Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Generation of quality-controlled SARS-CoV-2 variant stocks

Nature Protocols volume 18, pages 3821–3855 (2023)Cite this article

Subjects

Abstract

One of the main challenges in the fight against coronavirus disease 2019 (COVID-19) stems from the ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into multiple variants. To address this hurdle, research groups around the world have independently developed protocols to isolate these variants from clinical samples. These isolates are then used in translational and basic research—for example, in vaccine development, drug screening or characterizing SARS-CoV-2 biology and pathogenesis. However, over the course of the COVID-19 pandemic, we have learned that the introduction of artefacts during both in vitro isolation and subsequent propagation to virus stocks can lessen the validity and reproducibility of data. We propose a rigorous pipeline for the generation of high-quality SARS-CoV-2 variant clonal isolates that minimizes the acquisition of mutations and introduces stringent controls to detect them. Overall, the process includes eight stages: (i) cell maintenance, (ii) isolation of SARS-CoV-2 from clinical specimens, (iii) determination of infectious virus titers by plaque assay, (iv) clonal isolation by plaque purification, (v) whole-virus-genome deep-sequencing, (vi and vii) amplification of selected virus clones to master and working stocks and (viii) sucrose purification. This comprehensive protocol will enable researchers to generate reliable SARS-CoV-2 variant inoculates for in vitro and in vivo experimentation and will facilitate comparisons and collaborative work. Quality-controlled working stocks for most applications can be generated from acquired biorepository virus within 1 month. An additional 5–8 d are required when virus is isolated from clinical swab material, and another 6–7 d is needed for sucrose-purifying the stocks.

Key points

  • This protocol describes a method for generating single-clone viral stocks of severe acute respiratory syndrome coronavirus 2 from patient samples or from biorepositories.

  • The protocol improves on previous methods by introducing a number of stringent measures to prevent the introduction of artefacts during viral propagation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Workflow for the generation of quality-controlled working stocks of SARS-CoV-2 variants of concern.
Fig. 2: SARS-CoV-2 WA-1 plaque presentation before and after plaque purification.
Fig. 3: Characterization of SARS-CoV-2 plaque morphologies and titers obtained on different cell types.
Fig. 4: Presentation of the CPE of SARS-CoV-2 USA/NYU-VC-003/2020 on Vero E6 cells during virus isolation.
Fig. 5: Clonal virus isolation by plaque picking.
Fig. 6: Presentation of SARS-CoV-2 variant plaque morphology.
Fig. 7: Representative well for SARS-CoV-2 plaque assay scoring.
Fig. 8: Multiple sequence alignment and clone selection.

Similar content being viewed by others

Data availability

Source data are provided with this paper. Sequences for viruses used in this study are available at SARS-CoV-2 USA-WA1/2020, GISAID, EPI_ISL_404895.2; SARS-CoV-2 hCoV-19/England/204820464/2020, GISAID, EPI_ISL_683466; SARS-CoV-2 hCoV-19/South Africa/KRISP-K005325/2020, GISAID, EPI_ISL_678615; SARS-CoV-2 hCoV-19/Japan/TY7-503/2021, GISAID, EPI_ISL_877769; SARS-CoV-2 hCoV-19/USA/PHC658/2021, GenBank accession no. OL442162; SARS-CoV-2 hCoV-19/USA/CA-Stanford-106_S04/2022, GISAID, EPI_ISL_15196219; and SARS-CoV-2 USA/NYU-VC-003/2020, GenBank accession no. MT703677. Source data are provided with this paper.

References

  1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Available at https://covid19.who.int/ (2023).

  2. de Vries, M. et al. A comparative analysis of SARS-CoV-2 antivirals characterizes 3CLpro inhibitor PF-00835231 as a potential new treatment for COVID-19. J. Virol. 95, e01819-20 (2021).

    Article  PubMed Central  PubMed  Google Scholar 

  3. FDA. Emergency Use Authorization (Eua) of Paxlovid for Coronavirus Disease 2019 (Covid-19). Available at https://www.fda.gov/media/155051/download (2023).

  4. Samanovic, M. I. et al. Robust immune responses are observed after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2-experienced individuals. Sci. Transl. Med. 14, eabi8961 (2022).

