Abstract
Vesicle-associated viruses, or viral vesicles, are groups of virions packaged within or bound to the surface of small membrane sacs released from host cells. This structure has recently been identified as a common form of certain viral pathogens in host bodies and excreta and is known to enhance viral infectivity, virulence and persistence, raising health concerns if discharged into aquatic environments. However, the presence of vesicle-associated viruses in environments remains unexplored. Here we show that vesicle-associated viruses are abundant and diverse in real wastewater. Using a highly selective immunomagnetic method, we successfully isolated viral vesicles from municipal and hospital wastewater. Quantitative PCR revealed that a substantial fraction of human noroviruses in wastewater were vesicle-associated, accounting for 17% of norovirus GI and 45% of norovirus GII on average. Metagenomic analysis further showed that wastewater vesicles carry diverse bacteriophages as well as human, animal and plant viruses. These findings highlight the need to consider vesicle-associated viruses in wastewater treatment and public health protection.
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Data availability
All data generated or analysed during this study are included in this Article and its Supplementary Information. The raw sequencing reads were deposited in the NCBI database with BioProject number PRJNA1055674.
Code availability
The Linux code used in this study for bioinformatic analysis is publicly available via GitHub at https://github.com/sunyuepeng00/Vesicle-bioinformatics/blob/main/viral%20community.
References
Okoh, A. I., Sibanda, T. & Gusha, S. S. Inadequately treated wastewater as a source of human enteric viruses in the environment. Int. J. Environ. Res. Public Health 7, 2620–2637 (2010).
De Graaf, M., van Beek, J. & Koopmans, M. P. Human norovirus transmission and evolution in a changing world. Nat. Rev. Microbiol. 14, 421–433 (2016).
Norovirus burden and trends. CDC https://www.cdc.gov/norovirus/burden.html (2023).
Rzeżutka, A. & Cook, N. Survival of human enteric viruses in the environment and food. FEMS Microbiol. Rev. 28, 441–453 (2004).
Corpuz, M. V. A. et al. Viruses in wastewater: occurrence, abundance and detection methods. Sci. Total Environ. 745, 140910 (2020).
da Silva, A. K. et al. Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II. Appl. Environ. Microbiol. 73, 7891–7897 (2007).
Zhang, M. et al. Emerging pathogenic unit of vesicle-cloaked murine norovirus clusters is resistant to environmental stresses and UV254 disinfection. Environ. Sci. Technol. 55, 6197–6205 (2021).
Zhang, M., Ghosh, S., Li, M., Altan-Bonnet, N. & Shuai, D. Vesicle-cloaked rotavirus clusters are environmentally persistent and resistant to free chlorine disinfection. Environ. Sci. Technol. 56, 8475–8484 (2022).
Kotwal, G. & Cannon, J. L. Environmental persistence and transfer of enteric viruses. Curr. Opin. Virol. 4, 37–43 (2014).
Santiana, M. et al. Vesicle-cloaked virus clusters are optimal units for inter-organismal viral transmission. Cell Host Microbe 24, 208–220 (2018).
Chen, Y.-H. et al. Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell 160, 619–630 (2015).
Fu, Y. & Xiong, S. Exosomes mediate Coxsackievirus B3 transmission and expand the viral tropism. PLoS Pathog. 19, e1011090 (2023).
Smith, S. C., Krystofiak, E. & Ogden, K. M. Mammalian orthoreovirus can exit cells in extracellular vesicles. PLoS Pathog. 20, e1011637 (2024).
Baez-Navarro, C., Quevedo, I. R., López, S., Arias, C. F. & Iša, P. The association of human astrovirus with extracellular vesicles facilitates cell infection and protects the virus from neutralizing antibodies. J. Virol. 96, e00848–00822 (2022).
Feng, Z., Hirai-Yuki, A., McKnight, K. L. & Lemon, S. M. Naked viruses that aren’t always naked: quasi-enveloped agents of acute hepatitis. Annu. Rev. Virol. 1, 539–560 (2014).
Birge, R. et al. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ. 23, 962–978 (2016).
Martins, S. dT., & Alves, L. R. Extracellular vesicles in viral infections: two sides of the same coin? Front. Cell. Infect. Microbiol. 10, 593170 (2020).
Muraoka, S. et al. Assessment of separation methods for extracellular vesicles from human and mouse brain tissues and human cerebrospinal fluids. Methods 177, 35–49 (2020).
