The mutational dynamics of the SARS-CoV-2 virus in serial passages <i>in vitro</i>

Sissy Therese Sonnleitner; Stefanie Sonnleitner; Eva Hinterbichler; Hannah Halbfurter; Dominik B.C. Kopecky; Stephan Koblmüller; Christian Sturmbauer; Wilfried Posch; Gernot Walder

doi:10.1016/j.virs.2022.01.029

April 2022

Citation: Sissy Therese Sonnleitner, Stefanie Sonnleitner, Eva Hinterbichler, Hannah Halbfurter, Dominik B.C. Kopecky, Stephan Koblmüller, Christian Sturmbauer, Wilfried Posch, Gernot Walder. The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro .VIROLOGICA SINICA, 2022, 37(2) : 198-207. http://dx.doi.org/10.1016/j.virs.2022.01.029

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

a Dr. Gernot Walder GmbH, Medical Laboratory, Department of Virology, Ausservillgraten, 9931, Austria
b Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, 6020, Austria
c Institute of Biology, University of Graz, Graz, 8010, Austria

Corresponding author: Sissy Therese Sonnleitner, sissy.sonnleitner@infektiologie.tirol
Received Date: 26 September 2021
Accepted Date: 21 January 2022
Available online: 29 January 2022

Abstract

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.
- SARS-CoV-2
- , Whole genome sequencing (WGS)
- , Mutational dynamics
- , Adaptation
- , Serial passage in vitro