    Article  CAS  PubMed  Google Scholar 

  5. Samanovic, M. I. et al. Vaccine-acquired SARS-CoV-2 immunity versus infection-acquired immunity: a comparison of three COVID-19 vaccines. Vaccines (Basel) 10, 2152 (2022).

    Article  CAS  PubMed  Google Scholar 

  6. Kister, I. et al. Hybrid and vaccine-induced immunity against SAR-CoV-2 in MS patients on different disease-modifying therapies. Ann. Clin. Transl. Neurol. 9, 1643–1659 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Kister, I. et al. Cellular and humoral immunity to SARS-CoV-2 infection in multiple sclerosis patients on ocrelizumab and other disease-modifying therapies: a multi-ethnic observational study. Ann. Neurol. 91, 782–795 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Izmirly, P. M. et al. Evaluation of immune response and disease status in systemic lupus erythematosus patients following SARS–CoV-2 vaccination. Arthritis Rheumatol. 74, 284–294 (2022).

    Article  CAS  PubMed  Google Scholar 

  9. Ching, K. L. et al. ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection. PLoS Biol. 20, 1–25 (2022).

    Article  Google Scholar 

  10. Rona, G. et al. The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target. Cell Death Differ. 29, 285–292 (2022).

    Article  CAS  PubMed  Google Scholar 

  11. Rodriguez-Rodriguez, B. A. et al. A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8. Nat. Commun. 14, 3026 (2023).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Lokugamage, K. G. et al. Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV. J. Virol. 94, e01410-20 (2020).

    Article  PubMed Central  PubMed  Google Scholar 

  13. Yount, B. et al. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. USA 100, 12995–13000 (2003).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Drosten, C. et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1967–1976 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Scobey, T. et al. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. USA 110, 16157–16162 (2013).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Coleman, C. M. & Frieman, M. B. Growth and quantification of MERS-CoV infection. Curr. Protoc. Microbiol. 2015, 15E.2.1–15E.2.9 (2015).

    Google Scholar 

  17. Sun, F. et al. SARS-CoV-2 quasispecies provides an advantage mutation pool for the epidemic variants. Microbiol. Spectr. 9, e0026121 (2021).

    Article  PubMed  Google Scholar 

  18. Bal, A. et al. Detection and prevalence of SARS-CoV-2 co-infections during the Omicron variant circulation in France. Nat. Commun. 13, 6316 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Russell, C. D. et al. Co-infections, secondary infections, and antimicrobial use in patients hospitalised with COVID-19 during the first pandemic wave from the ISARIC WHO CCP-UK study: a multicentre, prospective cohort study. Lancet Microbe 2, e354–e365 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Singh, V., Upadhyay, P., Reddy, J. & Granger, J. SARS-CoV-2 respiratory co-infections: incidence of viral and bacterial co-pathogens. Int. J. Infect. Dis. 105, 617–620 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Swets, M. C. et al. SARS-CoV-2 co-infection with influenza viruses, respiratory syncytial virus, or adenoviruses. Lancet 399, 1463–1464 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Örd, M., Faustova, I. & Loog, M. The sequence at SpikeS1/S2 site enables cleavage by furin and phospho-regulation in SARS-CoV2 butnot in SARS-CoV1 or MERS-CoV. Sci. Rep. 10, (2020).

  23. Jackson, C.B., Farzan, M., Chen, B. & Choe, H. Mechanisms of SARS-CoV-2 entry intocells. Nat. Rev. Mol. Cell Biol. 23, (2022).

  24. Meng, B. et al. Altered TMPRSS2 usage by SARS-CoV-2Omicron impacts infectivity and fusogenicity. Nature 603, 706–714 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Koch, J. et al. TMPRSS2 expression dictates the entry route used by SARS‐CoV‐2 to infect host cells. EMBO J. 40, e107821 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Takeda, M. Proteolytic activation of SARS-CoV-2 spike protein. Microbiol. Immunol. 66, 15–23 (2022).

    Article  CAS  PubMed  Google Scholar 

  27. Klimstra, W. B. et al. SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients. J. Gen. Virol. 101, 1156–1169 (2020).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Vu, M. N. et al. QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis. Proc. Natl. Acad. Sci. USA 119, 1–7 (2022).

