Вопросы вирусологии. 2021; 66: 269-278
Мониторинг распространения вариантов SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) на территории Московского региона с помощью таргетного высокопроизводительного секвенирования
https://doi.org/10.36233/0507-4088-72Аннотация
Введение. С начала пандемического распространения инфекции COVID-19, вызываемой коронавирусом SARS-CoV-2, международное научное сообщество регулярно фиксирует появление мутаций этого патогена, потенциально способных повысить его контагиозность и/или вирулентность. В частности, с конца 2020 г. в мире обнаружено несколько вызывающих озабоченность вариантов, включая альфа (B.1.1.7), бета (B.1.351), гамма (P.1) и дельта (B.1.617.2). Однако существующие механизмы поиска мутаций и выявления штаммов не всегда бывают достаточно эффективными, поскольку лишь небольшая доля получаемых от пациентов образцов возбудителя может быть исследована на наличие генетических изменений, например методом полногеномного секвенирования из-за его высокой стоимости.
Материал и методы. В исследовании применён способ таргетного высокопроизводительного секвенирования нового (следующего) поколения (next generation sequencing, NGS) наиболее значимых регионов гена, кодирующего S-гликопротеин (шиповидный, spike) вируса SARS-CoV-2, для чего разработана соответствующая праймерная панель. В среднем на платформе Illumina на 1 образец приходилось около 50 тыс. парноконцевых прочтений длиной ≥150 п.н. С помощью описанной методики нами исследованы 579 случайных образцов, полученных у проживающих в Московском регионе пациентов с новой коронавирусной инфекцией с февраля по июнь 2021 г.
Результаты. В работе продемонстрирована динамика представленности в Российской Федерации ряда штаммов нового коронавируса и нескольких его мутаций на протяжении февраля–июня 2021 г. Установлено, что штамм дельта появился на территории Москвы и Московской области в мае текущего года, а в июне стал доминирующим, частично вытеснив другие разновидности вируса.
Обсуждение. Полученные данные представляют возможность определять принадлежность образцов к упомянутым и некоторым другим штаммам, а описанный подход может быть использован для стандартизации процедуры поиска новых и существующих разновидностей SARS-CoV-2. Методика делает возможным изучение большого количества образцов в короткие сроки, позволяя получать более детальное представление об эпидемиологической ситуации в регионе.
Список литературы
1. Zhou P., Yang X.L., Wang X.G., Hu B., Zhang L., Zhang W., et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579(7798): 270–3. https://doi.org/10.1038/s41586-020-2012-7
2. COVID-19 data in motion. Available at: https://coronavirus.jhu.edu (accessed July 24, 2021).
3. Chen P., Nirula A., Heller B., Gottlieb R.L., Boscia J., Morris J., et al. SARS-CoV-2 neutralizing antibody LY-CoV555 in outpatients with Covid-19. N. Engl. J. Med. 2021; 384(3): 229–37. https://doi.org/10.1056/nejmoa2029849
4. Weinreich D.M., Sivapalasingam S., Norton T., Ali S., Gao H., Bhore R., et al. REGN-COV2, a neutralizing antibody cocktail, in outpatients with Covid-19. N. Engl. J. Med. 2021; 384(3): 238–51. https://doi.org/10.1056/nejmoa2035002
5. Baden L.R., El Sahly H.M., Essink B., Kotloff K., Frey S., Novak R., et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 2021; 384(5): 403–16. https://doi.org/10.1056/nejmoa2035389
6. Polack F.P., Thomas S.J., Kitchin N., Absalon J., Gurtman A., Lockhart S., et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 2020; 383(27): 2603–15. https://doi.org/10.1056/nejmoa2034577
7. Jones I., Roy P. Sputnik V COVID-19 vaccine candidate appears safe and effective. Lancet. 2021; 397(10275): 642–3. https://doi.org/10.1016/s0140-6736(21)00191-4
8. Рыжиков А.Б., Рыжиков Е.А., Богрянцева М.П., Усова С.В., Даниленко Е.Д., Нечаева Е.А., и др. Простое слепое плацебо-контролируемое рандомизированное исследование безопасности, реактогенности и иммуногенности вакцины «ЭпиВакКорона» для профилактики COVID-19 на добровольцах в возрасте 18–60 лет (фаза I–II). Инфекция и иммунитет. 2021; 11(2): 283–96. https://doi.org/10.15789/2220-7619-ASB-1699
9. About Variants of the Virus that Causes COVID-19. Available at: https://www.cdc.gov/coronavirus/2019-ncov/Transmission/variant.html (accessed July 26, 2021).
