P13L
104. QOS50674 P13L
105. QOS50686 P13L
106. QOS50686 D225Y
107. QOS50722 P13L
108. QOS50734 P13L
109. QOS50746 P13L
110. QOS50746 S413I
111. QOS50758 S413I
112. QOS50758 P13L
113. QOS50770 P13L
114. QOS50782 P13L
115. QOS50818 P13L
116. QOS50830 P13L
117. QOS50853 P13L
118. QOS50865 P13L
119. QOS50889 Q9H
120. QOS50889 P199S
121. QOS50901 S202N
122. QOS50924 S202N
123. QOS50948 P13L
124. QOS50960 P13L
125. QOS50972 P13L
126. QOS50996 P13L
127. QOS51008 P13L
128. QOS51020 P13L
129. QOS51032 P13L
130. QOS51068 R209I
131. QOS51068 P367L
132. QOS51080 R203K
133. QOS51080 G204R
134. QOS51092 P13L
135. QOS51104 R203K
136. QOS51104 G204R
137. QOU99154 P14L
138. QOU99201 Q9H
139. QOU99201 P199S
140. QOU99223 Q9H
141. QOU99223 P199S
142. QOU99247 S202N
143. QOU99259 S202N
144. QOU99270 Q9H
145. QOU99270 P199S
146. QOU99281 Q9H
147. QOU99281 P199S
148. QOU99292 Q9H
149. QOU99292 P199S
150. QOU99303 Q9H
151. QOU99303 P199S

Results

Mutational identification

A total of 729 structural protein sequences were retrieved from the NCBI virus database for spike glycoproteins, nucleocapsid phosphoproteins, envelope proteins, and membrane glycoproteins submitted from Asian countries in the month of October 2020, along with four references sequences. The size of the different reference structural proteins, i.e., spikes glycoprotein, nucleocapsid phosphoprotein, envelope protein, and membrane glycoprotein being 1273, 419, 75, and 222 amino acids.

The sequences were viewed using Jalview after alignment to compare and detect the mutations among the Asian isolates with the Wuhan isolates with respect to structural proteins. Amongst the 729 sequences released from Asia, a total of 987 point mutations were detected in all four structural proteins (Figure 1). Among the 311 mutants, spike showed the highest mutations with 807 point mutations (Table 3), followed by nucleocapsid with 151 mutations (Table 2), while envelope showed 19 mutations (Table 1) and membrane showed only 10 point mutations (Table 4).

Table 3.

Mutational location after Multiple Sequence Alignment of SARS-CoV-2 surface glycoprotein sequence with position and sequence