    Article  Google Scholar 

  29. Funnell, S. G. P. et al. A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells. NPJ Vaccines 6, 83 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Johnson, B. A. et al. Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis. Nature 591, 293–299 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Chen, Y. et al. Genetic mutation of SARS-CoV-2 during consecutive passages in permissive cells. Virol. Sin. 36, 1073–1076 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Chung, H., Noh, J. Y., Koo, B. S., Hong, J. J. & Kim, H. K. SARS-CoV-2 mutations acquired during serial passage in human cell lines are consistent with several of those found in recent natural SARS-CoV-2 variants. Comput. Struct. Biotechnol. J. 20, 1925–1934 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Duerr, R. et al. Delta-Omicron recombinant escapes therapeutic antibody neutralization. iScience 26, 106075 (2023).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Sonnleitner, S. T. et al. The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro. Virol. Sin. 37, 198–207 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Matsuyama, S. et al. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc. Natl. Acad. Sci. USA 117, 7001–7003 (2020).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Sasaki, M. et al. SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells. PLoS Pathog. 17, e1009233 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Kalamvoki, M. & Norris, V. A defective viral particle approach to COVID-19. Cells 11, 302 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Girgis, S. et al. Evolution of naturally arising SARS-CoV-2 defective interfering particles. Commun. Biol. 5, 1–12 (2022).

    Article  Google Scholar 

  39. Mautner, L. et al. Replication kinetics and infectivity of SARS-CoV-2 variants of concern in common cell culture models. Virol. J. 19, 1–11 (2022).

    Article  Google Scholar 

  40. Mlcochova, P. et al. SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion. Nature 599, 114–119 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Willett, B. J. et al. SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway. Nat. Microbiol. 7, 1161–1179 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. McGrath, M. E. et al. SARS-CoV-2 variant spike and accessory gene mutations alter pathogenesis. Proc. Natl. Acad. Sci. USA 119, e2204717119 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Iwata-Yoshikawa, N. et al. Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways. Nat. Commun. 13, 6100 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Plavec, Z. et al. SARS-CoV-2 production, purification methods and UV inactivation for proteomics and structural studies. Viruses 14, 4–15 (2022).

    Article  Google Scholar 

  45. Bernard-Raichon, L. et al. Gut microbiome dysbiosis in antibiotic-treated COVID-19 patients is associated with microbial translocation and bacteremia. Nat. Commun. 13, 5926 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Samanovic, M. I. et al. Robust immune responses areobserved after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experiencedindividuals. 14, 1–31 (2022).

  47. Case, J. B., Bailey, A. L., Kim, A. S., Chen, R. E. & Diamond, M. S. Growth, detection, quantification, and inactivation of SARS-CoV-2. Virology 548, 39–48 (2020).

    Article  CAS  PubMed  Google Scholar 

  48. Xie, X. et al. Engineering SARS-CoV-2 using a reverse genetic system. Nat. Protoc. 16, 1761–1784 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Chaudhry, M. Z. et al. Rapid SARS-CoV-2 adaptation to available cellular proteases. J. Virol. 96, e0218621 (2022).

    Article  PubMed  Google Scholar 

  50. Baczenas, J. J. et al. Propagation of SARS-CoV-2 in Calu-3 cells to eliminate mutations in the furin cleavage site of spike. Viruses 13, 2434 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Yamada, S. et al. Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells. BMJ Open Respir. Res. 8, e000830 (2021).

    Article  PubMed  Google Scholar 

  52. Duerr, R. et al. Dominance of Alpha and Iota variants in SARS-CoV-2 vaccine breakthrough infections in New York City. J. Clin. Invest. 131, e152702 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. McAuley, J. et al. Optimal preparation of SARS-CoV-2 viral transport medium for culture. Virol. J. 18, 53 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  54. Rosenthal, S. H. et al. Development and validation of a high throughput SARS-CoV-2 whole-genome sequencing workflow in a clinical laboratory. Sci. Rep. 12, 2054 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  55. Laporte, M. et al. The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways. PLoS Pathog. 17, e1009500 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  56. Peacock, T. P. et al. The altered entry pathway and antigenic distance of the SARS-CoV-2 Omicron variant map to separate domains of spike protein. Preprint at https://www.biorxiv.org/content/10.1101/2021.12.31.474653v2 (2022).

  57. Rottem, S., Kosower, N. S. & Kornspan, J. D. Contamination of tissue cultures by mycoplasmas. In Biomedical Tissue Culture (eds. Ceccherini-Nelli, L. & Matteoli, B.) (IntechOpen, 2012).