10. Wang W.B., Liang Y., Jin Y.Q., Zhang J., Su J.G., Li Q.M. E484K mutation in SARS-CoV-2 RBD enhances binding affinity with hACE2 but reduces interactions with neutralizing antibodies and nanobodies: binding free energy calculation studies. bioRxiv. 2021; Preprint. https://doi.org/10.1101/2021.02.17.431566
11. Garcia-Beltran W.F., Lam E.C., St. Denis K., Nitido A.D., Garcia Z.H., Hauser B.M., et al. Circulating SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. medRxiv. 2021; Preprint. https://doi.org/10.1101/2021.02.14.21251704
12. Liu H., Wei P., Zhang Q., Chen Z., Aviszus K., Downing W., et al. 501Y.V2 and 501Y.V3 variants of SARS-CoV-2 lose binding to bamlanivimab in vitro. MAbs. 2021; 13(1): 1919285. https://doi.org/10.1080/19420862.2021.1919285
13. Yuan M., Huang D., Lee C.D., Wu N.C., Jackson A.M., Zhu X., et al. Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants. Science. 2021; eabh1139. https://doi.org/10.1126/science.abh1139
14. Ikegame S., Siddiquey M.N.A., Hung C.-T., Haas G., Brambilla L., Oguntuyo K.Y., et al. Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants. medRxiv. 2021.03.31.21254660. doi: https://doi.org/10.1101/2021.03.31.21254660
15. Gard N., Buzko O., Spilman P., Niazi K., Rabizadeh S., Soon- Shiong P. Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, potentially resulting in an escape mutant bioRxiv. 2021.01.13.426558. doi: https://doi.org/10.1101/2021.01.13.426558
16. Tian F., Tong B., Sun L., Shi S., Zheng B., Wang Z., et al. Mutation N501Y in RBD of spike protein strengthens the interaction between COVID-19 and its receptor ACE2. bioRxiv. 2021; Preprint. https://doi.org/10.1101/2021.02.14.431117
17. Хафизов К.Ф., Петров В.В., Красовитов К.В., Золкина М.В., Акимкин В.Г. Экспресс-диагностика новой коронавирусной инфекции с помощью реакции петлевой изотермической амплификации. Вопросы вирусологии. 2021; 66(1): 17–28. https://doi.org/10.36233/0507-4088-42
18. Gladkikh A., Dolgova A., Dedkov V., Sbarzaglia V., Kanaeva O., Popova A., et al. Characterization of a novel SARS-CoV-2 genetic variant with distinct spike protein mutations. Viruses. 2021; 13(6): 1029. https://doi.org/10.3390/v13061029
19. Klink G.V., Safina K.R., Garushyants S.K., Moldovan M., Nabieva E., Komissarov A.B., et al. Spread of endemic SARSCoV-2 lineages in Russia. medRxiv. 2021; Preprint. https://doi.org/10.1101/2021.05.25.21257695
20. 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 the SARS-CoV-2 outbreak in Russia. Nat. Commun. 2021; 12(1): 649. https://doi.org/10.1038/s41467-020-20880-z
21. Long S.W., Olsen R.J., Christensen P.A., Subedi S., Olson R., Davis J.J., et al. Sequence Analysis of 20,453 Severe Acute Respiratory Syndrome Coronavirus 2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern. Am. J. Pathol. 2021; 191(6): 983–92. https://doi.org/10.1016/j.ajpath.2021.03.004
22. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990; 215(3): 403–10. https://doi.org/10.1016/s0022-2836(05)80360-2
23. Li H., Durbin R. Fast and accurate short read alignment with Burrows– Wheeler transform. Bioinformatics. 2009; 25(14): 1754–60. https://doi.org/10.1093/bioinformatics/btp324
24. Bushnell B., Rood J., Singer E. BBMerge – Accurate paired shotgun read merging via overlap. PLoS One. 2017; 12(10): e0185056. https://doi.org/10.1371/journal.pone.0185056
25. Poplin R., Ruano-Rubio V., DePristo M.A., Fennell T.J., Carneiro M.O., Van der Auwera G.A., et al. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv. 2018; 201178. doi: https://doi.org/10.1101/201178
26. Humphrey W., Dalke A., Schulten K. VMD: visual molecular dynamics. J. Mol. Graph. 1996; 14(1): 33–8. https://doi.org/10.1016/0263-7855(96)00018-5
27. Lv Z., Deng Y.Q., Ye Q., Cao L., Sun C.Y., Fan C., et al. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody. Science. 2020; 369(6510): 1505–9. https://doi.org/10.1126/science.abc5881
28. Davies N.G., Abbott S., Barnard R.C., Jarvis C.I., Kucharski A.J., Munday J.D., et al. Estimated transmissibility and impact of SARSCoV-2 lineage B.1.1.7 in England. Science. 2021; 372(6538): eabg3055. https://doi.org/10.1126/science.abg3055
29. Expert comment on the ‘Delta plus’ variant (B.1.617.2 with the addition of K417N mutation). Available at: https://www.sciencemediacentre.org/expert-comment-on-the-delta-plus-variant-b-1-617-2-with-theaddition-of-k417n-mutation/ (accessed July 26, 2021).
