vuzbiochemiИзвестия вузов. Прикладная химия и биотехнологияProceedings of Universities. Applied Chemistry and Biotechnology2227-29252500-1558ИРНИТУ10.21285/2227-2925-2021-11-4-540-548vuzbiochemi-705Research ArticleФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯPHYSICOCHEMICAL BIOLOGYРазработка и испытание методик определения наличия в пробах воды ДНК вирусов папилломы человека 6-го и 16-го типовDevelopment and testing of procedures for detecting HPV 6 and HPV 16 DNA in water sampleshttps://orcid.org/0000-0002-6392-9365СтолбиковА. С.StolbikovA. S.

Алексей Сергеевич Столбиков, к.б.н., старший научный сотрудник, Сибирский институт физиологии и биохимии растений СО РАН, 664033, г. Иркутск, ул. Лермонтова, 132; доцент, Иркутский государственный университет

664003, г. Иркутск, ул. К. Маркса, 1

Aleksey S. Stolbikov, Cand. Sci. (Biology), Senior Researcher, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov St., Irkutsk, 664033; Associate Professor, Irkutsk State University, 1, K. Marx St., Irkutsk, 664003

valkir5@yandex.ruhttps://orcid.org/0000-0002-7602-7301СаляевР. К.SalyaevR. K.

Рюрик Константинович Саляев, д.б.н., член-корреспондент РАН, советник РАН

664033, г. Иркутск, ул. Лермонтова, 132

Ryurik K. Salyaev, Dr. Sci. (Biology), Corresponding Member of RAS, Advisor of RAS

132, Lermontov St., Irkutsk, 664033

salyaev@sifibr.irk.ruhttps://orcid.org/0000-0003-3480-9855РекославскаяН. И.RekoslavskayaN. I.

Наталья Игоревна Рекославская, д.б.н., главный научный сотрудник

664033, г. Иркутск, ул. Лермонтова, 132

Nataliya I. Rekoslavskaya, Dr. Sci. (Biology), Chief Researcher

132, Lermontov St., Irkutsk, 664033

rekoslavskaya@sifibr.irk.ruСибирский институт физиологии и биохимии растений СО РАН; Иркутский государственный университетРоссияSiberian Institute of Plant Physiology and Biochemistry, SB RAS; Irkutsk State UniversityRussian FederationСибирский институт физиологии и биохимии растений СО РАНРоссияSiberian Institute of Plant Physiology and Biochemistry, SB RASRussian Federation202106012022114540548Copyright © Столбиков А.С., Саляев Р.К., Рекославская Н.И., 20222022Столбиков А.С., Саляев Р.К., Рекославская Н.И.Stolbikov A.S., Salyaev R.K., Rekoslavskaya N.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vuzbiochemi.elpub.ru/jour/article/view/705

В данном исследовании авторами решались задачи разработки и испытания методик определения в пробах воды ДНК опасных вирусов папилломы человека 6-го и 16-го типов. Биоинформационными методами с помощью базы данных NCBI (National Center for Biotechnology Information) и программы BioEdit были изучены консервативные участки нуклеотидных последовательностей HPV6 L1 и HPV16 L1. Всего было исследовано 135 нуклеотидных последовательностей HPV6 L1 и 945 нуклеотидных последовательностей HPV16 L1. На выявленные консервативные участки нуклеотидных сиквенсов, с помощью специализированных программ (PerlPrimer v.1.1.21, FastPCR 6.6, Primer3Plus), было разработано 5 пар специфических праймеров. Опробовано несколько методик взятия проб из различных водных объектов, находящихся в районе пос. Листвянка (оз. Байкал). Образцы подвергались комплексной очистке от нерастворимых частиц и бактериального загрязнения, затем исследовались на наличие ДНК ВПЧ с помощью ПЦР-анализа с использованием праймеров, комплементарных генетическим последовательностям ВПЧ6 L1 и ВПЧ16 L1. В результате проведенных исследований в водных пробах были обнаружены ДНК ВПЧ 6-го и 16-го типов. Разработанные и опробованные методики взятия и исследования проб из различных водных источников Байкальской природной территории с последующим проведением ПЦР-анализа позволили получить положительные результаты присутствия опасных вирусов. Полагаем, что предложенные методики тестирования водных проб на наличие в них ВПЧ будут полезны при разработке эффективного мониторинга водных объектов и сточных вод не только Байкальского, но и других регионов.

