Results Detection of SARS-CoV-2 in suspected samples: Three of the 881 TS/nasal swab (NS) specimens from the suspected cases, tested positive for the SARS-CoV-2 using the real-time RT-PCR specific to E gene, RdRp (1), RdRp (2) and N gene. The Ct value of the E gene ranged from 19.8 to 34.5 for the TS/NS specimens. Detailed Ct values for the real-time RT-PCRs specific to the above-mentioned genes of the positive specimens are given in Table I. Blood samples were found to be negative for the SARS-CoV-2. Table I Real-time reverse transcription-polymerase chain reaction (RT-PCR) values for RdRp (1), RdRp (2), E gene and N gene, per cent genome coverage recovered and reads mapped for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive cases Positive cases Ct values for real-time RT-PCR for the confirmation of SARS-CoV-2 Relevant reads Total reads Genome length recovered (bp) Per cent genome coverage RdRp (1) RdRp (2) E gene N gene Rnase P internal control Case 1 33.33 27.93 34.5 33.90 Positive 20,096 5,615,846 29,854 99.83 Case 2 24.6 29 19.8 38 Positive 610 8,587,146 16,047 53.66 Case 3 34.17 32.64 28.98 36.35 Positive 11,296 1,405,038 29,851 99.83 Case 1 travelled from Wuhan, China, reached India on January 23, 2020 and further travelled to the final destination of Kerala on January 24. This individual developed cough on January 25 and further experienced a sore throat and mild fever and was admitted to the General Hospital, Thrissur, Kerala. The second case travelled from Wuhan and had close contact with case 1 during the travel to the final destination in India. Case 2 developed similar symptoms along with fever and diarrhoea on January 26, and the collected TS specimens were referred to the ICMR-NIV on January 28. The second case was hospitalized on January 30, in a medical college, Alappuzha, Kerala. The clinical sample (TS) was collected on January 31, 2020. Case 3 travelled from China to India, developed a runny nose on January 30 and was admitted to the General Hospital, Kasaragod, Kerala, on January 31, 2020. TS specimens were collected on January 31, 2020. NGS of SARS-CoV-2 from India - Phylogenetic analysis and molecular characterization: NGS analysis from the TS specimens retrieved two complete genome sequences from case 1 and case 3. The complete genomic sequence data for case 2 could not be recovered due to the lower kappa concentration of the sample and hence not included in the study for analysis. The FastQ files were reference mapped with the available Wuhan seafood pneumonia virus (Wuhan Hu-1) complete SARS-CoV-2 genome (accession number: NC 045512.2). The total reads which were mapped and the percentage of the genome recovered for the two cases are summarized in Table I. Analysis of the complete genome sequences of SARS-CoV-2 from the positive cases in India revealed that the percentage nt and aa differences between case 1 and case 3 were 0.038 and 0.10 per cent, respectively. The sequences of case 1 and case 3 diverged from the Wuhan-Hu1 sequence by 0.017 per cent nt and 0.041 per cent aa respectively. Indian SARS-CoV-2 clustered with the Sarbecovirus subgenus of the Betacoronavirus genus and was closest to the BatCoV RaTG13 sequence (96.09% nt)8. The phylogenetic comparison showed the clustering of the genome sequences of case 1 and case 3 with the existing sequences of the SARS-CoV-2 sequences (Fig. 1). The phylogeny revealed emerging heterogeneity within the SARS-CoV-2 sequences globally. The Indian SARS-CoV-2 viruses were positioned in different clusters. Fig. 1 Phylogenetic tree of the complete genomes of severe acute respiratory syndrome coronavirus 2 viruses. Indian viruses are shown in magenta font colour. Indian SARS-CoV-2 sequences showed two changes 408 Arg→Ile and 930 Ala→Val in the spike protein compared to the Wuhan Hu-1 sequence. The mutations were further mapped on the spike protein model of the Indian sequence (Supplementary Fig. 1 (available from http://www.ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_200_281471_sm6.pdf)). Deletion of a three-nucleotide stretch, encoding tyrosine residue at position 144, of the spike gene was also observed in the Indian SARS-CoV-2 from case 1 when compared to the other SARS-CoV-2 sequences. As noted in the earlier SARS-CoV-2 sequences, both the Indian sequences possessed the polybasic cleavage site (RRAR) in the spike protein at the junction of S1 and S2, the two subunits of the spike protein19. Epitope predictions: Thirty one linear B-cell epitopes were predicted by Bepipred in the Indian SARS-CoV-2, of which three were found to have a length of <6 amino acids and hence not considered. Linear epitopes were also predicted using the ABCpred prediction tool, which predicted 47 epitopes based on the threshold of 0.8. Regions common to both the prediction methods (n=17) were identified manually. The 17 epitopes were screened for their antigenicity using the VaxiJen v2.0 tool (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html), and nine of these epitopes were shortlisted. These epitopes were further screened for their ability to elicit an IFN-γ response, which was predicted using the IFNepitope tool. Finally, five epitopes, four in the S1 domain and one in the S2 domain, were predicted, which could possibly generate an immune response and suppress the IFN-γ response (Table II). N-linked glycosylation site prediction revealed that two putative glycosylation sites (with a low value for jury agreement) were present within the epitope stretch 328-344. Table II Linear B-cell epitopes predicted on the spike protein of the Indian severe acute respiratory syndrome coronavirus 2 Peptide Epitope probability Vaxigen score Interferon (IFN)-γ response# 243-HRSYLTPGDSSSGWTA-258 0.92 Antigen (0.602) Negative (1) 327-FPNITNLCPFGEVFNA-342 0.82 Antigen (0.606) Negative (−0.132) 404-EVIQIAPGQTGKIADY-419 0.86 Antigen (1.231) Negative (1) 413-TGKIADYNYKLPDDFT-428 0.84 Antigen (0.9642) Negative (−0.334) 1204-YEQYIKWPWYIWLGFI-1219 0.89 Antigen (0.951) Negative (1) Epitopes were predicted using a combination of the Bepipred server and the ABCpred prediction server. The antigenicity was predicted using the VaxiJen v2.0 tool. IFN-γ response was predicted using the INFepitope server. #Values in bracket show prediction score given by the software The discontinuous epitopes in the spike protein of the Indian SARS-CoV-2 were further identified using multiple methods, Ellipro and DiscoTope. Conformational epitopes based on these methods were mapped on the pre-fusion structure of the modelled Indian SARS-CoV-2 spike protein. The newly released structure of the SARS-CoV-2 spike protein was used as the template for modelling the Indian spike protein. Ramachandran plot statistics revealed 83.7 per cent of the residues to be in the core region, 14.4 per cent in the additionally allowed region and 0.5 per cent in the disallowed region. Four epitopes were predicted by Ellipro based on the PI threshold of 0.8 (Supplementary Table II (available from http:/ /www. ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_200_281471_sm7.pdf)). The result from the DiscoTope is presented in Supplementary Table III (available from http://www.ijmr.org.in/articles/ 2020/151/2/ images/ IndianJMedRes_2020_151_2_200_281471_sm8.pdf). The mapped conformational epitopes are depicted in Figure 2. For the purpose of comparison, the Indian S protein sequence was also modelled using the pre-fusion structure of SARS-CoV-1 (6ACC.PDB; 87.29% identity), and the results for the conformational epitopes predicted are in Supplementary Table IV (available from http://www.ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_200_28147 1_sm9.pdf and Supplementary Figure 2 (available from http://www.ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_200_281471_sm10.pdf). Supplementary Table II Conformational B-cell epitopes predicted by Ellipro based on the chains A, B and C of the Indian severe acute respiratory syndrome coronavirus 2 spike protein modelled structure (template used: 6VSB.PDB). Ellipro protusion Index threshold set to 0.8 cut-off Chain A Ellipro Score 1 D1137, P1138, L1139, Q1140, P1141, E1142, L1143, D1144 0.984 2 Y705, S706, N707, N708, S709, T1074, T1075, A1076, P1077, A1078, I1079, C1080, H1081, D1082, G1083, K1084, A1085, H1086, F1087, P1088, R1089, E1090, G1091, F1093, V1094, S1095, N1096, G1097, T1098, H1099, W1100, F1101, V1102, Y1108, E1109, P1110, Q1111, I1112, I1113, T1114, T1115, D1116, N1117, T1118, F1119, V1120, S1121, G1122, N1123, 0.906 3 N341, A342, T343, R344, F345, A346, S347, V348, Y349, A350, W351, N352, S397, F398, V399, I400, R401, E404, Q412, T413, G414, K415, I416, A417, D418, Y419, N420, Y421, K422, L423, S436, N437, N438, L439, D440, S441, K442, V443, G444, G445, N446, Y447, N448, Y449, L450, Y451, R452, L453, F454, R455, K456, S457, N458, L459, K460, P461, F462, E463, R464, D465, I466, S467, T468, E469, I470, Y471, Q472, A473, G474, S475, T476, P477, C478, N479, G480, V481, G483, F484, N485, C486, Y487, F488, P489, L490, Q491, S492, Y493, G494, F495, Q496, P497, T498, N499, G500, V501, G502, Y503, Q504, P505, R507 0.887 4 I68, H69, V70, S71, G72, T73, N74, G75, T76, K77, R78, S98, I100, C136, D138, F140, G142, Y143, H144, K145, N146, N147, K148, S149, W150, M151, E152, S153, E154, F155, R156, N183, F184, A241, L242, H243, R244, S245, Y246, L247, T248, P249, G250, D251, S252, S253, S254, G255, W256, T257, A258, G259, A260 0.876 Chain B 1 A704, Y705, S706, N707, N708, S709, F1073, T1074, T1075, A1076, P1077, A1078, I1079, C1080, H1081, D1082, G1083, K1084, A1085, H1086, F1087, P1088, R1089,E1090, G1091, V1092,F1093, V1094, S1095, N1096, G1097, T1098, H1099, W1100, F1101, V1102, T1103, Q1104, R1105, F1107, Y1108, E1109,P1110, Q1111, I1112, I1113, T1114, T1115, D1116, N1117, T1118, F1119, V1120, S1121, G1122, N1123, C1124, D1125, V1126, V1127, I1128, G1129, I1130, V1131, N1132, N1133, T1134, V1135, Y1136, D1137, P1138, L1139, Q1140,P1141,E1142, L1143, D1144 0.902 2 N341, A342, T343, F345, A346, S347, V348, Y349, A350, W351, V399, R401, G402, T413, G414, K415, D418, Y419, N420, Y421, K422, S436, N437, N438, L439, D440, S441, K442, V443, G444, G445, N446, Y447, N448, Y449, L450, Y451, R452, L453, F454, R455, K456, S457, N458, L459, K460, P461, E463, R464, D465, I466, S467, T468, E469, I470, Y471, Q472, A473, G474, S475, T476, P477, C478, N479, G480, V481, E482, G483, F484, N485, C486, Y487, F488, P489, L490, Q491, S492, Y493, G494, F495, Q496, P497, T498, N499, G500, V501, G502, Y503, Q504, P505 0.886 3 A67, I68, H69, V70, S71, G72, T73, N74, G75, T76, K77, R78, E96, K97, S98, N99, I100, R102, N122, A123, T124, N125, C136, N137, D138, P139, F140, L141, G142, Y143, H144, K145, N146, N147, K148, S149, W150, M151, E152, S153, E154, F155, L239, L240, A241, L242, H243, R244, S245, Y246, L247, T248, P249, G250, D251, S252, S253, S254, G255, W256, T257, A258, G259, A260, A261 0.869 Chain C 1 D1137, P1138, L1139, Q1140, P1141, E1142, L1143, D1144 0.984 2 Y705, S706, N707, N708, S709, T1074, T1075, A1076, P1077, A1078, I1079, C1080, H1081, D1082, G1083, K1084, A1085, H1086, F1087, P1088, R1089, E1090, G1091, F1093, V1094, S1095, N1096, G1097, T1098, H1099, W1100, F1101, V1102, Y1108, E1109, P1110, Q1111, I1112, I1113, T1114, T1115, D1116, N1117, T1118, F1119, V1120, S1121, G1122, N1123, C1124, D1125, V1126, V1127, I1128, G1129, I1130, V1131, N1132, N1133, T1134, V1135, Y1136 0.906 3 N341, A342, T343, R344, F345, A346, S347, V348, Y349, A350, W351, N352, S397, F398, V399, I400, R401, E404, Q412, T413, G414, K415, I416, A417, D418, Y419, N420, Y421, K422, L423, S436, N437, N438, L439, D440, S441, K442, V443, G444, G445, N446, Y447, N448, Y449, L450, Y451, R452, L453, F454, R455, K456, S457, N458, L459, K460, P461, F462, E463, R464, D465, I466, S467, T468, E469, I470, Y471, Q472, A473, G474, S475, T476, P477, C478, N479, G480, V481, G483, F484, N485, C486, Y487, F488, P489, L490, Q491, S492, Y493, G494, F495, Q496, P497, T498, N499, G500, V501, G502, Y503, Q504, P505, R507 0.887 4 I68, H69, V70, S71, G72, T73, N74, G75, T76, K77, R78, S98, I100, C136, D138, F140, G142, Y143, H144, K145, N146, N147, K148, S149, W150, M151, E152, S153, E154, F155, R156, N183, F184, A241, L242, H243, R244, S245, Y246, L247, T248, P249, G250, D251, S252, S253, S254, G255, W256, T257, A258, G259, A260 0.876 Fig. 2 Predicted conformational B-cell epitopes mapped on the pre-fusion structure of the modelled Indian severe acute respiratory syndrome coronavirus 2 spike protein using the pre-fusion structure of severe acute respiratory syndrome-coronavirus-2 (6VSB.PDB) (colour key: blue - epitopes 67-261; green - epitopes 341-507 based on the predicted epitopes as shown in Supplementary Table II). (A) Top view (B) Side view. Supplementary Table III Conformational B-cell epitopes predicted by the Discotope server based on the chain A, B and C of India severe acute respiratory syndrome coronavirus 2 spike protein modelled structure (template used 6VSB.PDB). Discotope threshold set to -3.7 cut-off CHAIN A 71S, 72G, 73T, 74N, 75G, 76T, 148K, 149S, 150W, 151M, 152E, 178E, 179G, 180K, 181Q, 209N, 245S, 247L, 248T, 250G, 252S, 253S, 440D, 441S, 442K, 443V, 444G, 445G, 446N, 447Y, 448N, 452R, 453L, 454F, 455R, 456K, 458N, 460K, 461P, 465D, 468T, 470I, 482E, 487Y, 488F, 489P, 490L, 491Q, 492S, 493Y, 494G, 495F, 496Q, 497P, 498T, 499N, 500G, 501V, 502G, 554N, 556K, 558L, 559P, 560F, 568A, 677N, 678S, 679P, 680R, 681R, 682A, 683R, 701N, 702S, 703V, 791P, 792I, 807P, 808S, 810P, 912N, 915Y, 916E, 1069Q, 1097G, 1099H, 1116D, 1137D, 1138P, 1139L, 1140Q, 1141P, 1142E, 1143L, 1144D CHAIN B 70V, 73T, 74N, 75G, 76T, 147N, 148K, 149S, 150W, 151M, 176D, 177L, 178E, 179G, 180K, 181Q, 182G, 183N, 212R, 244R, 245S, 246Y, 247L, 248T, 249P, 250G, 251D, 252S, 253S, 254S, 255G, 256W, 438N, 441S, 442K, 443V, 444G, 445G, 446N, 447Y, 454F, 456K, 457S, 458N, 460K, 467S, 476T, 492S, 494G, 495F, 496Q, 497P, 498T, 499N, 500G, 501V, 502G, 503Y, 554N, 556K, 558L, 559P, 676T, 677N, 678S, 679P, 680R, 701N, 702S, 703V, 714T, 791P, 792I, 807P, 808S, 810P, 912N, 915Y, 916E, 1069Q, 1109E, 1112I, 1116D, 1137D, 1138P, 1139L, 1140Q, 1141P, 1142E, 1143L, 1144D CHAIN C 72G, 73T, 74N, 75G, 97K, 98S, 143Y, 144H, 145K, 146N, 147N, 148K, 149S, 150W, 151M, 152E, 153S, 180K, 181Q, 182G, 183N, 184F, 209N, 252S, 253S, 441S, 442K, 443V, 444G, 445G, 446N, 447Y, 454F, 456K, 457S, 458N, 460K, 480G, 492S, 494G, 496Q, 497P, 498T, 499N, 500G, 501V, 502G, 503Y, 554N, 556K, 558L, 559P, 676T, 678S, 679P, 