S. No. Accession Mutated sequence and position
1. QJF74843 V367F
2. QJF74867 D614G
3. QOI53592 M153I
4. QMI57728 T95I
5. QMI57728 N185K
6. QOI53580 D614G
7. QOR64233 D614G
8. QOR64245 D614G
9. QOQ57012 D614G
10. QOQ57024 D614G
11. QOQ57060 D614G
12. QOR64233 A701T
13. QOR64233 P812L
14. QOR64245 P812L
15. QOQ57012 P812L
16. QOQ57060 P812L
17. QOR64233 H1083Q
18. QOR64245 H1083Q
19. QOQ57012 H1083Q
20. QOQ57024 H1083Q
21. QOQ57072 D614G
22. QOQ57084 D614G
23. QOQ57096 D614G
24. QOQ57108 D614G
25. QOQ57121 D614G
26. QOQ57108 A701T
27. QOQ57072 A701T
28. QOQ57096 P812L
29. QOQ57121 P812L
30. QOQ57096 H1083Q
31. QOQ57121 H1083Q
32. QOQ57108 H1083Q
33. QOQ72544 L54F
34. QOQ72556 L54F
35. QOQ72568 L54F
36. QOQ72544 D614G
37. QOQ72556 D614G
38. QOQ72568 D614G
39. QOQ72580 D614G
40. QOQ84795 D614G
41. QOQ72544 A701T
42. QOQ72556 P812L
43. QOQ72568 P812L
44. QOQ72580 P812L
45. QOQ72544 H1083Q
46. QOQ84826 D614G
47. QOR63434 D614G
48. QOR63446 D614G
49. QOR63458 D614G
50. QOR63470 D614G
51. QOR63434 P812L
52. QOR63470 P812L
53. QOQ53335 S305T
54. QOQ53335 C488R
55. QOR63482 D614G
56. QOQ57036 D614G
57. QOQ57048 D614G
58. QOR63506 D614G
59. QOQ53335 D614G
60. QOQ57048 A701T
61. QOQ57036 P812L
62. QOQ57048 P812L
63. QOQ57036 H1083Q
64. QOQ57048 H1083Q
65. QOQ53339 F2L
66. QOQ53339 V11I
67. QOQ53339 S13R
68. QOQ53339 Q14H
69. QOQ53339 R34H
70. QOQ53339 V42I
71. QOQ53339 R44K
72. QOQ53339 V47I
73. QOQ53339 F59I
74. QOQ53339 K77N
75. QOQ53339 D111N
76. QOQ53339 Q115H
77. QOQ53339 A123T
78. QOQ53339 N487I
79. QOQ53339 V512L
80. QOQ53339 A522P
81. QOQ53339 A262T
82. QOQ53339 Q677H
83. QOQ53336 G199R
84. QOQ53340 A262T
85. QOQ53338 C301S
86. QOQ53340 R328T
87. QOQ53337 R457T
88. QOQ53338 D614G
89. QOQ53340 D614G
90. QOQ53336 A684V
91. QOQ53336 A688P
92. QOQ53336 V705I
93. QOQ53337 H1048Y
94. QOQ53337 Q1180H
95. QOQ53337 K1181Q
96. QOQ53341 V11I
97. QOL24227 V11I
98. QOQ53341 R44K
99. QOQ53341 V47I
100. QOL24227 K77N
101. QOQ53341 K77N
102. QOQ53341 K97N
103. QOQ53341 D111N
104. QOL24227 D111N
105. QOL24227 R190K
106. QOL24227 D198E
107. QOL24225 E224K
108. QOL24225 D228N
109. QOL24226 E224K
110. QOL24226 D228N
111. QOL24227 A262T
112. QOQ53341 Q271H
113. QOQ53341 F275L
114. QOL24228 V407L
115. QOL24228 P412S
116. QOL24227 D427H
117. QOL24227 N440H
118. QOL24227 Q474P
119. QOL24228 D614G
120. QOL24227 D614G
121. QOQ53341 G669R
122. QOQ53341 Q675R
123. QOQ53341 Q677H
124. QOL24227 S686I
125. QOL24227 A688P
126. QOL24225 K790Q
127. QOL24226 K790Q
128. QOL24225 R815K
129. QOL24226 R815K
130. QOL24225 D820N
131. QOL24226 D820N
132. QOL24225 D830N
133. QOL24226 D830N
134. QOL24228 P863H
135. QOL24241 F2L
136. QOL24241 V11I
137. QOL24241 Q14H
