Журналов:     Статей:        

Журнал микробиологии, эпидемиологии и иммунобиологии. 2019; : 21-29

Включение сайт-специфических мутаций в РВ1-ген вирулентного штамма A/WSN/33 (H1N1) вируса гриппа А меняет его фенотипические характеристики

Кост В. Ю., Сухова О. А., Акопова И. И., Горбачёва Е. О., Лисовская К. В., Ртищев А. А., Маркушин С. Г.

https://doi.org/10.36233/0372-9311-2019-6-21-29

Аннотация

Цель – изучение изменений фенотипических характеристик вирулентного штамма А/WSN/33 вируса гриппа А под влиянием включения сайт-специфических мутаций в его РВ1-гене.

Материалы и методы. С помощью двухступенчатой полимеразной цепной реакции в РВ1-гене штамма А/WSN/33(H1N1) были включены ts-мутации, взятые из генома холодоадаптированных (ХА) штаммов – доноров аттенуации А/Энн Арбор/6/60 (H2N2), А/Ленинград/134/17/57 (H2N2) и А/Краснодар/101/35/59 (H2N2). Исследовали ts- и att-фенотип полученных сайт-специфических мутантов, иммуногенность, а также снижение массы тела у инфицированных мышей.

Результаты. Показано, что включение ts-мутаций из генома ХА штаммов – доноров аттенуации в РВ1-ген вирулентного штамма А/WSN/33 (H1N1) приводит к различным изменениям его фенотипических характеристик.

Обсуждение. Анализ генома ХА штаммов – доноров аттенуации вируса гриппа указывает на исключительно важное значение функциональных дефектов в РВ1-белке для формирования аттенуационного фенотипа вируса.

Заключение. Технологию сайт-специфического мутагенеза можно использовать для модификации РВ1-гена вирулентного штамма вируса гриппа А с целью конструирования живых гриппозных вакцин нового поколения.

Список литературы

1. Maassab H. Adaptation and growth characteristics of influenza virus at 25 degrees. C. Nature. 1967; 213(5076): 612-4. Doi: https://doi.org/10.1038/213612a0

2. Jin H., Zhou H., Lu B., Kemble G. Imparting temperature sensitivity and attenuation in ferrets to A/PuertoRico/8/34 influenza virus by transferring the genetic signature for temperature sensitivity from cold-adapted A/Ann Arbor/6/60. J. Virol. 2004; 78(2): 995-8. Doi: https://doi.org/10.1128/jvi.78.2.995-998.2004

3. Solórzano A., Ye J., Pérez D.R. Alternative live-attenuated influenza vaccines based on modifications in the polymerase genes protect against epidemic and pandemic flu. J. Virol. 2010; 84(9): 4587-96. Doi: https://doi.org/10.1128/JVI.00101-10

4. Cox A., Dewhurst S. A single mutation at PB1 residue 319 dramatically increases the safety of PR8 live attenuated influenza vaccine in a murine model without compromising vaccine efficacy. J. Virol. 2015; 90(5): 2702-5. Doi: https://doi.org/10.1128/JVI.02723-15

5. Song H., Nieto G., Perez D. A new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates. J. Virol. 2007; 81(17): 9238-48. Doi: https://doi.org/10.1128/JVI.00893-07

6. Zhou B., Li Yo., Speer S., Subba A., Lin X., Wentworth D.E. Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses. Vaccine. 2012; 30(24): 3691-702. Doi: https://doi.org/10.1016/j.vaccine.2012.03.025

7. Алексадрова Г.И., Климов А.И. Живая вакцина против гриппа. СПб.: Наука; 1994.

8. Рекстин А.Р., Дешева Ю.А., Лу Х., Александрова Г.И., Климов А.И., Кац Д.М. и др. Гетеросубтипический иммунный ответ и защита от высокопатогенного вируса гриппа А (Н5N1) у мышей, иммунизированных холодоадаптированным вирусом гриппа А/Ленинград.134/17/57 (Н2N2). Медицинская иммунология. 2005; 7(5-6): 503-10.