Electronic Supplementary Material

10.1016j.virs.2022.01.029-ESM.docx
References
1. Andreano, E., Piccini, G., Licastro, D., Casalino, L., Johnson, N.V., Paciello, I., Dal Monego, S., Pantano, E., Manganaro, N., Manenti, A., Manna, R., Casa, E., Hyseni, I., Benincasa, L., Montomoli, E., Amaro, R.E., McLellan, J.S., Rappuoli, R., 2020. SARSCoV-2 Escape bioRxiv.
2. Badua, C.L.D.C., Baldo, K.A.T., Medina, P.M.B., 2021. Genomic and proteomic mutation landscapes of SARS-CoV-2. J. Med. Virol. 93, 1702-1721.
3. Biebricher, C.K., Eigen, M., 2006. What is a quasispecies? Curr. Top. Microbiol. Immunol. 299, 1-31.
4. Brouwer, P.J.M., Caniels, T.G., van der Straten, K., Snitselaar, J.L., Aldon, Y., Bangaru, S., Torres, J.L., Okba, N.M.A., Claireaux, M., Kerster, G., Bentlage, A.E.H., van Haaren, M.M., Guerra, D., Burger, J.A., Schermer, E.E., Verheul, K.D., van der Velde, N., van der Kooi, A., van Schooten, J., van Breemen, M.J., Bijl, T.P.L., Sliepen, K., Aartse, A., Derking, R., Bontjer, I., Kootstra, N.A., Wiersinga, W.J., Vidarsson, G., Haagmans, B.L., Ward, A.B., de Bree, G.J., Sanders, R.W., van Gils, M.J., 2020. Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. Science 369, 643-650.
5. Cao, Y., Su, B., Guo, X., Sun, W., Deng, Y., Bao, L., Zhu, Q., Zhang, X., Zheng, Y., Geng, C., Chai, X., He, R., Li, X., Lv, Q., Zhu, H., Deng, W., Xu, Y., Wang, Y., Qiao, L., Tan, Y., Song, L., Wang, G., Du, X., Gao, N., Liu, J., Xiao, J., Su, X.D., Du, Z., Feng, Y., Qin, C., Jin, R., Xie, X.S., 2020. Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients' B cells. Cell 182, 73-84 e16.
6. Castaño-Rodriguez, C., Honrubia, J.M., Gutiérrez-Álvarez, J., DeDiego, M.L., Nieto-Torres, J.L., Jimenez-Guardeno, J.M., Regla-Nava, J.A., Fernandez-Delgado, R., Verdia-Báguena, C., Queralt-Martín, M., Kochan, G., Perlman, S., Aguilella, V.M., Sola, I., Enjuanes, L., 2018. Role of Severe acute respiratory syndrome coronavirus viroporins E, 3a, and 8a in replication and pathogenesis. mBio 9, e02325-17.
7. Chen, C.C., Krüger, J., Sramala, I., Hsu, H.J., Henklein, P., Chen, Y.M., Fischer, W.B., 2011. ORF8a of SARS-CoV forms an ion channel:experiments and molecular dynamics simulations. Biochim. Biophys. Acta 1808, 572-579.
8. Chrisman, B.S., Paskov, K., Stockham, N., Tabatabaei, K., Jung, J.Y., Washington, P., Varma, M., Sun, M.W., Maleki, S., Wall, D.P., 2021. Indels in SARS-CoV-2 occur at template-switching hotspots. BioData Min. 14, 20.
9. Elena, S.F., Sanjuán, R., 2005. Adaptive value of high mutation rates of RNA viruses:separating causes from consequences. J. Virol. 79, 11555-11558.
10. Franco-Muñoz, C.,Álvarez-Díaz, D.A., Laiton-Donato, K., Wiesner, M., Escandón, P., Usme-Ciro, J.A., Franco-Sierra, N.D., Flórez-Sánchez, A.C., Gómez-Rangel, S., Rodríguez-Calderon, L.D., Barbosa-Ramirez, J., Ospitia-Baez, E., Walteros, D.M., Ospina-Martinez, M.L., Mercado-Reyes, M., 2020. Substitutions in spike and nucleocapsid proteins of SARS-CoV-2 circulating in South America. Infect. Genet. Evol. 85, 104557.
11. Garushyants, S.K., Rogozin, I.B., Koonin, E.V., 2021. Insertions in SARS-CoV-2 Genome Caused by Template Switch and Duplications Give Rise to New Variants that Merit Monitoring. bioRxiv.
12. Gietl, S., Schönegger, C.M., Falk, M., Weiler, S., Obererlacher, S., Jansen, B., Sonnleitner, S.T., Walder, G., 2021. Home quarantine in COVID-19:a study on 50 consecutive patients in Austria. Clin. Med. 21, e9-e13.
13. Greaney, A.J., Loes, A.N., Crawford, K.H.D., Starr, T.N., Malone, K.D., Chu, H.Y., Bloom, J.D., 2021. Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies. Cell Host Microbe 29, 463-476 e6.
14. Guan, Y., Zheng, B.J., He, Y.Q., Liu, X.L., Zhuang, Z.X., Cheung, C.L., Luo, S.W., Li, P.H., Zhang, L.J., Guan, Y.J., Butt, K.M., Wong, K.L., Chan, K.W., Lim, W., Shortridge, K.F., Yuen, K.Y., Peiris, J.S., Poon, L.L., 2003. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276-278.
15. Harvey, W.T., Carabelli, A.M., Jackson, B., Gupta, R.K., Thomson, E.C., Harrison, E.M., Ludden, C., Reeve, R., Rambaut, A., Peacock, S.J., Robertson, D.L., Consortium, C.-G.U.C.-U., 2021. SARS-CoV-2 variants, spike mutations and immune escape. Nat. Rev. Microbiol. 19, 409-424.
16. Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q., Vinh, L.S., 2018. UFBoot2:improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518-522.
17. Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S., VandePol, S., 1982. Rapid evolution of RNA genomes. Science 215, 1577-1585.
18. Ju, B., Zhang, Q., Ge, J., Wang, R., Sun, J., Ge, X., Yu, J., Shan, S., Zhou, B., Song, S., Tang, X., Lan, J., Yuan, J., Wang, H., Zhao, J., Zhang, S., Wang, Y., Shi, X., Liu, L., Wang, X., Zhang, Z., Zhang, L., 2020. Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature 584, 115-119.
19. Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A., Jermiin, L.S., 2017. ModelFinder:fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587-589.
20. Kim, J.S., Jang, J.H., Kim, J.M., Chung, Y.S., Yoo, C.K., Han, M.G., 2020. Genome-wide identification and characterization of point mutations in the SARS-CoV-2 genome. Osong Public Health Res. Perspect. 11, 101-111.
21. Koenig, P.A., Das, H., Liu, H., Kümmerer, B.M., Gohr, F.N., Jenster, L.M., Schiffelers, L.D.J., Tesfamariam, Y.M., Uchima, M., Wuerth, J.D., Gatterdam, K., Ruetalo, N., Christensen, M.H., Fandrey, C.I., Normann, S., Todtmann, J.M.P., Pritzl, S., Hanke, L., Boos, J., Yuan, M., Zhu, X., Schmid-Burgk, J.L., Kato, H., Schindler, M., Wilson, I.A., Geyer, M., Ludwig, K.U., Hällberg, B.M., Wu, N.C., Schmidt, F.I., 2021. Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science 371, eabe6230.
22. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X:molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547-1549.
23. Lauring, A.S., Andino, R., 2010. Quasispecies theory and the behavior of RNA viruses. PLoS Pathog. 6, e1001005.
24. Liu, L., Wang, P., Nair, M.S., Yu, J., Rapp, M., Wang, Q., Luo, Y., Chan, J.F., Sahi, V., Figueroa, A., Guo, X.V., Cerutti, G., Bimela, J., Gorman, J., Zhou, T., Chen, Z., Yuen, K.Y., Kwong, P.D., Sodroski, J.G., Yin, M.T., Sheng, Z., Huang, Y., Shapiro, L., Ho, D.D., 2020. Potent neutralizing antibodies against multiple epitopes on SARSCoV-2 spike. Nature 584, 450-456.
25. Liu, Z., VanBlargan, L.A., Bloyet, L.M., Rothlauf, P.W., Chen, R.E., Stumpf, S., Zhao, H., Errico, J.M., Theel, E.S., Liebeskind, M.J., Alford, B., Buchser, W.J., Ellebedy, A.H., Fremont, D.H., Diamond, M.S., Whelan, S.P.J., 2021. Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization. Cell Host Microbe 29, 477-488 e4.
26. Lu, W., Zheng, B.J., Xu, K., Schwarz, W., Du, L., Wong, C.K., Chen, J., Duan, S., Deubel, V., Sun, B., 2006. Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release. Proc. Natl. Acad. Sci. U. S. A. 103, 12540-12545.
27. Majumdar, P., Niyogi, S., 2021. SARS-CoV-2 mutations:the biological trackway towards viral fitness. Epidemiol. Infect. 149, e110.
28. McCarthy, K.R., Rennick, L.J., Nambulli, S., Robinson-McCarthy, L.R., Bain, W.G., Haidar, G., Duprex, W.P., 2021. Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape. Science 371, 1139-1142.
29. Nguyen, L.T., Schmidt, H.A., von Haeseler, A., Minh, B.Q., 2015. IQ-TREE:a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268-274.
30. Nieto-Torres, J.L., Verdiá-Báguena, C., Jimenez-Guardeño, J.M., Regla-Nava, J.A., Castaño-Rodriguez, C., Fernandez-Delgado, R., Torres, J., Aguilella, V.M., Enjuanes, L., 2015. Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome. Virology 485, 330-339.
31. Nowak, M.A., 1992. What is a quasispecies? Trends Ecol. Evol. 7, 118-121. https://doi.org/10.1016/0169-5347(92)90145-2.
32. Okonechnikov, K., Golosova, O., Fursov, M., team, U., 2012. Unipro UGENE:a unified bioinformatics toolkit. Bioinformatics 28, 1166-1167.
33. Panzera, Y., Ramos, N., Frabasile, S., Calleros, L., Marandino, A., Tomás, G., Techera, C., Grecco, S., Fuques, E., Goñi, N., Ramas, V., Coppola, L., Chiparelli, H., Sorhouet, C., Mogdasy, C., Arbiza, J., Delfraro, A., P érez, R., 2021. A deletion in SARS-CoV-2 ORF7 identified in COVID-19 outbreak in Uruguay. Transbound Emerg. Dis. 68, 3075-3082.
34. Rogers, T.F., Zhao, F., Huang, D., Beutler, N., Burns, A., He, W.T., Limbo, O., Smith, C., Song, G., Woehl, J., Yang, L., Abbott, R.K., Callaghan, S., Garcia, E., Hurtado, J., Parren, M., Peng, L., Ramirez, S., Ricketts, J., Ricciardi, M.J., Rawlings, S.A., Wu, N.C., Yuan, M., Smith, D.M., Nemazee, D., Teijaro, J.R., Voss, J.E., Wilson, I.A., Andrabi, R., Briney, B., Landais, E., Sok, D., Jardine, J.G., Burton, D.R., 2020. Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science 369, 956-963.