The present study aims to develop and test procedures for detecting the DNA of dangerous human papillomaviruses (HPV) types 6 and 16 in water samples. The conserved segments of HPV 6 L1 and HPV 16 L1 nucleic acid sequences were studied using bioinformatic methods with the help of the NCBI (National Center for Biotechnology Information) database and the BioEdit program. A total of 135 nucleic acid sequences of HPV6 L1 and 945 nucleic acid sequences of HPV16 L1 were examined. Five pairs of specific primers were developed for the identified conserved segments of nucleic acid sequences using specialized programs (PerlPrimer v.1.1.21, FastPCR 6.6, and Primer3Plus). In addition, several procedures for collecting samples from various water bodies located near Listvyanka settlement (Lake Baikal) were tested. The samples were subjected to comprehensive purification from insoluble particles and bacterial contamination to be tested for the presence of HPV DNA via PCR analysis using primers complementary to the nucleic acid sequences of HPV6 L1 and HPV16 L1. The conducted studies revealed HPV 6 and HPV 16 DNA in the water samples. Due to the use of the developed and tested procedures for collecting and examining samples from various water sources in the Baikal Natural Territory followed by a PCR analysis, it was possible to detect the presence of dangerous viruses. The proposed procedure of testing water samples for the presence of HPV can be useful in developing effective monitoring of water bodies and wastewater both in Baikal and other regions.