138. QOL24241 R34C
139. QOL24241 Y37N
140. QOL24241 V42I
141. QOL24241 R44K
142. QOL24241 F65I
143. QOL78311 S94F
144. QOL78311 T95P
145. QOL24240 D111N
146. QOL24240 A282T
147. QOL78311 D568H
148. QOL78311 D614G
149. QOL24241 H655Y
150. QOL24240 Q675RR
151. QOL79057 S13N
152. QOL79057 D40E
153. QOL79057 V42L
154. QOL79057 S161F
155. QOL79057 S246N
156. QOL79057 D614G
157. QOL79058 D614G
158. QOL79058 R1019K
159. QOL79058 P1090L
160. QOL79059 V11F
161. QOL79059 R21K
162. QOL79135 R21K
163. QOL79135 A222V
164. QOL79061 K529I
165. QOL79059 D614G
166. QOL79135 D614G
167. QOL79061 E619K
168. QOL79061 G652R
169. QOL79061 Q677H
170. QOL79061 Y695N
171. QOL79061 V729A
172. QOL79136 D614G
173. QOL79137 V11I
174. QOL79137 Q115H
175. QOL79137 D614G
176. QOL79137 P863H
177. QOL79137 Q913H
178. QOL79137 I934T
179. QOL79333 C136W
180. QOL79333 N137Y
181. QOL79333 I203L
182. QOL21485 H207P
183. QOL20612 E224K
184. QOL21486 E224K
185. QOL21486 R237K
186. QOL21486 F238V
187. QOL21486 Q239P
188. QOL20612 T240S
189. QOL79332 A262T
190. QOL79333 L252P
191. QOL79332 D467N
192. QOL21486 A475V
193. QOL20612 Q506H
194. QOL20612 V510E
195. QOL20612 V512E
196. QOL20612 D614G
197. QOL21485 V511I
198. QOL79333 D568H
199. QOL79332 Q675R
200. QOL20612 V826G
201. QOL21485 V826G
202. QOL20612 I844M
203. QOL21486 I844F
204. QOL21486 R847K
205. QOL21486 D848N
206. QOL21486 C851W
207. QOL20612 R847K
208. QOL20612 D848N
209. QOL21535 T21I
210. QOL21535 V42A
211. QOL21535 V511E
212. QOL21535 C525W
213. QOL21535 K557R
214. QOL21535 R567K
215. QOL21535 N657I
216. QOL21535 Q677H
217. QOL21535 V722A
218. QOL21535 D737H
219. QOL21535 T768P
220. QOL21535 G769E
221. QOL21535 E780K
222. QOL21535 V781F
223. QOL21535 K790Q
224. QOL21535 I834J
225. QOL21536 E224K
226. QOL21536 V826G
227. QOL21536 Y837N
228. QOL21536 R847K
229. QOL21536 D848N
230. QOL21536 C851W
231. QOL21536 L858F
232. QOL21681 D228N
233. QOL21681 E780K
234. QOL21681 D808N
235. QOL21681 S816P
236. QOL21681 D820N
237. QOL21720 T22I
238. QOL21720 V213G
239. QOL21720 E224K
240. QOL21720 C538W
241. QOL21720 K825Q
242. QOL10078 R402I
243. QOL10078 V407L
244. QOL10078 P412S
245. QOL10078 D614G
246. QOL10078 P863H
247. QOJ86680 T22I
248. QOK36756 Y160I
249. QOK36756 N185I
250. QOK36756 Y200N
251. QOK36756 D228Y
252. QOJ86685 L226I
253. QOJ86685 V227M
254. QOJ86685 D228I
255. QOJ86685 L249S
256. QOJ86685 T250F
257. QOK36765 E224K
258. QOK36765 T240S
259. QOJ86685 A263S
260. QOJ86684 A262T
261. QOJ86684 D287Y
262. QOJ86684 V289E
263. QOK36765 A522T
264. QOK36766 V511I
265. QOJ86685 P464L
266. QOJ86685 T500I
267. QOJ86685 N501R
268. QOJ86685 R509K
269. QOJ86685 V511E
270. QOJ86685 S514P
271. QOJ86685 H519I
272. QOJ86685 P521Q
273. QOJ86685 A522P
274. QOJ86685 T523P
275. QOJ86685 P527T
276. QOJ86685 D568N
277. QOJ86685 D627N
278. QOJ86684 E583A
279. QOJ86684 D586E
280. QOJ86684 T602K
281. QOJ86684 N603K
282. QOJ86684 N606Y
283. QOJ86684 C617R
284. QOJ86684 G652R
285. QOJ86684 E661K
286. QOJ86685 E661D
287. QOJ86684 A672P
288. QOK36766 Q675R
289. QOK36766 N824K
290. QOK36766 K825Q
291. QOK36766 V826M
292. QOK36765 R847K
293. QOK36765 D848N
294. QOK36765 P863H
295. QOJ86680 P863H
296. QOJ86680 T874P
297. QOJ86680 L877M
298. QOK36756 P863H
299. QOK36767 V11I
300. QOK36767 R44K
301. QOK36767 A684V
302. QOK36767 V705I
303. QOK51484 V11I
304. QOK51484 K77N
305. QOK51484 D111N
306. QOK51484 Q115H
307. QOK51484 V159L
308. QOK51484 K187Q
309. QOK51484 R190K
310. QOK51484 K206N
311. QOK51484 A262T
312. QOK51484 D614G
313. QOK51484 V620F
314. QOK51484 G648R
315. QOK51484 A706R
316. QOL00258 V11I
317. QOL00258 K77N
318. QOL00258 D111N
319. QOL00258 Q271H
320. QOL00258 S443C
321. QOL00258 G669R
322. QOL00258 Q677H
323. QOL00258 Q675R
324. QOL00259 R44I
325. QOL00259 K77N
326. QOL00259 D111N
327. QOL00259 R158L
328. QOL00259 S162I
329. QOL00259 E169Q
330. QOL00259 Q173H
331. QOL00259 Q183H
332. QOL00259 D614G
333. QOL00259 S686T
334. QOL00259 E702Q
335. QOL00259 V705I
336. QOL00259 I714L
337. QOL00263 V11I
338. QOL00263 K77N
339. QOL00263 Q115H
340. QOL00263 H146P
341. QOL00263 E1569D
342. QOL00263 D614G
343. QOL00263 S750T
344. QOL00264 V11I
345. QOL00264 V42I
346. QOL00264 K77N
347. QOL00264 D111N
348. QOL00264 H207L
349. QOL00264 A262T
350. QOL00264 N487I
351. QOL00264 V512L
352. QOL00264 A522P
353. QOL00264 G669R
354. QOL00264 Q677H
355. QOL00264 Q675R
356. QOL00468 R44I
357. QOL00468 K77N
358. QOL00468 D111N
359. QOL00468 R158L
360. QOL00468 S162I
361. QOL00468 E169Q
362. QOL00468 Q173H
363. QOL00468 V407L
364. QOL00468 R408I
365. QOL00468 A411T
366. QOL00468 D614G
367. QOL00468 D627N
368. QOL00468 A653P
369. QOL00468 N658H
370. QOL00468 I664L
371. QOL00469 V11I
372. QOL00469 S13R
373. QOL00469 T29Q
374. QOL00469 R34H
375. QOL00469 V42I
376. QOL00469 R44K
377. QOL00469 V47I
378. QOL00469 F59I
379. QOL00469 F65I
380. QOL00469 K77N
381. QOL00469 D111N
382. QOL00469 S112T
383. QOL00469 Q115H
384. QOL00469 A123T
385. QOL00469 A262T
386. QOL00469 N487I
387. QOL00469 V512L
388. QOL00469 A522P
389. QOL00469 E661D
390. QOL00469 G669R
391. QOL00469 A672P
392. QOL00469 Q677H
393. QOL00501 E224K
394. QOL00501 I235V
395. QOL00501 K814Q
396. QOL00501 D830N
397. QOL00501 P863H
398. QOL00502 V11I
399. QOL00502 V47I
400. QOL00502 R78K
401. QOL00502 K77N
402. QOL00502 D111N
403. QOL00502 Q115H
404. QOL00502 E156D
405. QOL00502 R158P
406. QOL00502 G648R