  58. Watanabe, I. & Okada, S. Effects of temperature on growth rate of cultured mammalian cells (L5178Y). J. Cell Biol. 32, 309–323 (1967).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  59. Clements, G. B. Selection of biochemically variant, in some cases mutant, mammalian cells in culture. Adv. Cancer Res. 21, 273–390 (1975).

    Article  CAS  PubMed  Google Scholar 

  60. Stacey, G. N. & Masters, J. R. Cryopreservation and banking of mammalian cell lines. Nat. Protoc. 3, 1981–1989 (2008).

    Article  CAS  PubMed  Google Scholar 

  61. La Scola, B. et al. Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards. Eur. J. Clin. Microbiol. Infect. Dis. 39, 1059–1061 (2020).

    Article  PubMed Central  PubMed  Google Scholar 

  62. Francis, R. et al. High-speed large-scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled to high-content screening. Clin. Microbiol. Infect. 27, 128.e1–128.e7 (2021).

    Article  CAS  PubMed  Google Scholar 

  63. Sonnleitner, S. T. et al. An in vitro model for assessment of SARS-CoV-2 infectivity by defining the correlation between virus isolation and quantitative PCR value: isolation success of SARS-CoV-2 from oropharyngeal swabs correlates negatively with Cq value. Virol. J. 18, 71 (2021).

    Article  PubMed  Google Scholar 

  64. Sung, A. et al. Isolation of SARS-CoV-2 in viral cell culture in immunocompromised patients with persistently positive RT-PCR results. Front. Cell. Infect. Microbiol. 12, 804175 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  65. Rhoads, D. et al. College of American Pathologists (CAP) Microbiology Committee perspective: caution must be used in interpreting the cycle threshold (Ct) value. Clin. Infect. Dis. 72, e685–e686 (2021).

    Article  CAS  PubMed  Google Scholar 

  66. Potter, R. F. et al. Evaluation of PCR cycle threshold values by patient population with the quidel lyra SARS-CoV-2 assay. Diagn. Microbiol. Infect. Dis. 101, 115387 (2021).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  67. Arora, D. J. S., Tremblay, P., Bourgault, R. & Boileau, S. Concentration and purification of influenza virus from allantoic fluid. Anal. Biochem. 144, 189–192 (1985).

    Article  CAS  PubMed  Google Scholar 

  68. Mbiguino, A. & Menezes, J. Purification of human respiratory syncytial virus: superiority of sucrose gradient over percoll, renografin, and metrizamide gradients. J. Virol. Methods 31, 161–170 (1991).

    Article  CAS  PubMed  Google Scholar 

  69. Putnak, R. et al. Development of a purified, inactivated, dengue-2 virus vaccine prototype in Vero cells: immunogenicity and protection in mice and rhesus monkeys. J. Infect. Dis. 174, 1176–1184 (1996).

    Article  CAS  PubMed  Google Scholar 

  70. Ali, A. & Roossinck, M. J. Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation. J. Virol. Methods 141, 84–86 (2007).

    Article  CAS  PubMed  Google Scholar 

  71. Hankaniemi, M. M. et al. Optimized production and purification of Coxsackievirus B1 vaccine and its preclinical evaluation in a mouse model. Vaccine 35, 3718–3725 (2017).

    Article  CAS  PubMed  Google Scholar 

  72. Leibowitz, J., Kaufman, G. & Liu, P. Coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus. Curr. Protoc. Microbiol. Chapter 15, Unit 15E.1 (2011).

    PubMed  Google Scholar 

  73. Su, S. et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol. 24, 490–502 (2016).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  74. Heguy, A. et al. Amplification artifact in SARS-CoV-2 Omicron sequences carrying P681R mutation, New York, USA. Emerg. Infect. Dis. 28, 881–883 (2022).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  75. Gribble, J. et al. The coronavirus proofreading exoribonuclease mediates extensive viral recombination. PLoS Pathog. 17, 1–28 (2021).

    Article  Google Scholar 

  76. Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 20, 1160–1166 (2018).

    Article  Google Scholar 

  77. Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  78. Los Alamos National Laboratory. HIV Sequence Database. Highlighter. Available at https://www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter_top.html (2022).