680R, 681R, 682A, 683R, 684S, 685V, 701N, 702S, 714T, 791P, 792I, 807P, 808S, 912N, 915Y, 916E, 1069Q, 1072N, 1098T, 1112I, 1116D, 1138P, 1139L, 1140Q, 1141P, 1142E, 1143L, 1144D Supplementary Table IV Conformational B-cell epitopes predicted using the modelled structure of the spike protein (template used: 6ACC.PDB; 87.29% identity). (A) Ellipro server (using a protusion Index threshold of 0.9) (B) DiscoTope server A. Ellipro epitope prediction Epitope number Epitope residues Epitope score 1 244-RSYLTPGDSSSGW-256 0.95 2 347S, 349Y, 419Y, 441-SKVGGNYNYLYRLFR-455, 457S, 465-DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN -499 0.943 3 1074 TTAPAICHDGKAHFPR 1089, 1094-VSNGTHWFV-1102, 1110- PQIITTDNTFVSGNCDVVIGIVNNTV-1135 0.934 4 144-HKNNKSWMESE-154 0.912 5 72-GTNGTK-77 0.907 B. DiscoTope epitope prediction G72, T73, N74, G75, K145, N146, N147, K148, S149, L174, E178, K180, Q181, G182, 183, V211, S245, Y246, L247, T248, P249, G250, D251, S252, S253, K415, N438, S441, K442, V443, G444, G445, N446, Y447, N448, K456, S457, N458, K460, A473, G474, S475, S492, G494, Q496, P497, T498, N499, G500, V501, Y503, N554, K556, L558, P559, I567, Q675, T676, N677, S678, P679, R680, R681, A682, R683, S702, V703, A704, Y705, T714, P791, P807, S808, K809, P810, E916, Q1069, E1070 T-cell epitope prediction revealed 105 strong binding epitopes capable of binding to different HLA types using the NetCTL1.2 software based on the threshold of 0.4. Twelve of these were shortlisted, considering a binding efficiency of >0.5 nM and capable of eliciting IFN-γ response (Table III). Table III Spike protein peptides capable of binding to major histocompatibility complex (MHC) class I predicted using NetCTL server Peptide Vaxijen Interferon (IFN)-γ response CTLPred Score (ANN/SVM) MHC restriction 89-GVYFASTEK-97 0.711 Positive (1) 0.58/0.986 HLA-A*1101, HLA-A3, HLA-A*3101, HLA-A68.1, HLA-B*2705 166-FEYVSQPFL-174 0.632 Positive (0.087) 0.65/0.184 HLA-A2, HLA-A*0201, HLA-A*0205, HLA-A2.1, HLA-B*2702, HLA-B*2705, HLA-B*3701, HLA-B40, HLA-B*4403, HLA-B*5301, HLA-B*5401, HLA-B*51, HLA-B60, HLA-B61, HLA-Cw*0301, H2-Kb, H2-Kk, 256-WTAGAAAYY-264 0.630 Positive (0.576) 0.82/0.544 HLA-A1, HLA-B*2702, HLA-B*3501, HLA-B*4403, HLA-B*5301, HLA-B*5401, HLA-B*51, HLA-B*5801, HLA-B62, HLA-Cw*0702 348-VYAWNRKRI-356 0.500 Positive (0.499) 0.93/0.497 HLA-A24, HLA-B*5101, HLA-B*5102, HLA-B*5103, HLA-B*51, HLA-Cw*0401, H2-Db, H2-Kd, H2-Kk 503-YQPYRVVVL-511 0.596 Positive (0.292) 0.40/0.596 HLA-A*0201, HLA-A*0205, HLA-A24, HLA-B14, HLA-B*2702, HLA-B*2705, HLA-B*3902, HLA-B*5201, HLA-B*5301, HLA-B*5401, HLA-B*51, HLA-B60, HLA-B62, HLA-B7, HLA-B8, HLA-Cw*0401, HLA-Cw*0602, H2-Dd, H2-Kb, H2-Ld 510-VLSFELLHA-518 1.077 Positive (0.268) 0.86/0.276 HLA-A*0201, HLA-A*0205, HLA-A3, HLA-B*5301, HLA-B*51, HLA-B62 825-TLADAGFIK-833 0.578 Positive (0.014) 0.75/0.992 HLA-A1, HLA-A*1101, HLA-A3, HLA-A*3101, HLA-A68.1, HLA-A20, HLA-B*2705 1058-VVFLHVTYV-1066 1.512 Positive (1) 0.77/0.779 HLA-A2, HLA-A*0201, HLA-A*0205, HLA-A68.1, HLA-A2.1, HLA-B14, HLA-B*5101, HLA-B*5102, HLA-B*5103, HLA-B*5201, HLA-B*5301, HLA-B*5401, HLA-B*51 1210-WPWYIWLGF-1218 1.495 Positive (0.221) 0.68/0.0695 HLA-B*2702, HLA-B*2705, HLA-B*3501, HLA-B*3801, HLA-B*5101, HLA-B*5102, HLA-B*5201, HLA-B*5301, HLA-B*5401, HLA-B*51, HLA-B*5801, HLA-B62, HLA-B*0702, HLA-Cw*0401, HLA-Cw*0702.H2-Ld Threshold of >0.7 nM was used for increased specificity of the prediction. The peptides were reconfirmed using CTLPred server using default parameters. The peptides that were classified as epitopes were further checked for their antigenicity score using the VaxiJen v2.0 tool