407. QOL00502 G652R
408. QOL00502 S758T
409. QOL00502 V785L
410. QOL00502 K814N
411. QOL00502 K854N
412. QOL00503 P9Q
413. QOL00503 V11I
414. QOL00503 S13R
415. QOL00503 R102T
416. QOL00503 L18P
417. QOL00503 Y37N
418. QOL00503 N74H
419. QOL00503 G103R
420. QOL00503 K77N
421. QOL00503 R78K
422. QOL00503 K97N
423. QOL00503 A262T
424. QOL00503 A653P
425. QOL00503 N658H
426. QOL00503 I664L
427. QOL00503 R408T
428. QOL00503 G413R
429. QOL00503 D427N
430. QOL00503 D614G
431. QOL00503 D627N
432. QOJ75919 V11I
433. QOJ75919 L176F
434. QOJ75919 L179P
435. QOJ75919 A262T
436. QOJ75919 Q498Y
437. QOJ75919 P499T
438. QOJ75919 T716P
439. QOJ75919 I726F
440. QOJ75920 S438C
441. QOJ75920 N544T
442. QOJ75920 H655Y
443. QOJ75920 V705F
444. QOJ75920 Q836H
445. QOJ75921 R21K
446. QOJ75921 R44K
447. QOJ75921 D111N
448. QOJ75921 S112P
449. QOJ75921 A262T
450. QOJ75921 T302A
451. QOJ75921 D614G
452. QOJ75922 A262T
453. QOJ75922 N536K
454. QOJ75922 T791P
455. QOJ75922 K811N
456. QOJ75922 T827A
457. QOJ75923 N460I
458. QOJ75923 K462N
459. QOJ75923 P463L
460. QOJ75923 I468L
461. QOJ75923 G838D
462. QOJ75923 G842A
463. QOJ75923 D848A
464. QOJ75924 E132V
465. QOJ75924 V159I
466. QOJ75924 S162N
467. QOJ75924 E191D
468. QOJ75924 I197T
469. QOJ75924 Q173F
470. QOJ75924 M177I
471. QOJ75924 C1043G
472. QOJ75924 G485S
473. QOJ75924 L513F
474. QOJ75924 D614G
475. QOJ75924 G838A
476. QOJ75924 T998P
477. QOJ75924 Q1011H
478. QOJ75924 A1015P
479. QOJ75924 D178Y
480. QOJ75924 V193L
481. QOJ75924 K187N
482. QOJ75924 N188H
483. QOJ75924 R190I
484. QOJ75924 F194L
485. QOJ75925 Q564H
486. QOJ75925 F565C
487. QOJ75925 Q1180P
488. QOJ75926 T22I
489. QOJ75926 P225R
490. QOJ75926 I231L
491. QOJ75926 R237S
492. QOJ75926 G667A
493. QOJ75926 A672P
494. QOJ75926 Q677H
495. QOJ75926 L242P
496. QOJ75926 R246S
497. QOJ75926 A688T
498. QOJ75927 F65Y
499. QOJ75927 V120D
500. QOJ75927 V127D
501. QOJ75927 F133L
502. QOJ75927 F135L
503. QOJ75927 P139T
504. QOJ75927 C166S
505. QOJ75928 L244F
506. QOJ75928 L533F
507. QOJ75928 D809K
508. QOJ75929 V11I
509. QOJ75929 T22I
510. QOJ75929 A262T
511. QOJ75929 N30I
512. QOJ75929 H66L
513. QOJ75929 N487I
514. QOJ75929 N74H
515. QOJ75929 N641K
516. QOJ75930 D614G
517. QOJ75931 V11I
518. QOJ75931 H245Q
519. QOJ75931 S247N
520. QOJ75931 D614G
521. QOJ75931 R685G
522. QOJ75932 D614G
523. QOJ75932 W1102C
524. QOJ75932 N1108K
525. QOJ75932 T1116N
526. QOJ75933 D614G
527. QOJ75934 D614G
528. QOJ75934 T827I
529. QOJ75934 L828F
530. QOJ75935 V11I
531. QOJ75935 S13R
532. QOJ75935 Q14H
533. QOJ75935 D614G
534. QOJ75935 R682P
535. QOJ75935 S686N
536. QOJ75935 E702K
537. QOJ75935 N710T
538. QOJ75935 I714T
539. QOJ75936 F194V
540. QOJ75936 D614G