  79. V’kovski, P., Kratzel, A., Steiner, S., Stalder, H. & Thiel, V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat. Rev. Microbiol. 19, 155–170 (2021).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Mehul Suthar from Emory University and the NIH/NIAID SARS-CoV-2 Assessment of Viral Evolution (SAVE) program and Dr. Benjamin Pinsky from Stanford University for providing us with Delta and Omicron variant isolates. We thank the Office of Science & Research High-Containment Laboratories at the NYU Grossman School of Medicine for their support in the completion of this research. The NYU Genome Technology Core is partially supported by NYU Cancer Center support grant P30CA016087. Research was further supported by the following grants from the NIH: R01AI143639 to M.D. and AI148574 to M.J.M. Work was further supported by The Vilcek Institute of Graduate Biomedical Sciences, by the NIH National Center for Advancing Translational Sciences (NCATS) through Grant Award Number UL1TR001445 and by NYU Grossman School of Medicine Startup funds.

Author information

Authors and Affiliations

  1. Department of Microbiology, NYU Grossman School of Medicine, New York, NY, USA

    Maren de Vries, Grace O. Ciabattoni, Bruno A. Rodriguez-Rodriguez, Keaton M. Crosse, Ralf Duerr & Meike Dittmann

  2. High Containment Laboratories—Office of Science and Research, NYU Langone Health, New York, NY, USA

    Dominick Papandrea & Ludovic Desvignes

  3. Department of Medicine, NYU Grossman School of Medicine, New York, NY, USA

    Marie I. Samanovic, Mark J. Mulligan, Ludovic Desvignes & Ralf Duerr

  4. NYU Langone Vaccine Center, NYU Grossman School of Medicine, New York, NY, USA

    Marie I. Samanovic, Mark J. Mulligan & Ralf Duerr

  5. Genome Technology Center, Office of Science and Research, NYU Langone Health, New York, NY, USA

    Dacia Dimartino, Christian Marier & Adriana Heguy

  6. Department of Pathology, NYU Grossman School of Medicine, New York, NY, USA

    Adriana Heguy

Authors
  1. Maren de Vries
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Grace O. Ciabattoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bruno A. Rodriguez-Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Keaton M. Crosse
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dominick Papandrea
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marie I. Samanovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dacia Dimartino
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christian Marier
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mark J. Mulligan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Adriana Heguy
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ludovic Desvignes
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ralf Duerr
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Meike Dittmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.d.V. and M.D. developed the protocol. M.d.V., M.D., D.D., C.M. and R.D. wrote the manuscript. G.O.C., B.A.R.-R., K.M.C. and M.d.V. performed viral experiments. D.D. and C.M. performed the whole-viral-genome deep sequencing. R.D. performed sequence analysis. M.I.S. and M.J.M provided swab material for isolation and repository viruses. D.P. and L.D. assisted in the development of the protocol and supervised risk management of BSL-3 laboratory work. All authors read and edited the manuscript.

Corresponding author

Correspondence to Meike Dittmann.

Ethics declarations

Competing interests

M.J.M. reports the following potential competing interests: laboratory research and clinical trials contract funding for vaccines or monoclonal antibodies against SARS-CoV-2 with Lilly, Pfizer and Sanofi and personal fees for Scientific Advisory Board service from Merck, Meissa Vaccines, Inc. and Pfizer. All remaining authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Pragya D. Yadav and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

de Vries, M. et al. J. Virol. 95, e01819-20 (2021): https://doi.org/10.1128/JVI.01819-20

Ching, K. L. et al. PLoS Biol. 20, 1–25 (2022): https://doi.org/10.1371/journal.pbio.3001754

Rodriguez-Rodriguez, B. A. et al. Nat. Commun. 14, 3026 (2023): https://doi.org/10.1038/s41467-023-38783-0

Supplementary information

Source data

Source Data Fig. 2

Whole-well images of plaque assays

Source Data Fig. 3

Scans of plaque assay plates for panel a

Source Data Fig. 3

Raw data of titer determination in panel b

Source Data Fig. 4

Whole-well images of CPE

Souce Data Fig. 6

Scans of plaque assay plates

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

de Vries, M., Ciabattoni, G.O., Rodriguez-Rodriguez, B.A. et al. Generation of quality-controlled SARS-CoV-2 variant stocks. Nat Protoc 18, 3821–3855 (2023). https://doi.org/10.1038/s41596-023-00897-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-023-00897-6

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology