CHARACTERIZATION OF CYANOPHAGES ISOLATED FROM FRESHWATER BODIES IN KYIV REGION, UKRAINE
DOI: https://doi.org/10.17721/1728.2748.2025.103.22-27
Keywords:
freshwater ecosystems, cyanophages, Caudoviricetes, gp20Abstract
Background. Cyanophages, which specifically infect cyanobacteria, are among the most ecologically significant bacteriophages in aquatic ecosystems. These viruses typically possess icosahedral capsids containing double-stranded DNA and have tails that vary in length and morphology depending on the cyanophage species. This study aims to characterize cyanophages and describe their morphological and molecular features based on virus isolates obtained from freshwater bodies in the Kyiv region between July and September 2024.
Methods. Cyanophage isolation and concentration, transmission electron microscopy (TEM), polymerase chain reaction (PCR), gel electrophoresis and sequencing.
Results. The primary isolation and identification of cyanophages from freshwater bodies in the Kyiv region were carried out. Electron microscopy revealed podo- and myovirus-like phage particles of varying sizes and morphological characteristics, suggesting their affiliation with the class Caudoviricetes. A PCR product of the expected size was successfully obtained using CPS1-CPS4 primers, which target the portal protein gene, amplifying a fragment of the gp20 gene from three isolates. Molecular analysis using PCR targeting the gp20 gene provided genetic evidence supporting the cyanophage identity of the isolates. A BLASTn search against the NCBI GenBank database revealed that the sequenced amplicon has the highest similarity (80.48 % and 80.14% identity) with fragments of the gp20 gene from uncultivated cyanophages (accession numbers KY082133.1 and MT596572.1).
Conclusions. These findings represent the first molecular investigation of cyanophages in Ukraine, expanding current knowledge of the morphological diversity and genetic characteristics of freshwater cyanophages. TEM analysis confirmed the presence of tailed virions with podo and myovirus-like morphology, consistent with representatives of the class Caudoviricetes. The amplicons obtained from the gp 20 gene fragment isolates in this study are related to previously reported T4-like cyanophages, while also representing distinct lineages within this group.
Furthermore, the results provide a foundation for future studies aimed at characterizing the ecological roles of these viruses and exploring their potential practical use in the biocontrol of harmful cyanobacterial blooms.
References
Abbasi, M., & Alam, M. (2025). Understanding the roles of viruses as key players in environmental dynamics and ecosystem functioning. Discovery Viruses, 2(10), Article 10. https://doi.org/10.1007/s44370-025-00015-y
Acuña-Alonso, C., Álvarez, X., Valero, E., & Pacheco, F. A. L. (2022). Modelling of threats that affect cyano-HABs in an eutrophicated reservoir: First phase towards water security and environmental governance in watersheds. Science of the Total Environment, 809, Article 152119. https://doi.org/10.1016/j.scitotenv.2021.152119
Biedunkova, O., & Kuznietsov, P. (2024). Investigation of the formation and variability of dissolved inorganic carbon and dissolved organic carbon in the water of a small river (on the example of the Styr River, Ukraine). Environmental Monitoring and Assessment, 196(1), Article 1115. https://doi.org/10.1007/s10661-024-13309-3