30. Weekly epidemiological update on COVID-19 – 22 June 2021. Available at: https://www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19---22-june-2021 (accessed July 24, 2021).
Problems of Virology. 2021; 66: 269-278
Monitoring the spread of the SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) variants in the Moscow region using targeted high-throughput sequencing
https://doi.org/10.36233/0507-4088-72Abstract
Introduction. Since the outbreak of the COVID-19 pandemic caused by SARS-CoV-2 novel coronavirus, the international community has been concerned about the emergence of mutations altering some biological properties of the pathogen like increasing its infectivity or virulence. Particularly, since the end of 2020, several variants of concern have been identified around the world, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). However, the existing mechanism of detecting important mutations are not always effective enough, since only a relatively small part of all pathogen samples can be examined by whole genome sequencing due to its high cost.
Material and methods. In this study, we have designed special primer panel and used it for targeted highthroughput sequencing of several significant S-gene (spike) regions of SARS-CoV-2. The Illumina platform averaged approximately 50,000 paired-end reads with a length of ≥150 bp per sample. This method was used to examine 579 random samples obtained from COVID-19 patients in Moscow and the Moscow region from February to June 2021.
Results. This study demonstrated the dynamics of distribution of several SARS-CoV-2 strains and its some single mutations. It was found that the Delta strain appeared in the region in May 2021, and became prevalent in June, partially displacing other strains.
Discussion. The obtained results provide an opportunity to assign the viral samples to one of the strains, including the previously mentioned in time- and cost-effective manner. The approach can be used for standardization of the procedure of searching for mutations in individual regions of the SARS-CoV-2 genome. It allows to get a more detailed data about the epidemiological situation in a region.
References
1. Zhou P., Yang X.L., Wang X.G., Hu B., Zhang L., Zhang W., et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579(7798): 270–3. https://doi.org/10.1038/s41586-020-2012-7
2. COVID-19 data in motion. Available at: https://coronavirus.jhu.edu (accessed July 24, 2021).
3. Chen P., Nirula A., Heller B., Gottlieb R.L., Boscia J., Morris J., et al. SARS-CoV-2 neutralizing antibody LY-CoV555 in outpatients with Covid-19. N. Engl. J. Med. 2021; 384(3): 229–37. https://doi.org/10.1056/nejmoa2029849
4. Weinreich D.M., Sivapalasingam S., Norton T., Ali S., Gao H., Bhore R., et al. REGN-COV2, a neutralizing antibody cocktail, in outpatients with Covid-19. N. Engl. J. Med. 2021; 384(3): 238–51. https://doi.org/10.1056/nejmoa2035002
5. Baden L.R., El Sahly H.M., Essink B., Kotloff K., Frey S., Novak R., et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 2021; 384(5): 403–16. https://doi.org/10.1056/nejmoa2035389
6. Polack F.P., Thomas S.J., Kitchin N., Absalon J., Gurtman A., Lockhart S., et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 2020; 383(27): 2603–15. https://doi.org/10.1056/nejmoa2034577
7. Jones I., Roy P. Sputnik V COVID-19 vaccine candidate appears safe and effective. Lancet. 2021; 397(10275): 642–3. https://doi.org/10.1016/s0140-6736(21)00191-4
8. Ryzhikov A.B., Ryzhikov E.A., Bogryantseva M.P., Usova S.V., Danilenko E.D., Nechaeva E.A., i dr. Prostoe slepoe platsebo-kontroliruemoe randomizirovannoe issledovanie bezopasnosti, reaktogennosti i immunogennosti vaktsiny «EpiVakKorona» dlya profilaktiki COVID-19 na dobrovol'tsakh v vozraste 18–60 let (faza I–II). Infektsiya i immunitet. 2021; 11(2): 283–96. https://doi.org/10.15789/2220-7619-ASB-1699
9. About Variants of the Virus that Causes COVID-19. Available at: https://www.cdc.gov/coronavirus/2019-ncov/Transmission/variant.html (accessed July 26, 2021).