541. QOJ75936 F759S
542. QOJ75936 N764T
543. QOJ75936 E702K
544. QOJ75936 V705F
545. QOJ75936 T739S
546. QOJ75936 I742L
547. QOJ75936 T747P
548. QOJ75936 G757R
549. QOJ75937 L560P
550. QOJ75937 D614G
551. QOJ75938 G648R
552. QOJ75938 E661D
553. QOJ75938 G669R
554. QOJ75938 A672P
555. QOJ75938 Q677H
556. QOJ75939 D614G
557. QOJ75940 N440H
558. QOJ75940 F486L
559. QOJ75940 D614G
560. QOJ75941 A262P
561. QOJ75941 Y266N
562. QOJ75941 D614G
563. QOJ42687 V3D
564. QOJ42687 L10I
565. QOJ42687 V11I
566. QOJ42687 Q14H
567. QOJ42687 N544T
568. QOJ42687 E702K
569. QOJ42687 V705J
570. QOJ42687 W64C
571. QOJ42687 T385S
572. QOJ42687 E619K
573. QOJ42687 S686T
574. QOJ42687 A688P
575. QOJ42687 S689N
576. QOJ42687 Y695N
577. QOJ42687 S721T
578. QOJ42687 E725K
579. QOJ42687 L727P
580. QOJ42687 V729A
581. QOJ42687 M731K
582. QOJ42687 T22I
583. QOJ42687 Y28N
584. QOJ42687 T29P
585. QOJ42687 K41Q
586. QOE90911 Y38C
587. QOE90911 F86S
588. QOE90911 T95I
589. QOE90911 E96G
590. QOE90911 D290N
591. QOE90911 L296F
592. QOE90911 L303V
593. QOE90911 S151G
594. QOE90911 N185K
595. QOE90911 P272S
596. QOE90911 A288P
597. QOE90912 A222P
598. QOE90912 V227I
599. QOE90912 K424N
600. QOE90912 P863H
601. QOE90912 K535S
602. QOE90912 I1183F
603. QOE90912 D1199Y
604. QOE90914 D614G
605. QOE90914 Y636F
606. QOE90914 L650S
607. QOE90914 P863H
608. QOE90915 H656Y
609. QOE83920 K77N
610. QOE83920 A262T
611. QOE83920 N542T
612. QOE83920 D614G
613. QOE83920 V620F
614. QOE83972 P225T
615. QOE83972 A262T
616. QOE83972 N487I
617. QOE83972 D614G
618. QOE83972 V620I
619. QOE83972 P863H
620. QOE83973 Q115H
621. QOE83973 E156D
622. QOE83973 V171L
623. QOE83973 K182N
624. QOE83973 A262T
625. QOE83973 D614G
626. QOE83973 S758I
627. QOE83973 P863H
628. QOE83973 N925Y
629. QOE83973 G932A
630. QOE83973 T941P
631. QOE83974 S13R
632. QOE83974 L18P
633. QOE83974 A262T
634. QOE83974 P863H
635. QOE83974 I931T
636. QOE83974 G932A
637. QOE83974 S943T
638. QOE83974 Y37N
639. QOE83974 I100T
640. QOE83974 R408T
641. QOE83974 D427N
642. QOE83974 Q580H
643. QOE83974 Y636F
644. QOE83974 N641I
645. QOE83974 G648R
646. QOE83975 K535E
647. QOE83975 H824N
648. QOE83975 G842C
649. QOE83975 V1177E
650. QOE84134 I231L
651. QOE84134 T240N
652. QOE84134 N280D
653. QOE84134 A288P
654. QOE84134 T302A
655. QOE84134 V736L
656. QOE84134 N751H
657. QOE84134 S816A
658. QOE84134 D820N
659. QOE84134 I788F
660. QOE84134 K811N
661. QOE84134 S813P
662. QOE84134 K814Q
663. QOE84221 F106L
664. QOE84221 Q134P
665. QOE84221 N148T
666. QOE84221 A262T
667. QOE84221 P863H
668. QOE84221 V510E
669. QOE84221 K535E
670. QOE84221 N969H
671. QOE84221 F486L
672. QOE84221 N487I
673. QOE84221 Q493P
674. QOE84221 N501K