Bilous, O. P., Nezbrytska, I., Zhezherya, V., Dubniak, S., Batoh, S., Kazantsev, T., Polishchuk, O., Zhezherya, T., Leontieva, T., & Cantonati, M. (2023). Interactions between aquatic plants and cyanobacterial blooms in freshwater reservoir ecosystems. Water, 15(4), Article 672. https://doi.org/ 10.3390/w15040672
Brenner, S., & Horne, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochimica et Biophysica Acta, 34(1), 103–110. https://doi.org/10.1016/0006-3002(59)90237-9
Brum, J. R., Cesar Ignacio-Espinoza, J., Roux, S., Doulcier, G., Acinas, S. G., Alberti, A., Chaffron, S., Cruaud, C., de Vargas, C., Gasol, J. M., Gorsky, G., Gregory, A. C., Guidi, L., Hingamp, P., Iudicone, D., Not, F., Ogata, H., Pesant, S., Poulos, B. T., … & Sullivan, M. B. (2015). Patterns and ecological drivers of ocean viral communities. Science, 348(6237), Article 1261498. https://doi.org/10.1126/science.1261498
Fuller, N. J., Wilson, W. H., Joint, I. R., & Mann, N. H. (1998). Occurrence of a sequence in marine cyanophages similar to that of T4 g20 and its application to PCR-based detection and quantification techniques. Applied and Environmental Microbiology, 64(6), 2051–2060. https://doi.org/10.1128/AEM.64.6.2051-2060.1998
Guixa-Boixereu, N., Lysnes, K., & Pedrós-Alió, C. (1999). Viral lysis and bacterivory during a phytoplankton bloom in a coastal water microcosm. Applied and Environmental Microbiology, 65(5), 1949–1958. https://doi.org/ 10.1128/AEM.65.5.1949-1958.1999
Guo, Y., Dong, X., Li, H., Tong, Y., Liu, Z., & Jin, J. (2024). Cyanophage engineering for algal blooms control. Viruses, 16(11), Article 1745. https://doi.org/10.3390/v16111745
Ho, J. C., Michalak, A. M., & Pahlevan, N. (2019). Widespread global increase in intense lake phytoplankton blooms since the 1980s. Nature, 574(7780), 667–670. https://doi.org/10.1038/s41586-019-1648-7
Hou, X., Feng, L., Dai, Y., Hu, C., Gibson, C. E., Li, Z., & Lee, Z. (2022). Global mapping reveals increase in lacustrine algal blooms over the past decade. Nature Geoscience, 15(2), 130–134. https://doi.org/10.1038/ s41561-021-00887-x
Huisman, J., Codd, G. A., Paerl, H. W., Ibelings, B. W., Verspagen, J. M. H., & Visser, P. M. (2018). Cyanobacterial blooms. Nature Reviews Microbiology, 16(8), 471–483. https://doi.org/10.1038/s41579-018-0040-1
Huo, D., Gan, N., Geng, R., Cao, Q., Song, L., Yu, G., & Li, R. (2021). Cyanobacterial blooms in China: Diversity, distribution, and cyanotoxins. Harmful Algae, 109, Article 102106. https://doi.org/10.1016/j.hal. 2021.102106
Kaloudis, T., Hiskia, A., & Triantis, T. M. (2022). Cyanotoxins in bloom: Ever-increasing occurrence and global distribution of freshwater cyanotoxins from planktic and benthic cyanobacteria. Toxins, 14(4), Article 264. https://doi.org/10.3390/toxins14040264
Ladyka, M., & Starodubtsev, V. (2022). Water reservoirs and the war in Ukraine: Environmental problems. EUREKA: Life Sciences, 6, 36–43. https://doi.org/10.21303/2504-5695.2022.002664
Lawrence, J. E., & Steward, G. F. (2010). Purification of viruses by centrifugation. In S. W. Wilhelm, M. G. Weinbauer, & C. A. Suttle (Eds.), Manual of aquatic viral ecology (pp. 166–181). American Society of Limnology and Oceanography. https://doi.org/10.4319/mave.2010.978-0-9845591-0-7.166
Liu, Y., Demina, T. A., Roux, S., Aiewsakun, P., Kazlauskas, D., Eloe-Fadrosh, E. A., & Oksanen, H. M. (2021). Diversity, taxonomy, and evolution of archaeal viruses of the class Caudoviricetes. PLOS Biology, 19(11), Article e3001442. https://doi.org/10.1371/journal.pbio.3001442
