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Журнал микробиологии, эпидемиологии и иммунобиологии. 2020; 97: 511-517

Генетическая вариабельность SARS-CoV-2 в биологических образцах от пациентов г. Москвы

Сперанская А. С., Каптелова В. В., Самойлов А. Е., Бухарина А. Ю., Шипулина О. Ю., Корнеенко Е. В., Акимкин В. Г.

https://doi.org/10.36233/0372-9311-2020-97-6-1

Аннотация

Сосуществование субпопуляций SARS-CoV-2 с различными вариантами генома внутри организма одного пациента — один из обсуждаемых в настоящее время феноменов. В данной работе мы провели высокопроизводительное секвенирование и сборку полных геномов вирусов из образцов, которые представляли собой мазки или аутопсийный материал от пациентов с диагнозом СOVID-19, предварительно подтвержденным методом полимеразной цепной реакции в реальном времени (Ct = 10,4–19,8). Подготовку образцов к секвенированию проводили с помощью протокола SCV-2000bp. Полученные данные проверяли на присутствие в образце более чем одного генетического варианта SARS-CoV-2. Варианты нуклеотидных замен, покрытие для каждого варианта, а также координаты вариабельной позиции в референсном геноме определяли с помощью инструментов программы «CLC Genomics Workbench». При поиске вариабельных нуклеотидных позиций исходили из предположения, что в образце имеются 2 генетических варианта (не более), для вероятности определяемого варианта использовали пороговое значение ≥ 90%. Также игнорировали варианты, которые были представлены менее чем 20% прочтений от общего покрытия. Полученные результаты показали, что в 5 образцах имеется вариабельность, т.е. содержится несколько генетических вариантов SARS-CoV-2. В 4 образцах оба найденных варианта геномов различались лишь в одной нуклеотидной позиции. В пятом образце были найдены более существенные различия: сразу 3 вариабельных позиции и одна делеция длиной в 3 нуклеотида. Наше исследование показывает возможность сосуществования различных генетических вариантов SARS-CoV-2 в организме пациента.
Список литературы

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12. Kireev D.E., Lopatukhin A.E., Murzakova A.V., Pimkina E.V., Speranskaya A.S., Neverov A.D., et al. Evaluating the accuracy and sensitivity of detecting minority HIV-1 populations by Illumina next-generation sequencing. J. Virol. Methods. 2018; 261: 40–5. https://doi.org/10.1016/j.jviromet.2018.08.001

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16. van der Kuyl A.C., Cornelissen M. Identifying HIV-1 dual infections. Retrovirology. 2007; 4: 67. https://doi.org/10.1186/1742-4690-4-67

17. Weinberg A., Bloch K.C., Li S., Tang Y.W., Palmer M., Tyler K.L. Dual infections of the central nervous system with Epstein‐Barr virus. J. Infect. Dis. 2005; 191(2): 234–7. https://doi.org/10.1086/426402

18. Hashim H.O., Mohammed M.K., Mousa M.J., Abdulameer H.H., Alhassnawi A.T., Hassan S.A., et al. Unexpected co-infection with different strains of SARS-CoV-2 in patients with COVID-19. Preprints.org. 2020. Preprint. https://doi.org/10.20944/preprints202009.0375.v1

19. Liu S., Shen J., Fang S., Li K., Liu J., Yang L., et al. Genetic spectrum and distinct evolution patterns of SARS-CoV-2. Front. Microbiol. 2020; 11: 593548. https://doi.org/10.3389/fmicb.2020.593548

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21. Samoilov A., Kaptelova V.V., Bukharina A.Y., Shipulina O.Y., Korneenko E.V., Lukyanov A.V. et al. Change of dominant strain during dual SARS-CoV-2 infection: preprint. medRxiv. Preprint. 2020. https://doi.org/10.1101/2020.11.29.20238402

22. Ilmjärv S., Abdul F., Acosta-Gutiérrez S., Estarellas C., Galdadas I., Casimir M., et al. Epidemiologically most successful SARS-CoV-2 variant: concurrent mutations in RNA-dependent RNA polymerase and spike protein. medRxiv. Preprint. 2020. https://doi.org/10.1101/2020.08.23.20180281

23. Speranskaya A., Kaptelova V., Valdokhina A., Bulanenko V., Samoilov A., Korneenko E., et al. SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing. bioRxiv. Preprint. 2020. https://doi.org/10.1101/2020.08.04.234880

24. Kaptelova V.V., Speranskaya A.S. Protocol for SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing v1. https://dx.doi.org/10.17504/protocols.io.bn77mhrn

25. Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–20. https://doi.org/10.1093/bioinformatics/btu170

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

27. Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012; 9(4): 357–9. https://doi.org/10.1038/nmeth.1923

28. Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. https://doi.org/10.1093/bioinformatics/btp352

29. Quinlan A.R., Hall I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–2. https://doi.org/10.1093/bioinformatics/btq033

30. Romano M., Ruggiero A., Squeglia F., Maga G., Berisio R. A structural view of SARS-CoV-2 RNA replication machinery: RNA synthesis, proofreading and final capping. Cells. 2020; 9(5): 1267. https://doi.org/10.3390/cells9051267

31. Zhao Z., Li H., Wu X., Zhong Y., Zhang K., Zhang Y.P., et al. Moderate mutation rate in the SARS coronavirus genome and its implications. BMC Evol. Biol. 2004; 4: 21. https://doi.org/10.1186/1471-2148-4-21

Journal of microbiology, epidemiology and immunobiology. 2020; 97: 511-517

Genetic variability of SARS-CoV-2 in biological samples from patients in Moscow

Speranskaya A. S., Kaptelova V. V., Samoilov A. E., Bukharina A. Yu., Shipulina O. Yu., Korneenko E. V., Akimkin V. G.

https://doi.org/10.36233/0372-9311-2020-97-6-1

Abstract

Currently, a lot of attention is given to SARS-CoV-2 subpopulations and their coexistence with different genomic variants within the same patient. In this study, we performed next-generation whole-genome sequencing and assembly of viruses from samples representing swabs or autopsy specimens obtained from patients diagnosed with СOVID-19, which were initially confirmed by the real-time polymerase chain reaction (Ct = 10.4–19.8). Samples were prepared for sequencing by using the SCV-2000bp protocol. The obtained data were checked for presence of more than one SARS-CoV-2 genetic variants in a sample. Variants of nucleotide substitutions, coverage for each variant, and location of the variable position in the reference genome were detected with tools incorporated in the CLC Genomics Workbench program. In our search for variable nucleotide positions, we assumed that the sample had two genetic variants (not more); the threshold value ≥ 90% was set for probability of the identified variant. Variants represented by less than 20% of the reads in the total coverage were not taken into consideration. The obtained results showed that 5 samples had variability, i.e. they had several genetic variants of SARS-CoV-2. In 4 samples, both of the detected genomic variants differed only in one nucleotide position. The fifth sample demonstrated more substantial differences: a total of 3 variable positions and one three-nucleotide deletion. Our study shows that different genetic variants of SARS-CoV-2 can coexist within the same patient.
References

1. Tang X., Wu C., Li X., Song Y., Yao X., Wu X., et al. On the origin and continuing evolution of SARS-CoV-2. Natl. Sci. Rev. 2020; 7(6): 1012–23. https://doi.org/10.1093/nsr/nwaa036

2. Komissarov A.B., Safina K.R., Garushyants S.K., Fadeev A.V., Sergeeva M.V., Ivanova A.A., et al. Genomic epidemiology of the early stages of SARS-CoV-2 outbreak in Russia. medRxiv. Preprint. 2020. https://doi.org/10.1101/2020.07.14.20150979

3. Sýkorová E., Fajkus J., Mezníková M., Lim K.Y., Neplechová K., Blattner F.R., et al. Minisatellite telomeres occur in the family Alliaceae but are lost in Allium. Am. J. Bot. 2006; 93(6): 814–23. https://doi.org/10.3732/ajb.93.6.814

4. Nyayanit D., Yadav P.D., Kharde R., Shete-Aich A. Quasispecies analysis of the SARS-CoV-2 from representative clinical samples: A preliminary analysis. Indian J. Med. Res. 2020; 152(1): 105. https://doi.org/10.4103/ijmr.ijmr_2251_20

5. Jary A., Leducq V., Malet I., Marot S., Klement-Frutos E., Teyssou E., et al. Evolution of viral quasispecies during SARSCoV-2 infection. Clin. Microbiol. Infect. 2020; 26(11): 1560. e1-1560.e4. https://doi.org/10.1016/j.cmi.2020.07.032

6. Chaudhry M.Z., Eschke K., Grashoff M., Abassi L., Kim Y., Brunotte L., et al. SARS-CoV-2 quasispecies mediate rapid virus evolution and adaptation. bioRxiv. Preprint. 2020. https://doi.org/10.1101/2020.08.10.241414

7. Xu D., Zhang Z., Wang F.S. SARS-associated coronavirus quasispecies in individual patients. N. Engl. J. Med. 2004; 350(13): 1366–7. https://doi.org/10.1056/nejmc032421

8. Park D., Huh H.J., Kim Y.J., Son D.S., Jeon H.J., Im E.H., et al. Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus. Mol. Case Stud. 2016; 2(6): a001214. https://doi.org/10.1101/mcs.a001214

9. Kuipers J., Batavia A.A., Jablonski K.P., Bayer F., Borgsmüller N., Dondi A., et al. Within-patient genetic diversity of SARS-CoV-2. bioRxiv. Preprint. 2020. https://doi.org/10.1101/2020.10.12.335919

10. McElroy K., Zagordi O., Bull R., Luciani F., Beerenwinkel N. Accurate single nucleotide variant detection in viral populations by combining probabilistic clustering with a statistical test of strand bias. BMC Genomics. 2013; 14(1): 501. https://doi.org/10.1186/1471-2164-14-501