вирус папилломы человекаВПЧ6ВПЧ16ПЦР-анализбиоинформационные методыhuman papillomavirusHPV6HPV16PCR analysisbioinformatic methodsИсследование выполнено при финансовой поддержке РФФИ и Правительства Иркутской области в рамках научного проекта № 20-44-380001.The reported study was funded by RFBR and the Government of the Irkutsk Region, project no. 20-44-380001.ReferencesКостин А. А., Старинский В. В., Самсонов Ю. В., Асратов А. Т. Анализ статистических данных о злокачественных новообразованиях, ассоциированных с вирусом папилломы человека // Исследования и практика в медицине. 2016. Т. 3. N 1. С. 66–78. https://doi.org/10.17709/2409-2231-2016-3-1-9.Kostin A. A., Starinskiy V. V., Samsonov Y. V., Asratov A. T. The analysis of statistical data on malignant neoplasms associated with human papillomavirus. Issledovaniya i Praktika v Meditsine = Research'n Practical Medicine Journal. 2016;3(1):66–78. (In Russian). https://doi.org/10.17709/2409-2231-2016-3-1-9.Bosch F. X., Broker T. R., Forman D., Moscicki A.-B., Gillison M. L., Doorbar J., et al. Comprehensive control of human papillomavirus infections and related diseases // Vaccine. 2013. Vol. 31, suppl. 7. P. H1–H31. https://doi.org/10.1016/j.vacci-ne.2013.10.003.Bosch F. X., Broker T. R., Forman D., Moscicki A.-B., Gillison M. L., Doorbar J., et al. Comprehensive control of human papillomavirus infections and related diseases // Vaccine. 2013. Vol. 31, suppl. 7. P. H1–H31. https://doi.org/10.1016/j.vacci-ne.2013.10.003.Ferlay J., Colombet M., Soerjomataram I., Mathers C., Parkin D. M., Piñeros M., et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods // International Journal of Cancer. 2019. Vol. 144, no. 8. P. 1941–1953. https://doi.org/10.1002/ijc.31937.Ferlay J., Colombet M., Soerjomataram I., Mathers C., Parkin D. M., Piñeros M., et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. International Journal of Cancer. 2019;144(8):1941–1953. https://doi.org/10.1002/ijc.31937.Злокачественные новообразования в России в 2015 году (заболеваемость и смертность) / под ред. А. Д. Каприна, В. В. Старинского, В. Г. Петровой. М.: Изд-во МНИОИ им. П. А. Герцена, 2017. 250 с.Kaprin A. D., Starinskii V. V., Petrova G. V. (eds.) Malignant neoplasms in Russia in 2015 (morbidity and mortality). Moscow: MNIOI im. P. A. Gertsena; 2017. 250 p. (In Russian).McLaughlin-Drubin M. E., Munger K. Oncogenic activities of human papillomaviruses // Virus Research. 2009. Vol. 143, no. 2. P. 195–208. https://doi.org/10.1016/j.virusres.2009.06.008.McLaughlin-Drubin M. E., Munger K. Oncogenic activities of human papillomaviruses. Virus Research. 2009;143(2):195–208. https://doi.org/10.1016/j.virusres.2009.06.008.Chesson H. W., Blandford J. M., Gift T. L., Tao G., Irwin K. L. The estimated direct medical cost of sexually transmitted diseases among American youth, 2000 // Perspectives on Sexual and Reproductive Health. 2004. Vol. 36, no. 1. P. 11–19. https://doi.org/10.1363/psrh.36.11.04.Chesson H. W., Blandford J. M., Gift T. L., Tao G., Irwin K. L. The estimated direct medical cost of sexually transmitted diseases among American youth, 2000. Perspectives on Sexual and Reproductive Health. 2004;36(1):11–19. https://doi.org/10.1363/psrh.36.11.04.Ryndock E. J., Meyers C. A risk for nonsexual transmission of human papillomavirus? // Expert Review of Anti-infective Therapy. 2014. Vol. 12, no. 10. P. 1165–1170. https://doi.org/10.1586/14787210.2014.959497.Ryndock E. J., Meyers C. A risk for nonsexual transmission of human papillomavirus? Expert Review of Anti-infective Therapy. 2014;12(10): 1165–1170. https://doi.org/10.1586/14787210.2014.959497.Meyers J., Ryndock E., Conway M. J., Meyers C., Robison R. Susceptibility of high-risk human papillomavirus type 16 to clinical disinfecttants // Journal of Antimicrobial Chemotherapy. 2014. Vol. 69, no. 6. P. 1546–1550. https://doi.org/10.1093/jac/dku006.Meyers J., Ryndock E., Conway M. J., Meyers C., Robison R. Susceptibility of high-risk human papillomavirus type 16 to clinical disinfectants. Journal of Antimicrobial Chemotherapy. 2014;69(6):1546–1550. https://doi.org/10.1093/jac/dku006.Di Bonito P., Libera S. D., Petricca S., Iaconelli M., Sanguinetti M., Graffeo R., et al. A large spectrum of alpha and beta papillomaviruses are detected in human stool samples // Journal of General Virology. 2015. Vol. 96, no. 3. P. 607–613. https://doi.org/10.1099/vir.0.071787-0.Di Bonito P., Libera S. D., Petricca S., Iaconelli M., Sanguinetti M., Graffeo R., et al. A large spectrum of alpha and beta papillomaviruses are detected in human stool samples. Journal of General Virology. 2015;96(3):607–613. https://doi.org/10.1099/vir.0.071787-0.Fratini M., Di Bonito P., La Rosa G. Oncogenic Papillomavirus and Polyomavirus in Water Environments: Is There a Potential for Waterborne Transmission? // Food and Environmental Virology. 2014. Vol. 6, no. 1. P. 1–12. https://doi.org/10.1007/s12560-013-9134-0.Fratini M., Di Bonito P., La Rosa G. Oncogenic Papillomavirus and Polyomavirus in Water Environments: Is There a Potential for Waterborne Transmission? Food and Environmental Virology. 2014;6(1):1–12. https://doi.org/10.1007/s12560-013-9134-0.Symonds E. M., Griffin D. W., Breitbart M. Eukaryotic Viruses in Wastewater Samples from the United States // Applied and Environmental Microbiology. 