Nakai, W. et al. A novel affinity-based method for the isolation of highly purified extracellular vesicles. Sci. Rep. 6, 33935 (2016).
Maestre-Carballa, L. et al. Insights into the antibiotic resistance dissemination in a wastewater effluent microbiome: bacteria, viruses and vesicles matter. Environ. Microbiol. 21, 4582–4596 (2019).
Veerman, R. E. et al. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. J. Extracell. Vesicles 10, e12128 (2021).
Miyanishi, M. et al. Identification of Tim4 as a phosphatidylserine receptor. Nature 450, 435–439 (2007).
McNamara, R. P. & Dittmer, D. P. Modern techniques for the isolation of extracellular vesicles and viruses. J. Neuroimmune Pharmacol. 15, 459–472 (2020).
Momen-Heravi, F. Isolation of extracellular vesicles by ultracentrifugation. in Extracellular Vesicles: Methods and Protocols (eds Kuo, W. P. & Jia, S.) 25–32 (Springer, 2017).
Bobrie, A., Colombo, M., Krumeich, S., Raposo, G. & Théry, C. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J. Extracell. Vesicles 1, 18397 (2012).
Owusu, I. A., Quaye, O., Passalacqua, K. D. & Wobus, C. E. Egress of non-enveloped enteric RNA viruses. J. Gen. Virol. 102, 001557 (2021).
Tsen, S.-W. D. et al. Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser. Virol. J. 11, 20 (2014).
Lu, F., Wang, J., Song, M. & Dai, X. The inhibitory effect of resveratrol from Reynoutria japonica on MNV-1, a human norovirus surrogate. Food Environ. Virol. 16, 241–252 (2024).
Ghosh, S. et al. Enteric viruses replicate in salivary glands and infect through saliva. Nature 607, 345–350 (2022).
Altan-Bonnet, N., Perales, C. & Domingo, E. Extracellular vesicles: vehicles of en bloc viral transmission. Virus Res. 265, 143–149 (2019).
Ahmed, S. M. et al. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect. Dis. 14, 725–730 (2014).
CaliciNet data – norovirus US outbreak map. CDC https://www.cdc.gov/norovirus/reporting/calicinet/data.html (2023).
Doerflinger, S. Y. et al. Membrane alterations induced by nonstructural proteins of human norovirus. PLoS Pathog. 13, e1006705 (2017).
Royet, A. et al. Nonstructural protein 4 of human norovirus self-assembles into various membrane-bridging multimers. J. Biol. Chem. 300, 107724 (2024).
Bok, K. et al. Evolutionary dynamics of GII. 4 noroviruses over a 34-year period. J. Virol. 83, 11890–11901 (2009).
Eftim, S. E. et al. Occurrence of norovirus in raw sewage—a systematic literature review and meta-analysis. Water Res. 111, 366–374 (2017).
Chen, J., Madhiyan, M., Moor, K. J., Chen, H. & Shuai, D. Kinetics and mechanisms of solar UVB disinfection of vesicle-cloaked murine norovirus clusters and free noroviruses. Environ. Sci. Technol. 59, 2461–2472 (2025).
He, Z., Wang, D., Chen, J., Hu, X. & Shuai, D. Peroxide disinfection of vesicle-cloaked murine norovirus clusters: vesicle membranes protect viruses from inactivation. Environ. Sci. Technol. 59, 6488–6501 (2025).
Rajko-Nenow, P. et al. Norovirus genotypes present in oysters and in effluent from a wastewater treatment plant during the seasonal peak of infections in Ireland in 2010. Appl. Environ. Microbiol. 79, 2578–2587 (2013).
Nordgren, J., Matussek, A., Mattsson, A., Svensson, L. & Lindgren, P.-E. Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant. Water Res. 43, 1117–1125 (2009).
Number of suspected or confirmed norovirus outbreaks reported by NoroSTAT-participating states per week, 2012–2025. CDC https://www.cdc.gov/norovirus/php/reporting/norostat-data.html (2025).
Movahed, N. et al. Turnip mosaic virus components are released into the extracellular space by vesicles in infected leaves. Plant Physiol. 180, 1375–1388 (2019).
Yang, X., Li, Y. & Wang, A. Research advances in potyviruses: from the laboratory bench to the field. Annu. Rev. Phytopathol. 59, 1–29 (2021).