35. Rohaim, M.A., El Naggar, R.F., Clayton, E., Munir, M., 2021. Structural and functional insights into non-structural proteins of coronaviruses. Microb. Pathog. 150, 104641.
36. Salinas, N.R., Little, D.P., 2014. 2matrix:a utility for indel coding and phylogenetic matrix concatenation(1.). Appl. Plant Sci. 2, apps.1300083.
37. Seydoux, E., Homad, L.J., MacCamy, A.J., Parks, K.R., Hurlburt, N.K., Jennewein, M.F., Akins, N.R., Stuart, A.B., Wan, Y.H., Feng, J., Whaley, R.E., Singh, S., Boeckh, M., Cohen, K.W., McElrath, M.J., Englund, J.A., Chu, H.Y., Pancera, M., McGuire, A.T., Stamatatos, L., 2020. Analysis of a SARS-CoV-2-infected individual reveals development of potent neutralizing antibodies with limited somatic mutation. Immunity 53, 98-105 e5.
38. Simmons, M.P., Ochoterena, H., 2000. Gaps as characters in sequence-based phylogenetic analyses. Syst. Biol. 49, 369-381.
39. Singer, J., Gifford, R., Cotten, M., Robertson, D., 2020. CoV-GLUE:A Web Application for Tracking SARS-CoV-2 Genomic Variation. https://doi.org/10.20944/preprints202006.0225.v1.
40. Siu, K.L., Yuen, K.S., Castaño-Rodriguez, C., Ye, Z.W., Yeung, M.L., Fung, S.Y., Yuan, S.,~Chan, C.P., Yuen, K.Y., Enjuanes, L., Jin, D.Y., 2019. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC. Faseb. J. 33, 8865-8877.
41. Sonnleitner, S.T., Dorighi, J., Jansen, B., Schönegger, C., Gietl, S., Koblmüller, S., Sturmbauer, C., Posch, W., Walder, G., 2021. 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.
42. Verkhivker, G.M., Agajanian, S., Oztas, D.Y., Gupta, G., 2021. Comparative perturbationbased modeling of the SARS-CoV-2 spike protein binding with host receptor and neutralizing antibodies:structurally adaptable Allosteric communication hotspots define spike sites targeted by global circulating mutations. Biochemistry 60, 1459-1484.
43. Vignuzzi, M., Stone, J.K., Andino, R., 2005. Ribavirin and lethal mutagenesis of poliovirus:molecular mechanisms, resistance and biological implications. Virus Res. 107, 173-181.
44. Walls, A.C., Park, Y.J., Tortorici, M.A., Wall, A., McGuire, A.T., Veesler, D., 2020. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 183, 1735.
45. Wec, A.Z., Wrapp, D., Herbert, A.S., Maurer, D.P., Haslwanter, D., Sakharkar, M., Jangra, R.K., Dieterle, M.E., Lilov, A., Huang, D., Tse, L.V., Johnson, N.V., Hsieh, C.L., Wang, N., Nett, J.H., Champney, E., Burnina, I., Brown, M., Lin, S., Sinclair, M., Johnson, C., Pudi, S., Bortz, R., Wirchnianski, A.S., Laudermilch, E., Florez, C., Fels, J.M., O'Brien, C.M., Graham, B.S., Nemazee, D., Burton, D.R., Baric, R.S., Voss, J.E., Chandran, K., Dye, J.M., McLellan, J.S., Walker, L.M., 2020. Broad neutralization of SARS-related viruses by human monoclonal antibodies. Science 369, 731-736.
46. Wu, Y., Wang, F., Shen, C., Peng, W., Li, D., Zhao, C., Li, Z., Li, S., Bi, Y., Yang, Y., Gong, Y., Xiao, H., Fan, Z., Tan, S., Wu, G., Tan, W., Lu, X., Fan, C., Wang, Q., Liu, Y., Zhang, C., Qi, J., Gao, G.F., Gao, F., Liu, L., 2020. A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science 368, 1274-1278.
47. Zhang, D., Gao, F., Jakovlić, I., Zou, H., Zhang, J., Li, W.X., Wang, G.T., 2020. PhyloSuite:an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol. Ecol. Resour. 20, 348-355.
48. Zheng, J., 2020. SARS-CoV-2:an emerging coronavirus that causes a global threat. Int. J. Biol. Sci. 16, 1678-1685.
49. Zost, S.J., Gilchuk, P., Case, J.B., Binshtein, E., Chen, R.E., Nkolola, J.P., Schäfer, A., Reidy, J.X., Trivette, A., Nargi, R.S., Sutton, R.E., Suryadevara, N., Martinez, D.R., Williamson, L.E., Chen, E.C., Jones, T., Day, S., Myers, L., Hassan, A.O., Kafai, N.M., Winkler, E.S., Fox, J.M., Shrihari, S., Mueller, B.K., Meiler, J., Chandrashekar, A., Mercado, N.B., Steinhardt, J.J., Ren, K., Loo, Y.M., Kallewaard, N.L., McCune, B.T., Keeler, S.P., Holtzman, M.J., Barouch, D.H., Gralinski, L.E., Baric, R.S., Thackray, L.B., Diamond, M.S., Carnahan, R.H., Crowe, J.E., 2020. Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature 584, 443-449.
Proportional views