Development of pyrosequencing-based assay for genotyping of different coronavirus variants
- Authors: Chistyakova A.K.1, Stepanova E.A.1, Isakova-Sivak I.N.1, Rudenko L.G.1
-
Affiliations:
- Institute of Experimental Medicine
- Issue: Vol 22, No 2 (2022)
- Pages: 97-114
- Section: Original research
- URL: https://journals.eco-vector.com/MAJ/article/view/108988
- DOI: https://doi.org/10.17816/MAJ108988
- ID: 108988
Cite item
Abstract
BACKGROUND: The rapid spread of SARS-CoV-2 virus, which caused the COVID-19 pandemic, and the emergence of new co-circulating antigenic variants require the development and update of subtyping kits and protocols. Pyrosequencing-based protocols are promising approach for detection of single nucleotide polymorphisms.
AIM: In this study we designed the assays for genotyping Variants of Concern of the SARS-CoV-2 coronavirus using polymerase chain reaction, followed by determination of the virus variant in the sample by pyrosequencing.
MATERIALS AND METHODS: Pyrosequencing assays were designed based on alignment of SARS-CoV-2 sequences. Testing was performed using RNA of SARS-CoV-2 viruses of different lineages (alpha, beta, gamma, delta, and omicron). Pyrosequencing was performed using the PyroMark Q24 system.
RESULTS: The protocols of sample preparation and pyrosequencing were developed and tested for sequencing of regions encoding substitutions in amino acid positions: L18F, T19R, T20N; A67V, Δ69-70; G142D, Δ143-145; Δ156-157, R158G; Δ242-244; K417N/T; L452R; S477N, T478K, E484A/K/Q; H655Y; N679K, P681H/R. The specificity of the system was also evaluated in reactions with a negative control sample (RNA isolated from human nasal swab).
CONCLUSIONS: In this study, we developed and initially tested protocol for detecting coronavirus variants (alpha, beta, gamma, delta, and omicron) from samples collected from cell culture, based on the PCR technique, followed by genotyping of the variants by pyrosequencing with PyroMark Q24. The developed protocols may be adjusted to the current epidemiological situation by increasing the number of detectable sites.
Keywords
Full Text
About the authors
Anna K. Chistyakova
Institute of Experimental Medicine
Author for correspondence.
Email: anna.k.chistiakova@gmail.com
ORCID iD: 0000-0001-9541-5636
SPIN-code: 8852-4103
Scopus Author ID: 57226491888
Student
Russian Federation, Saint PetersburgEkaterina A. Stepanova
Institute of Experimental Medicine
Email: fedorova.iem@gmail.com
ORCID iD: 0000-0002-8670-8645
SPIN-code: 8010-3047
Cand. Sci. (Biol.), Leading Research Associate, Department of Virology named after A.A. Smorodintsev
Russian Federation, Saint PetersburgIrina N. Isakova-Sivak
Institute of Experimental Medicine
Email: isakova.sivak@iemspb.ru
ORCID iD: 0000-0002-2801-1508
SPIN-code: 3469-3600
Scopus Author ID: 23973026600
Dr. Sci. (Biol.), Head of Laboratory of Immunology and Prevention of Viral Infections, A.A. Smorodintsev Department of Virology
Russian Federation, Saint PetersburgLarisa G. Rudenko
Institute of Experimental Medicine
Email: vaccine@mail.ru
ORCID iD: 0000-0002-0107-9959
SPIN-code: 4181-1372
Scopus Author ID: 7005033248
MD, Dr. Sci. (Med.), Professor, Head of Department of Virology, A.A. Smorodintsev Department of Virology
Russian Federation, Saint PetersburgReferences
- Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579:265–269. doi: 10.1038/s41586-020-2202-3
- Hadfield J, Megill C, Bell SM, et al. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 2018;34(23):4121–4123. doi: 10.1093/bioinformatics/bty407
- Koelle K, Martin MA, Antia R, et al. The changing epidemiology of SARS-CoV-2. Science. 2022;375(6585):1116–1121. doi: 10.1126/science.abm4915
- Magazine N, Zhang T, Wu Y, et al. Mutations and evolution of the SARS-CoV-2 spike protein. Viruses. 2022;14(3):640. doi: 10.3390/v14030640
- WHO. Tracking SARS-CoV-2 variants [Internet]. Available from: https://www.who.int/emergencies/emergency-health-kits/trauma-emergency-surgery-kit-who-tesk-2019/tracking-SARS-CoV-2-variants. Accessed: Nov 25, 2021.
- Mengist HM, Kombe Kombe AJ, Mekonnen D, et al. Mutations of SARS-CoV-2 spike protein: Implications on immune evasion and vaccine-induced immunity. Semin Immunol. 2021;55:101533. doi: 10.1016/j.smim.2021.101533
- Vogels CBF, Breban MI, Ott IM, et al. Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2. PLoS Biol. 2021;19(5):e3001236. doi: 10.1371/journal.pbio.3001236
- Kreutz M, Schock G, Kaiser J, et al. PyroMark® instruments, chemistry, and software for pyrosequencing® analysis. Methods Mol Biol. 2015;1315:17–27. doi: 10.1007/978-1-4939-2715-9_2
- Levine M, Sheu TG, Gubareva LV, Mishin VP. Detection of hemagglutinin variants of the pandemic influenza A (H1N1) 2009 virus by pyrosequencing. J Clin Microbiol. 2011;49(4):1307–1312. doi: 10.1128/JCM.02424-10
- Couturier BA, Bender JM, Schwarz MA, et al. Oseltamivir-resistant influenza A 2009 H1N1 virus in immunocompromised patients. Influenza Other Respir Viruses. 2010;4(4):199–204. doi: 10.1111/j.1750-2659.2010.00144.x
- Mok C-K, Chang S-C, Chen G-W, et al. Pyrosequencing reveals an oseltamivir-resistant marker in the quasispecies of avian influenza A (H7N9) virus. J Microbiol Immunol Infect. 2015;48(4):465–469. doi: 10.1016/j.jmii.2013.09.010
- Porter E, Tasker S, Day MJ, et al. Amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis. Vet Res. 2014;45(1):49. doi: 10.1186/1297-9716-45-49
- DeVries A, Wotton J, Lees C, et al. Neuraminidase H275Y and hemagglutinin D222G mutations in a fatal case of 2009 pandemic influenza A (H1N1) virus infection. Influenza Other Respir Viruses. 2012;6(6):e85–e88. doi: 10.1111/j.1750-2659.2011.00329.x
- Hodcroft EB. 2021. CoVariants: SARS-CoV-2 Mutations and Variants of Interest [Internet]. Available from: https://covariants.org. Accessed: Nov 25, 2021.
- Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045
- Duan L, Zheng Q, Zhang H, et al. The SARS-CoV-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens. Front Immunol. 2020;11:576622. doi: 10.3389/fimmu.2020.576622
- Qi Y, Fan H, Qi X, et al. A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus. Virus Res. 2014;179:119–124. doi: 10.1016/j.virusres.2013.10.026
- Deng Y-M, Caldwell N, Barr IG. Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing. PLoS One. 2011;6(8):e23400. doi: 10.1371/journal.pone.0023400
- Deng Y-M, Iannello P, Caldwell N, et al. The use of pyrosequencer-generated sequence-signatures to identify the influenza B-lineage and the subclade of the B/Yamataga-lineage viruses from currently circulating human influenza B viruses. J Clin Virol. 2013;58(1):94–99. doi: 10.1016/j.jcv.2013.04.018
- Lau H, Deng Y-M, Xu X, et al. Rapid detection of new B/Victoria-lineage haemagglutinin variants of influenza B viruses by pyrosequencing. Diagn Microbiol Infect Dis. 2019;93(4):311–317. doi: 10.1016/j.diagmicrobio.2018.11.003
- Tsiatis AC, Norris-Kirby A, Rich RG, et al. Comparison of sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn. 2010;12:425–432. doi: 10.2353/jmoldx.2010.090188
- Shcherbik S, Pearce N, Balish A, et al. Generation and characterization of live attenuated influenza A(H7N9) candidate vaccine virus based on Russian donor of attenuation. PLoS One. 2015;10(9):e0138951. doi: 10.1371/journal.pone.0138951
- Vogels CBF, Breban MI, Alpert T, et al. PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern. medRxiv. 2021. doi: 10.1101/2021.01.28.21250486
- Kemp SA, Collier DA, Datir RP, et al. SARS-CoV-2 evolution during treatment of chronic infection. Nature. 2021;592(7853):277–282. doi: 10.1038/s41586-021-03291-y
- McCallum M, De Marco A, Lempp FA, et al. N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2. Cell. 2021;184(9):2332–2347.e16. doi: 10.1016/j.cell.2021.03.028
- Starr TN, Greaney AJ, Hilton SK, et al. Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding. Cell. 2020;182(5):1295–1310.e20. doi: 10.1016/j.cell.2020.08.012
- Liu Z, VanBlargan LA, Bloyet L-M, et al. Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization. Cell Host Microbe. 2021;29(3):477–488.e4. doi: 10.1016/j.chom.2021.01.014
- Greaney AJ, Loes AN, Crawford KHD, et al. Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies. Cell Host Microbe. 2021;29(3):463–476.e6. doi: 10.1016/j.chom.2021.02.003
- Nonaka CKV, Franco MM, Gräf T, et al. Genomic evidence of SARS-CoV-2 reinfection involving E484K spike mutation, Brazil. Emerg Infect Dis. 2021;27(5):1522–1524. doi: 10.3201/eid2705.210191
- Braun KM, Moreno GK, Halfmann PJ, et al. Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck. PLoS Pathog. 2021;17(2):e1009373. doi: 10.1371/journal.ppat.1009373
- Mustafa Z, Kalbacher H, Burster T. Occurrence of a novel cleavage site for cathepsin G adjacent to the polybasic sequence within the proteolytically sensitive activation loop of the SARS-CoV-2 Omicron variant: The amino acid substitution N679K and P681H of the spike protein. PLoS One. 2022;17(4):e0264723. doi: 10.1371/journal.pone.0264723