11. Fahnøe U., Pedersen A.G., Dräger C., Orton R.J., Blome S., Höper D., et al. Creation of functional viruses from non-functional cDNA clones obtained from an RNA virus population by the use of ancestral reconstruction. PLoS One. 2015; 10(10): e0140912. https://doi.org/10.1371/journal.pone.0140912

12. Kireev D.E., Lopatukhin A.E., Murzakova A.V., Pimkina E.V., Speranskaya A.S., Neverov A.D., et al. Evaluating the accuracy and sensitivity of detecting minority HIV-1 populations by Illumina next-generation sequencing. J. Virol. Methods. 2018; 261: 40–5. https://doi.org/10.1016/j.jviromet.2018.08.001

13. Lapovok I.A., Lopatukhin A.E., Kireev D.E. Dvoinaya VICh-infektsiya: epidemiologiya, klinicheskaya znachimost' i diagnostika. Infektsionnye bolezni. 2019; 17(2): 81–7. [Lapovok I.A., Lopatukhin A.E., Kireev D.E. Dual HIV infection: epidemiology, clinical significance, and diagnosis. Infektsionnye bolezni. 2019; 17(2): 81–7. (in Russ.)] https://doi.org/10.20953/1729-9225-2019-2-81-87

14. Falchi A., Arena C., Andreoletti L., Jacques J., Leveque N., Blanchon T., et al. Dual infections by influenza A/H3N2 and B viruses and by influenza A/H3N2 and A/H1N1 viruses during winter 2007, Corsica Island, France. J. Clin. Virol. 2008; 41(2): 148–51. https://doi.org/10.1016/j.jcv.2007.11.003

15. Semple M.G., Cowell A., Dove W., Greensill J., McNamara P.S., Halfhide C., et al. Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis. J. Infect. Dis. 2005; 191(3): 382–6. https://doi.org/10.1086/426457

16. van der Kuyl A.C., Cornelissen M. Identifying HIV-1 dual infections. Retrovirology. 2007; 4: 67. https://doi.org/10.1186/1742-4690-4-67

17. Weinberg A., Bloch K.C., Li S., Tang Y.W., Palmer M., Tyler K.L. Dual infections of the central nervous system with Epstein‐Barr virus. J. Infect. Dis. 2005; 191(2): 234–7. https://doi.org/10.1086/426402

18. Hashim H.O., Mohammed M.K., Mousa M.J., Abdulameer H.H., Alhassnawi A.T., Hassan S.A., et al. Unexpected co-infection with different strains of SARS-CoV-2 in patients with COVID-19. Preprints.org. 2020. Preprint. https://doi.org/10.20944/preprints202009.0375.v1

19. Liu S., Shen J., Fang S., Li K., Liu J., Yang L., et al. Genetic spectrum and distinct evolution patterns of SARS-CoV-2. Front. Microbiol. 2020; 11: 593548. https://doi.org/10.3389/fmicb.2020.593548

20. Gudbjartsson D.F., Helgason A., Jonsson H., Magnusson O.T., Melsted P., Norddahl G.L., et al. Spread of SARS-CoV-2 in the Icelandic population. N. Engl. J. Med. 2020; 382(24): 2302–15. https://doi.org/10.1056/nejmoa2006100

21. Samoilov A., Kaptelova V.V., Bukharina A.Y., Shipulina O.Y., Korneenko E.V., Lukyanov A.V. et al. Change of dominant strain during dual SARS-CoV-2 infection: preprint. medRxiv. Preprint. 2020. https://doi.org/10.1101/2020.11.29.20238402

22. Ilmjärv S., Abdul F., Acosta-Gutiérrez S., Estarellas C., Galdadas I., Casimir M., et al. Epidemiologically most successful SARS-CoV-2 variant: concurrent mutations in RNA-dependent RNA polymerase and spike protein. medRxiv. Preprint. 2020. https://doi.org/10.1101/2020.08.23.20180281

23. Speranskaya A., Kaptelova V., Valdokhina A., Bulanenko V., Samoilov A., Korneenko E., et al. SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing. bioRxiv. Preprint. 2020. https://doi.org/10.1101/2020.08.04.234880

24. Kaptelova V.V., Speranskaya A.S. Protocol for SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing v1. https://dx.doi.org/10.17504/protocols.io.bn77mhrn

25. Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–20. https://doi.org/10.1093/bioinformatics/btu170

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

27. Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012; 9(4): 357–9. https://doi.org/10.1038/nmeth.1923

28. Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. https://doi.org/10.1093/bioinformatics/btp352

29. Quinlan A.R., Hall I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–2. https://doi.org/10.1093/bioinformatics/btq033

30. Romano M., Ruggiero A., Squeglia F., Maga G., Berisio R. A structural view of SARS-CoV-2 RNA replication machinery: RNA synthesis, proofreading and final capping. Cells. 2020; 9(5): 1267. https://doi.org/10.3390/cells9051267

31. Zhao Z., Li H., Wu X., Zhong Y., Zhang K., Zhang Y.P., et al. Moderate mutation rate in the SARS coronavirus genome and its implications. BMC Evol. Biol. 2004; 4: 21. https://doi.org/10.1186/1471-2148-4-21