2009. Vol. 75, no. 5. P. 1402–1409. https://doi.org/10.1128/AEM.01899-08.Symonds E. M., Griffin D. W., Breitbart M. Eukaryotic Viruses in Wastewater Samples from the United States. Applied and Environmental Microbiology. 2009;75(5):1402–1409. https://doi.org/10.1128/AEM.01899-08.Hamza H, Hamza I. A. Oncogenic papillomavirus and polyomavirus in urban sewage in Egypt // Science of The Total Environment. 2018. Vol. 610–611. P. 1413–1420. https://doi.org/10.1016/j.scitotenv.2017.08.218.Hamza H, Hamza I. A. Oncogenic papillomavirus and polyomavirus in urban sewage in Egypt. Science of The Total Environment. 2018;610– 611:1413–1420. https://doi.org/10.1016/j.scitotenv.2017.08.218.Cantalupo P. G., Calgua B., Zhao G., Hundesa A., Wier A. D., Katz J. P., et al. Raw sewage harbors diverse viral populations // mBio. 2011. Vol. 2, no. 5. e00180-11. https://doi.org/10.1128/mBio.00180-11.Cantalupo P. G., Calgua B., Zhao G., Hundesa A., Wier A. D., Katz J. P., et al. Raw sewage harbors diverse viral populations. mBio. 2011;2(5): e00180-11. https://doi.org/10.1128/mBio.00180-11.Di Bonito P., Iaconelli M., Gheit T., Tommasino M., Della Libera S., Bonadonna L., et al. Detection of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput screening of infectious agents // Water Research. 2017. Vol. 123. P. 549–555. https://doi.org/10.1016/j.watres.2017.06.088.Di Bonito P., Iaconelli M., Gheit T., Tommasino M., Della Libera S., Bonadonna L., et al. Detection of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput screening of infectious agents. Water Research. 2017;123;549–555. https://doi.org/10.1016/j.watres.2017.06.088.La Rosa G., Della Libera S., Petricca S., Iaconelli M., Briancesco R., Paradiso R., et al. First detection of papillomaviruses and polyomaviruses in swimming pool waters: unrecognized recreational water-related pathogens? // Journal of Applied Microbiology. 2015. Vol. 119, no. 6. P. 1683–1691. https://doi.org/10.1111/jam.12925.La Rosa G., Della Libera S., Petricca S., Iaconelli M., Briancesco R., Paradiso R., et al. First detection of papillomaviruses and polyomaviruses in swimming pool waters: unrecognized recreational water-related pathogens? Journal of Applied Microbiology. 2015;119(6):1683–1691. https://doi.org/10.1111/jam.12925.Bonadonna L., La Rosa G. A review and update on waterborne viral diseases associated with swimming pools // International Journal of Environmental Research and Public Health. 2019. Vol. 16, no. 2. Article number 166. https://doi.org/10.3390/ijerph16020166.Bonadonna L., La Rosa G. A review and update on waterborne viral diseases associated with swimming pools. International Journal of Environmental Research and Public Health. 2019;16(2). Article number 166. https://doi.org/10.3390/ijerph16 020166.Iaconelli M., Petricca S., D Libera S., Di Bonito P., La Rosa G. First detection of human papillomaviruses and human polyomaviruses in river Waters in Italy // Food and Environmental Virology. 2015. Vol. 7, no. 4. P. 309–315. https://doi.org/10.1007/s12560-015-9203-7.Iaconelli M., Petricca S., D Libera S., Di Bonito P., La Rosa G. First detection of human papillomaviruses and human polyomaviruses in river Waters in Italy. Food and Environmental Virology. 2015;7(4):309–315. https://doi.org/10.1007/s12560-015-9203-7.La Rosa G., Fratini M., Accardi L., D’Oro G., Della Libera S., Muscillo M., et al. Mucosal and cutaneous human papillomaviruses detected in raw sew-ages // PLOS ONE. 2013. Vol. 8, no. 1. P. e52391. https://doi.org/10.1371/journal.pone.0052391.La Rosa G., Fratini M., Accardi L., D’Oro G., Della Libera S., Muscillo M., et al. Mucosal and cutaneous human papillomaviruses detected in raw sewages. PLoS ONE. 2013;8(1):e52391. https://doi.org/10.1371/journal.pone.0052391.Злокачественные новообразования в России в 2014 году (заболеваемость и смертность) / под ред. А. Д. Каприна, В. В. Старинского, В. Г. Петровой. М.: Изд-во МНИОИ им. П. А. Герцена, 2016. 250 с.Kaprin A. D., Starinskii V. V., Petrova G. V. (eds.) Malignant neoplasms in Russia in 2014 (morbidity and mortality). Moscow: MNIOI im. P. A. Gertsena; 2016. 250 p. (In Russian).De Roda Husman A. M., Walboomers J. M., van den Brule A. J., Meijer C. J., Snijders P. J. The use of general primers GP5 and GP6 elongated at their 3 ends with adjacent highly conserved sequences improves HPV detection // Journal of General Virology. 1995. Vol. 76, no. 4. P. 1057–1062. https://doi.org/10.1099/0022-1317-76-4-1057.De Roda Husman A. M., Walboomers J. M., van den Brule A. J., Meijer C. J., Snijders P. J. The use of general primers GP5 and GP6 elongated at their 3 ends with adjacent highly conserved sequences improves HPV detection. Journal of General Virology. 1995;76(4):1057–1062. https://doi.org/10.1099/0022-1317-76-4-1057.Qu W., Jiang G., Cruz Y., Chang C. J., Ho G. Y., Klein R. S., et al. PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems // Journal of Clinical Microbiology. 1997. Vol. 35, no. 6. P. 1304–1310. https://doi.org/10.1128/JCM.35.6.1304-1310.1997Qu W., Jiang G., Cruz Y., Chang C. J., Ho G. Y., Klein R. S., et al. PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems. Journal of Clinical Microbiology. 1997;35(6):1304–1310. https://doi.org/10.1128/JCM.35.6.1304-1310.1997.

The authors declare that there are no conflicts of interest present.