675. QOE84222 A262T
676. QOE84222 S459Y
677. QOE84222 D614G
678. QOE84223 P225S
679. QOE84223 L229F
680. QOE84223 I231L
681. QOE84223 P863H
682. QOE84223 K1181Q
683. QOE84223 D820N
684. QOE84223 L821R
685. QOE84223 F823C
686. QOE84223 R815K
687. QOE84223 S816T
688. QOE84223 I818F
689. QOE84223 G798C
690. QOE84223 K814M
691. QOE84223 G838A
692. QOE84223 G842R
693. QOE84223 D843E
694. QOE84223 V826L
695. QOE84223 A829E
696. QOE84223 D830H
697. QOE84223 I834T
698. QOE84224 D398N
699. QOE84224 K535G
700. QOE84224 E702K
701. QOE84224 V705F
702. QOE84224 E725K
703. QOE84224 T736P
704. QOE84224 D775N
705. QOE84224 P863H
706. QOE84224 Y837N
707. QOE84224 A879P
708. QOE84224 E918Q
709. QOU99158 D614V
710. QOU99170 D614V
711. QOU99296 P1263L
712. QOS50441 K77M
713. QOS50451 A701S
714. QOS50594 D614G
715. QOS50606 D614G
716. QOS50630 D614G
717. QOS50654 D614G
718. QOS50654 D936H
719. QOS50678 D627P
720. QOS50726 K77M
721. QOS50750 I624T
722. QOS50834 F135L
723. QOS50845 M731I
724. QOS50952 K77M
725. QOS50964 K77M
726. QOS50928 D614G
727. QOS50928 K1045N
728. QOS50988 K77M
729. QOS51000 K77M
730. QOS51012 K77M
731. QOS51024 K77M
732. QOS51036 K77M
733. QOS51024 D88H
734. QOS50988 D1084Y
735. QOS51000 D1084Y
736. QOS51012 D1084Y
737. QOS51024 D1084Y
738. QOS51036 D1084Y
739. QOS51060 D614G
740. QOS51072 D614G
741. QOS51096 D614G
742. QOS51108 D614G
743. QOS51120 D614G
744. QKM75377 D614G
745. QKM75389 D614G
746. QKM75401 D614G
747. QKM75413 D614G
748. QKM75425 D614G
749. QKM75377 T547I
750. QKM75413 V213L
751. QKM75473 I569S
752. QKM75437 D614G
753. QKM75449 D614G
754. QKM75461 D614G
755. QKM75473 D614G
756. QKM75485 D614G
757. QKM75485 F797C
758. QKM75485 G799S
759. QKM75497 D614G
760. QKM75509 D614G
761. QKM75521 D614G
762. QKM75533 D614G
763. QKM75545 D614G
764. QKM75556 D614G
765. QKM75567 D614G
766. QKM75579 D614G
767. QKM75591 D614G
768. QKM75603 D614G
769. QKM75579 H146Q
770. QKM75591 H146Q
771. QKM75556 Y680F
772. QKM75567 G1167V
773. QKM75615 D614G
774. QKM75627 D614G
775. QKM75639 D614G
776. QKM75651 D614G
777. QKM75663 D614G
778. QKM75639 N211Y
779. QKM75651 N211Y
780. QKM75627 G769V
781. QKM75675 D614G
782. QKM75687 D614G
783. QKQ30528 D614G
784. QKQ30540 D614G
785. QKQ30552 D614G
786. QKQ30528 G142V
787. QKM75687 N211Y
788. QKQ30552 N211Y
789. QKQ30552 T236S
790. QKM75675 S256L
791. QKM75675 V1264M
792. QKQ30564 D614G
793. QKQ30576 D614G
794. QLA10238 D614G
795. QLA10250 D614G
796. QLA10262 D614G
797. QKQ30576 N211V
798. QLA10274 D614G
799. QLA10286 D614G
800. QLA10298 D614G
801. QLA10310 D614G
802. QLA10322 D614G
803. QLA10298 G75V
804. QLA10322 V1068F
805. QLA10334 D614G
806. QLA10346 D614G
807. QLA10346 H146Q