9. Rudenko L.G., Arden N.H., Grigorieva E., Naychin A., Rekstin A., Klimov A.I., et al. Immunogenicity and efficacy of Russian live attenuated and US inactivated influenza vaccines used alone and in combination in nursing home residents. Vaccine. 2000; 19(2-3): 308-18. Doi: https://doi.org/10.1016/s0264-410x(00)00153-5

10. Гендон Ю.З., Маркушин С.Г., Цфасман Т.М., Акопова И.И., Ахматова Н.К., Коптяева И.Б. Новые холодоадаптированные штаммы-доноры аттенуации для живых вакцин против гриппа. Вопросы вирусологии. 2013; 58(1): 11-7.

11. Hoffmann E., Neumann G., Kawaoka Y., Hobom G., Webster R.G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl. Acad. Sci. USA. 2000; 97(11): 6108-13. Doi: https://doi.org/10.1073/pnas.100133697

12. Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008;3(11):e3647.

13. Pflug A., Guilligay D., Reich S., Cusack S. Structure of influenza A polymerase bound to the viral RNA promoter. Nature. 2014; 516(7531): 355-60. Doi: https://doi.org/10.1038/nature14008

14. Da Costa B., Sausset A., Munier S., Ghounaris A., Naffakh N., Le Goffic R., et al. Temperature sensitive mutants in the influenza A virus RNA polymerase: alterations in the PA linker reduce nuclear targeting of the PB1-PA dimer and result in viral attenuation. J. Virol. 2015; 89(12): 3802-5. Doi: https://doi.org/10.1128/JVI.00589-15

15. Biswas S.K., Nayak D.P. Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. J. Virol. 1994; 68(3): 1819-26.

16. Cox N.J., Kitame F., Kendal A.P., Maassab H.F., Naeve C. Identification changes in the cold-adapted live attenuated influenza vaccine strain A/Ann Arbor/6/60 1988 (H2N2). Virology. 1988; 167(2): 554-67.

Journal of microbiology, epidemiology and immunobiology. 2019; : 21-29

Inclusion of site-specific mutations in the PB1-gene of a virulent A/WSN/33 (H1N1) strain of influenza A virus changes its phenotypic characteristics

Kost V. Yu., Sukhova O. A., Akopova I. I., Gorbacheva E. O., Lisovskaya K. V., Rtishchev A. A., Markushin S. G.

https://doi.org/10.36233/0372-9311-2019-6-21-29

Abstract

Aim. Study of changes in the phenotypic characteristics of the virulent A/WSN/33 (H1N1) strain of influenza A virus under the influence of the inclusion of site-specific mutations in the PB1-gene of this strain.

Materials and methods. Using a two-step polymerase reaction in the PB1 gene of A/ WSN/33 (H1N1) strain were included ts mutations taken from the genome of attenuated CA donors-strains: A/Ann Arbor/6/60 (H2N2), A/Leningrad 134/17/57 (H2N2) and A/ Krasnodar/101/35/59. Ts-phenotype, att-phenotype, immunogenicity, as well as weight loss in mice infected with these mutants were studied in the obtained site-specific mutants.

Results. It was shown that the inclusion of ts mutations from the genome of CA donorsstrains of attenuation in the PB1 gene of the virulent A/WSN/33 (H1N1) strain leads to a change in the phenotypic characteristics of this strain to different degrees.

Discussion. Analysis of the genome of CA strains- donors of attenuation of influenza virus indicates the crucial importance of the presence of functional defects in the PB1– protein for the formation of the attenuation phenotype of the virus.

Conclusion. The technology of site-specific mutagenesis canbe successfully used to modify the PB1 gene of a virulent influenza A virus strain in order to construct a new generation of live influenza vaccines.