Meng, L. H., Ke, F., Zhang, Q. Y., & Zhao, Z. (2023). Biological and genomic characteristics of MaMV-DH01, a novel freshwater Myoviridae cyanophage strain. Microbiology Spectrum, 11(1), Article e0288822. https://doi.org/10.1128/spectrum.02888-22
Nadel, O., Hanna, R., Rozenberg, A., Shitrit, D., Tahan, R., Pekarsky, I., & Lindell, D. (2024). Oceanic photosynthesis is directly affected by cyanophage NblA proteins [Preprint]. bioRxiv. https://doi.org/10.1101/ 2024.11.10.622831
Nezbrytska, I., Bilous, O., Sereda, T., Ivanova, N., Pohorielova, M., Shevchenko, T., & Zhezherya, T. (2024). Effects of war-related human activities on microalgae and macrophytes in freshwater ecosystems: A case study of the Irpin River Basin, Ukraine. Water, 16(24), Article 3604. https://doi.org/10.3390/w16243604
Rousso, B. Z., Bertone, E., Stewart, R., & Hamilton, D. P. (2020). A systematic literature review of forecasting and predictive models for cyanobacteria blooms in freshwater lakes. Water Research, 182, Article 115959. https://doi.org/10.1016/j.watres.2020.115959
Shumilova, O., Tockner, K., Sukhodolov, A., Khilchevskyi, V., De Meester, L., Gessner, M. O., & Brovkin, V. (2023). Impact of the Russia–Ukraine armed conflict on water resources and water infrastructure. Nature Sustainability, 6(5), 578–586. https://doi.org/10.1038/s41893-023-01068-x
Simmonds, P., Adriaenssens, E. M., Lefkowitz, E. J., Oksanen, H. M., Siddell, S. G., Zerbini, F. M., Alfenas-Zerbini, P., Aylward, F. O., Dempsey, D. M., Dutilh, B. E., Freitas-Astúa, J., García, M. L., Hendrickson, R. C., Hughes, H. R., Junglen, S., Krupovic, M., Kuhn, J. H., Lambert, A. J., Łobocka, M., …, & Varsani, A. (2024). Changes to virus taxonomy and the ICTV Statutes ratified by the International Committee on Taxonomy of Viruses (2024). Archives of Virology, 169(8), Article 236. https://doi.org/10.1007/s00705-024-06143-y
Song, L., Jia, Y., Qin, B., Li, R., Carmichael, W. W., Gan, N., & Sukenik, A. (2023). Harmful cyanobacterial blooms: Biological traits, mechanisms, risks, and control strategies. Annual Review of Environment and Resources, 48, 123–153. https://doi.org/10.1146/annurev-environ-112320-081653
Suttle, C. A. (2007). Marine viruses – major players in the global ecosystem. Nature Reviews Microbiology, 5(10), 801–812. https://doi.org/ 10.1038/nrmicro1750
Thurber, R. V., Haynes, M., Breitbart, M., Wegley, L., & Rohwer, F. (2009). Laboratory procedures to generate viral metagenomes. Nature Protocols, 4(4), 470–483. https://doi.org/10.1038/nprot.2009.10
Van Twest, R., & Kropinski, A. M. (2009). Bacteriophage enrichment from water and soil. In M. R. Clokie, & A. M. Kropinski (Eds.). Bacteriophages (Vol. 501, pp. 15–21). Humana Press. https://doi.org/10.1007/ 978-1-60327-164-6_2
Wang, Z., Zhang, C.-M., Li, C.-X., Li, Y.-F., Mou, X., & You, R. (2022). Evaluation of adsorption-elution methods with positively and negatively charged membranes for virus concentration in municipal wastewater: New insight into effects of dissolved organic matter. Journal of Environmental Chemical Engineering, 10(3), Article 107875. https://doi.org/10.1016/j. jece.2022.107875
Zhong, Y., Chen, F., Wilhelm, S. W., Poorvin, L., & Hodson, R. E. (2002). Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20. Applied and Environmental Microbiology, 68(4), 1576–1584. https://doi.org/10.1128/AEM.68.4.1576-1584.2002
Zhu, Y., Shang, J., Peng, C., & Sun, Y. (2022). Phage family classification under Caudoviricetes: A review of current tools using the latest ICTV classification framework. Frontiers in Microbiology, 13, Article 1032186. https://doi.org/10.3389/fmicb.2022.1032186