Kocholata, M., Jan, M., Martinec, J. & Malinska, H. A. Plant extracellular vesicles and their potential in human health research, the practical approach. Physiol. Res. 71, 327 (2022).
Ambrosone, A. et al. Plant extracellular vesicles: Current landscape and future directions. Plants 12, 4141 (2023).
Sabar, M. A., Honda, R. & Haramoto, E. CrAssphage as an indicator of human-fecal contamination in water environment and virus reduction in wastewater treatment. Water Res. 221, 118827 (2022).
Toyofuku, M., Nomura, N. & Eberl, L. Types and origins of bacterial membrane vesicles. Nat. Rev. Microbiol. 17, 13–24 (2019).
Sohlenkamp, C. & Geiger, O. Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiol. Rev. 40, 133–159 (2016).
Roier, S. et al. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nat. Commun. 7, 10515 (2016).
Ye, Y., Ellenberg, R. M., Graham, K. E. & Wigginton, K. R. Survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater. Environ. Sci. Technol. 50, 5077–5085 (2016).
Silverman, A. I. & Boehm, A. B. Systematic review and meta-analysis of the persistence of enveloped viruses in environmental waters and wastewater in the absence of disinfectants. Environ. Sci. Technol. 55, 14480–14493 (2021).
Belliot, G., Lavaux, A., Souihel, D., Agnello, D. & Pothier, P. Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants. Appl. Environ. Microbiol. 74, 3315–3318 (2008).
Jiang, M. et al. Evaluation of the impact of concentration and extraction methods on the targeted sequencing of human viruses from wastewater. Environ. Sci. Technol. 58, 8239–8250 (2024).
Tsugawa, H. et al. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nat. Methods 12, 523–526 (2015).
Rosario, K., Symonds Erin, M., Sinigalliano, C., Stewart, J. & Breitbart, M. Pepper mild mottle virus as an indicator of fecal pollution. Appl. Environ. Microbiol. 75, 7261–7267 (2009).
Schrader, C., Schielke, A., Ellerbroek, L. & Johne, R. PCR inhibitors—occurrence, properties and removal. J. Appl. Microbiol. 113, 1014–1026 (2012).
Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009).
Aw, T. G., Wengert, S. & Rose, J. B. Metagenomic analysis of viruses associated with field-grown and retail lettuce identifies human and animal viruses. Int. J. Food Microbiol. 223, 50–56 (2016).
Krueger, F. Trim Galore: a wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files, with some extra functionality for MspI-digested RRBS-type (Reduced Representation Bisufite-Seq) libraries. Babraham Bioinformatics http://www.bioinformatics.babraham.ac.uk/projects/trim_galore (2012).
Nurk, S., Meleshko, D., Korobeynikov, A. & Pevzner, P. A. metaSPAdes: a new versatile metagenomic assembler. Genome Res. 27, 824–834 (2017).
Bağcı, C., Patz, S. & Huson, D. H. DIAMOND+ MEGAN: fast and easy taxonomic and functional analysis of short and long microbiome sequences. Curr. Protoc. 1, e59 (2021).
Acknowledgements
The project was supported by the United States Environmental Protection Agency grant R840258. We thank the UC Riverside Metabolomics Core for performing the lipidomic analysis, the George Washington University Nanofabrication and Imaging Center for performing TEM imaging, and the high-performance computing cluster at the George Washington University for performing bioinformatic analysis.
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Y.Su.: investigation, visualization, methodology, writing –original draft, formal analysis, data curation; H.Z.: investigation, visualization, methodology; X.T.: methodology; M.H.: visualization, methodology; Y.Z.: visualization, methodology; H.W.: methodology, writing—review and editing; M.S.: methodology; T.G.A., N.A.-B., J.B.R., D.S. and Y.Sh.: supervision, conceptualization, writing—review and editing, funding acquisition.
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Source data for Fig. 1 (yield, particle size distribution and purity of different vesicle isolation methods), Fig. 2 (vesicle-to-total ratios of human norovirus) and Fig. 3 (time-series variation of human norovirus).
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Sun, Y., Zhang, H., Han, M. et al. Discovery of emerging vesicle-associated viruses in wastewater and implications for engineering interventions. Nat Water (2026). https://doi.org/10.1038/s44221-026-00608-x
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DOI: https://doi.org/10.1038/s44221-026-00608-x