References

1. Maassab H. Adaptation and growth characteristics of influenza virus at 25 degrees. C. Nature. 1967; 213(5076): 612-4. Doi: https://doi.org/10.1038/213612a0

2. Jin H., Zhou H., Lu B., Kemble G. Imparting temperature sensitivity and attenuation in ferrets to A/PuertoRico/8/34 influenza virus by transferring the genetic signature for temperature sensitivity from cold-adapted A/Ann Arbor/6/60. J. Virol. 2004; 78(2): 995-8. Doi: https://doi.org/10.1128/jvi.78.2.995-998.2004

3. Solórzano A., Ye J., Pérez D.R. Alternative live-attenuated influenza vaccines based on modifications in the polymerase genes protect against epidemic and pandemic flu. J. Virol. 2010; 84(9): 4587-96. Doi: https://doi.org/10.1128/JVI.00101-10

4. Cox A., Dewhurst S. A single mutation at PB1 residue 319 dramatically increases the safety of PR8 live attenuated influenza vaccine in a murine model without compromising vaccine efficacy. J. Virol. 2015; 90(5): 2702-5. Doi: https://doi.org/10.1128/JVI.02723-15

5. Song H., Nieto G., Perez D. A new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates. J. Virol. 2007; 81(17): 9238-48. Doi: https://doi.org/10.1128/JVI.00893-07

6. Zhou B., Li Yo., Speer S., Subba A., Lin X., Wentworth D.E. Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses. Vaccine. 2012; 30(24): 3691-702. Doi: https://doi.org/10.1016/j.vaccine.2012.03.025

7. Aleksadrova G.I., Klimov A.I. Zhivaya vaktsina protiv grippa. SPb.: Nauka; 1994.

8. Rekstin A.R., Desheva Yu.A., Lu Kh., Aleksandrova G.I., Klimov A.I., Kats D.M. i dr. Geterosubtipicheskii immunnyi otvet i zashchita ot vysokopatogennogo virusa grippa A (N5N1) u myshei, immunizirovannykh kholodoadaptirovannym virusom grippa A/Leningrad.134/17/57 (N2N2). Meditsinskaya immunologiya. 2005; 7(5-6): 503-10.

9. Rudenko L.G., Arden N.H., Grigorieva E., Naychin A., Rekstin A., Klimov A.I., et al. Immunogenicity and efficacy of Russian live attenuated and US inactivated influenza vaccines used alone and in combination in nursing home residents. Vaccine. 2000; 19(2-3): 308-18. Doi: https://doi.org/10.1016/s0264-410x(00)00153-5

10. Gendon Yu.Z., Markushin S.G., Tsfasman T.M., Akopova I.I., Akhmatova N.K., Koptyaeva I.B. Novye kholodoadaptirovannye shtammy-donory attenuatsii dlya zhivykh vaktsin protiv grippa. Voprosy virusologii. 2013; 58(1): 11-7.

11. Hoffmann E., Neumann G., Kawaoka Y., Hobom G., Webster R.G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl. Acad. Sci. USA. 2000; 97(11): 6108-13. Doi: https://doi.org/10.1073/pnas.100133697

12. Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008;3(11):e3647.

13. Pflug A., Guilligay D., Reich S., Cusack S. Structure of influenza A polymerase bound to the viral RNA promoter. Nature. 2014; 516(7531): 355-60. Doi: https://doi.org/10.1038/nature14008

14. Da Costa B., Sausset A., Munier S., Ghounaris A., Naffakh N., Le Goffic R., et al. Temperature sensitive mutants in the influenza A virus RNA polymerase: alterations in the PA linker reduce nuclear targeting of the PB1-PA dimer and result in viral attenuation. J. Virol. 2015; 89(12): 3802-5. Doi: https://doi.org/10.1128/JVI.00589-15

15. Biswas S.K., Nayak D.P. Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. J. Virol. 1994; 68(3): 1819-26.

16. Cox N.J., Kitame F., Kendal A.P., Maassab H.F., Naeve C. Identification changes in the cold-adapted live attenuated influenza vaccine strain A/Ann Arbor/6/60 1988 (H2N2). Virology. 1988; 167(2): 554-67.