Table 4.

Mutational location after Multiple Sequence Alignment of SARS-CoV-2 membrane glycoprotein sequence with position and sequence

Sl. No. Accession Mutated sequence and position
1. QOR64248 S214R
2. QOQ57027 S214R
3. QOQ57075 S214R
4. QOQ72547 S214R
5. QOQ84829 S214R
6. QOQ57051 S214R
7. QKM75606 A2V
8. QKM75618 A2V
9. QKQ30543 A2V
10. QLA10253 A2V

Table 5.

Showing the antigenicity and allergenicity of different structural protein of SARS-CoV-2

Structural protein Antigenicity Allergenicity
Surface glycoprotein 0.4696 Non-allergen
Envelope protein 0.6025 Non-allergen
Nucleocapsid phosphoprotein 0.5059 Non-allergen
Membrane glycoprotein 0.5102 Non-allergen

Assessment of antigenicity and allergenicity

VaxiJen v2.0 server was used to predict the antigenicity of all four structural proteins of SARS-CoV-2. A peptide to be used in vaccine production must bind with the B-cell and T-cell receptors and enhance the cell's immune response. This estimation indicated that envelope protein was the most antigenic one among the four with an antigenicity of 0.6025, followed by membrane glycoprotein, having antigenicity of 0.5102. In contrast, the antigenicity of nucleocapsid phosphoprotein ranked third with a value of 0.5059, and surface glycoprotein was the least antigenic with antigenicity of 0.4696 as shown in Table 5. The allergenicity of these proteins was also estimated to discern whether these antigenic peptides were allergenic or not. A good vaccine should be non-allergenic to the host, and it should not produce any IgE-mediated immune responses in the host. The allergenicity analysis revealed that all four structural proteins were non-allergenic to the host and could be used as potent vaccine targets.

Discussion

On 11th March 2020, the WHO announced COVID-19 as a global pandemic due to its rapid global spread to numerous countries, and declared it as a serious threat to public health across the world14. Therefore, antiviral therapeutics or candidate vaccines were imminently necessary to curb SARS-CoV-2 infections. The virus primarily caused acute to severe respiratory illness and pneumonia in humans. The symptoms of COVID-19 began within two days, and could continue up to 14 days.

The emergence of a huge number of novel mutations in the genome of SARS-CoV-2 may interrupt ongoing vaccine development and trial strategies in different parts of the world. Regular monitoring of mutations has been crucial in tracking and tracing the circulation of this virus among individuals and in different geographical locations. SARS-CoV-2 (Wuhan-Hu-1), complete genome sequence, was deposited in the NCBI Gene bank15 in January 2020.

RNA viruses, including SARS-CoV-2, exhibit a high frequency of novel point mutations which are supposedly beneficial for the viruses to adapt and evolve in changing climatic conditions, thereby enhancing their potential transmission worldwide16, 17. The mutation is considered as the essential natural selection phenomenon and most often select for those traits of the virus that are a pre-requisite for survival in the highly dynamic host environment. These characteristic features of viruses (e.g. SARS-CoV-2) could complicate the ongoing efforts of researchers to combat this contagious disease because the high frequency of viral mutations induces drug resistance and rapid immune evasion18, 19.

The results of our study revealed the presence of a total of 987 mutations from 729 sequences from Asia countries in October. Spike glycoproteins were found to be highly mutagenic amongst the structural proteins, followed by nucleocapsid (151 mutations). Meanwhile, envelope protein showed 19 mutations and only 10 point mutations were found in the membrane proteins. Furthermore, we estimated the antigenicity and allergenicity of these structural proteins to design and develop potentially potent candidate vaccines against SARS-CoV-2. Among the structural proteins, envelope (E) was found to be highly antigenic and least allergenic, followed by nucleocapsid (N), membrane (M), and spike (S) protein of SARS-CoV-2. Antigenicity and allergenicity are the essential criteria to develop efficacious antiviral therapeutics or vaccines against COVID-19.

The mutations cause alterations in the structural proteins of the SARS-CoV-2 virus and, therefore, help in evading the host immunity. The occurrence of the high frequency of mutations creates a barrier in the development of antiviral drugs. Our results shown in this article, here presents a snap shot picture of an enormously changing situation due to SARS-CoV-2.

Conclusions

In the present study, the occurrence of novel mutations in the different structural proteins of the SARS-CoV-2 virus provides further insight into the identification and magnitude of virulence properties of virus strains, from a large repertoire of strains. Our findings might be useful in the development of effective therapeutic strategies against all types of SARS-CoV-2 strains.

Abbreviations

COVID-19: coronavirus disease 2019

E: envelope

M: membrane

N: nucleocapsid

S: spike

SARS: Severe acute respiratory syndrome

Acknowledgments

Not applicable

Author’s contributions

All authors read and approved the final manuscript.

Funding

None

Availability of data and materials

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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