Identification of conserved SARS-CoV-2 gene regions for diagnostic tool development through whole-genome sequencing

Abstract

The high mutation frequency of the SARS-CoV-2 spike (S) gene undermines the reliability of S-based diagnostics. Identifying conserved protein epitopes that remain stable across SARS-CoV-2 variants is paramount in resource-limited settings. This study aims to identify conserved gene regions of SARS-CoV-2 using whole-genome sequencing and immunoinformatics to support the development of monoclonal antibody-based rapid diagnostics. SARS-CoV-2 positive samples (Ct ≤ 30) collected in Ethiopia (June–August 2022) from a retrospective study (n = 70) were sequenced using Illumina NextSeq-550. Conserved regions were identified via Multiple Alignment using Fast Fourier Transform, entropy plots, and mutation profiling. B-cell epitopes were predicted and assessed for antigenicity and cross-reactivity. Structural modeling was conducted using AlphaFold and visualization tools. High-quality whole-genome sequencing of 63 SARS-CoV-2 samples revealed conserved regions in the nucleocapsid gene across major variants. Three surface-exposed, antigenic, and non-allergenic B-cell epitopes were identified with low cross-reactivity to common human coronaviruses. Structural modeling and docking confirmed their accessibility and strong antibody binding, supporting their potential for robust diagnostic applications. In conclusion, conserved N protein epitopes identified in this study are promising targets for developing robust, variant-resistant rapid diagnostic tools.

Keywords

SARS-CoV-2 / Conserved gene region / Diagnostic tool / Monoclonal antibody-based approach / Ethiopia

References

[1] Zhou R, Li F, Chen F, et al. Viral dynamics in asymptomatic patients with COVID-19. Int J Infect Dis. 2020; 96:288-290. doi: 10.1016/j.ijid.2020.05.030

[2] Jiang HW, Zhang HN, Meng QF, et al. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cell Mol Immunol. 2020; 17(9):998-1000. doi: 10.1038/s41423-020-0514-8

[3] Chen Z, Zhang Z, Zhai X, et al. Rapid and sensitive detection of anti-SARS-CoV-2 IgG using lanthanide-doped nanoparticles-based lateral flow immunoassay. Anal Chem. 2020; 92(10):7226-7231. doi: 10.1021/acs.analchem.0c00784

[4] Planas D, Veyer D, Baidaliuk A, et al. Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. Nature. 2021; 596(7871):276-280. doi: 10.1038/s41586-021-03777-9

[5] Zahradník J, Marciano S, Shemesh M, et al. SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution. Nat Microbiol. 2021; 6(9):1188-1198. doi: 10.1038/s41564-021-00954-4

[6] Tegally H, Wilkinson E, Giovanetti M, et al. Detection of a SARS-CoV-2 variant of concern in South Africa. Nature. 2021; 592(7854):438-443. doi: 10.1038/s41586-021-03402-9

[7] Xie C, Jiang L, Huang G, et al. Comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests. Int J Infect Dis. 2020; 93:264-267. doi:10.1016/j.ijid.2020.02.050

[8] Naz A, Shahid F, Butt TT, Awan FM, Ali A, Malik A. Designing multi-epitope vaccines to combat emerging coronavirus disease 2019 (COVID-19) by employing immuno-informatics approach. Front Immunol. 2020; 11:1663. doi: 10.3389/fimmu.2020.01663

[9] Chen J, Wang R, Wang M, Wei GW. Mutations strengthened SARS-CoV-2 infectivity. J Mol Biol. 2020; 432(19):5212-5226. doi: 10.1016/j.jmb.2020.07.009

[10] Cosar B, Karagulleoglu ZY, Unal S, et al. SARS-CoV-2 mutations and their viral variants. Cytokine Growth Factor Rev. 2022; 63:10-22. doi: 10.1016/j.cytogfr.2021.06.001

[11] Sisay A, Abera A, Dufera B, et al. Diagnostic accuracy of three commercially available one-step RT-PCR assays for the detection of SARS-CoV-2 in resource-limited settings. PLoS ONE. 2022; 17(1):e0262178. doi: 10.1371/journal.pone.0262178

[12] Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal. 2011; 17(1):10-12. doi: 10.14806/ej.17.1.200

[13] National Center for Biotechnology Information nucleotide database, 2024, PQ140559 to PP069552. Available from: https://www.ncbi.nlm.nih.gov/nucleotide/ [Last accessed on August 12, 2024].

[14] GISAID EpiCoV database, 2024; EPI_ISL_19147618 to EPI_ISL_19302657. Available from: https://gisaid.org/ [Last accessed on August 12, 2024].

[15] Chang CK, Hou MH, Chang CF, Hsiao CD, Huang TH. The SARS coronavirus nucleocapsid protein: forms and functions. Antiviral Res. 2014; 103:39-50. doi: 10.1016/j.antiviral.2013.12.009

[16] Harvey WT, Carabelli AM, Jackson B, et al. SARS-CoV-2 variants, spike mutations and immune escape. Nat Rev Microbiol. 2021; 19(7):409-424. doi: 10.1038/s41579-021-00573-0

[17] Korber B, Fischer WM, Gnanakaran S, et al. Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell. 2020; 182(4):812-827.e19. doi: 10.1016/j.cell.2020.06.043

[18] Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020; 579(7798):265-269. doi: 10.1038/s41586-020-2008-3

[19] Mercatelli D, Giorgi FM. Geographic and genomic distribution of SARS-CoV-2 mutations. Front Microbiol. 2020; 11:1800. doi: 10.3389/fmicb.2020.01800

[20] Ahmed SF, Quadeer AA, McKay MR. Preliminary identification of potential vaccine targets for the COVID-19 coronavirus (SARS-CoV-2) based on SARS-CoV immunological studies. Viruses. 2020; 12(3):254. doi: 10.3390/v12030254

[21] Grifoni A, Weiskopf D, Ramirez SI, et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell. 2020; 181(7):1489-1501.e15. doi: 10.1016/j.cell.2020.05.015

[22] Jespersen MC, Peters B, Nielsen M, Marcatili P. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes. Nucleic Acids Res. 2017; 45(W1):W24-W29. doi: 10.1093/nar/gkx346

[23] Sette A, Crotty S. Adaptive immunity to SARS-CoV-2 and COVID-19. Cell. 2021; 184(4):861-880. doi: 10.1016/j.cell.2021.01.007

[24] Johnson BA, Zhou Y, Lokugamage KG, et al. Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis. PLoS Pathog. 2022; 18(10):e1010627. doi: 10.1371/journal.ppat.1010627

[25] Zhang L, Jackson CB, Mou H, et al. SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity. Nat Commun. 2020; 11:6013. doi: 10.1038/s41467-020-19808-4