10. Wang W.B., Liang Y., Jin Y.Q., Zhang J., Su J.G., Li Q.M. E484K mutation in SARS-CoV-2 RBD enhances binding affinity with hACE2 but reduces interactions with neutralizing antibodies and nanobodies: binding free energy calculation studies. bioRxiv. 2021; Preprint. https://doi.org/10.1101/2021.02.17.431566
11. Garcia-Beltran W.F., Lam E.C., St. Denis K., Nitido A.D., Garcia Z.H., Hauser B.M., et al. Circulating SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. medRxiv. 2021; Preprint. https://doi.org/10.1101/2021.02.14.21251704
12. Liu H., Wei P., Zhang Q., Chen Z., Aviszus K., Downing W., et al. 501Y.V2 and 501Y.V3 variants of SARS-CoV-2 lose binding to bamlanivimab in vitro. MAbs. 2021; 13(1): 1919285. https://doi.org/10.1080/19420862.2021.1919285
13. Yuan M., Huang D., Lee C.D., Wu N.C., Jackson A.M., Zhu X., et al. Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants. Science. 2021; eabh1139. https://doi.org/10.1126/science.abh1139
14. Ikegame S., Siddiquey M.N.A., Hung C.-T., Haas G., Brambilla L., Oguntuyo K.Y., et al. Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants. medRxiv. 2021.03.31.21254660. doi: https://doi.org/10.1101/2021.03.31.21254660
15. Gard N., Buzko O., Spilman P., Niazi K., Rabizadeh S., Soon- Shiong P. Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, potentially resulting in an escape mutant bioRxiv. 2021.01.13.426558. doi: https://doi.org/10.1101/2021.01.13.426558
16. Tian F., Tong B., Sun L., Shi S., Zheng B., Wang Z., et al. Mutation N501Y in RBD of spike protein strengthens the interaction between COVID-19 and its receptor ACE2. bioRxiv. 2021; Preprint. https://doi.org/10.1101/2021.02.14.431117
17. Khafizov K.F., Petrov V.V., Krasovitov K.V., Zolkina M.V., Akimkin V.G. Ekspress-diagnostika novoi koronavirusnoi infektsii s pomoshch'yu reaktsii petlevoi izotermicheskoi amplifikatsii. Voprosy virusologii. 2021; 66(1): 17–28. https://doi.org/10.36233/0507-4088-42
18. Gladkikh A., Dolgova A., Dedkov V., Sbarzaglia V., Kanaeva O., Popova A., et al. Characterization of a novel SARS-CoV-2 genetic variant with distinct spike protein mutations. Viruses. 2021; 13(6): 1029. https://doi.org/10.3390/v13061029
19. Klink G.V., Safina K.R., Garushyants S.K., Moldovan M., Nabieva E., Komissarov A.B., et al. Spread of endemic SARSCoV-2 lineages in Russia. medRxiv. 2021; Preprint. https://doi.org/10.1101/2021.05.25.21257695
20. 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 the SARS-CoV-2 outbreak in Russia. Nat. Commun. 2021; 12(1): 649. https://doi.org/10.1038/s41467-020-20880-z
21. Long S.W., Olsen R.J., Christensen P.A., Subedi S., Olson R., Davis J.J., et al. Sequence Analysis of 20,453 Severe Acute Respiratory Syndrome Coronavirus 2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern. Am. J. Pathol. 2021; 191(6): 983–92. https://doi.org/10.1016/j.ajpath.2021.03.004
22. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990; 215(3): 403–10. https://doi.org/10.1016/s0022-2836(05)80360-2
23. Li H., Durbin R. Fast and accurate short read alignment with Burrows– Wheeler transform. Bioinformatics. 2009; 25(14): 1754–60. https://doi.org/10.1093/bioinformatics/btp324
24. Bushnell B., Rood J., Singer E. BBMerge – Accurate paired shotgun read merging via overlap. PLoS One. 2017; 12(10): e0185056. https://doi.org/10.1371/journal.pone.0185056
25. Poplin R., Ruano-Rubio V., DePristo M.A., Fennell T.J., Carneiro M.O., Van der Auwera G.A., et al. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv. 2018; 201178. doi: https://doi.org/10.1101/201178
26. Humphrey W., Dalke A., Schulten K. VMD: visual molecular dynamics. J. Mol. Graph. 1996; 14(1): 33–8. https://doi.org/10.1016/0263-7855(96)00018-5
27. Lv Z., Deng Y.Q., Ye Q., Cao L., Sun C.Y., Fan C., et al. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody. Science. 2020; 369(6510): 1505–9. https://doi.org/10.1126/science.abc5881
28. Davies N.G., Abbott S., Barnard R.C., Jarvis C.I., Kucharski A.J., Munday J.D., et al. Estimated transmissibility and impact of SARSCoV-2 lineage B.1.1.7 in England. Science. 2021; 372(6538): eabg3055. https://doi.org/10.1126/science.abg3055
29. Expert comment on the ‘Delta plus’ variant (B.1.617.2 with the addition of K417N mutation). Available at: https://www.sciencemediacentre.org/expert-comment-on-the-delta-plus-variant-b-1-617-2-with-theaddition-of-k417n-mutation/ (accessed July 26, 2021).
30. Weekly epidemiological update on COVID-19 – 22 June 2021. Available at: https://www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19---22-june-2021 (accessed July 24, 2021).
