Food and Environmental Virology

, Volume 10, Issue 3, pp 297–304 | Cite as

Persistence of Viruses by qPCR Downstream of Three Effluent-Dominated Rivers in the Western United States

  • Hannah P. Sassi
  • Koiya D. Tuttle
  • Walter Q. Betancourt
  • Masaaki Kitajima
  • Charles P. GerbaEmail author
Original Paper


This study was designed to determine the quantitative polymerase chain reaction (qPCR) signal persistence of viruses in three effluent-dominated streams. Samples were collected from the effluent outfall of three wastewater treatment plants in the Western United States and downstream at different locations. All samples were tested for the presence of pepper mild mottle virus (PMMoV), adenoviruses, norovirus GI and GII, Aichi virus, and enteroviruses using qPCR. PMMoV was detected most frequently in 54/57 (94.7%) samples, followed by adenoviruses which was detected in 21/57 (36.8%) samples. PMMoV was detected at all locations downstream and up to 32 km from the discharge point. This study demonstrated that the detection signal of PMMoV was able to persist in wastewater discharges to a greater degree than human enteric viruses in effluent-dominated rivers.


Pepper mild mottle virus Wastewater Enteric virus Persistence Effluent-dominated river Quantitative PCR 



The authors would like to acknowledge Dr. Floris Van Ogtrop from the University of Sydney School of Life and Environmental Science for his input in data analyses and interpretation.


  1. Ashbolt, N. J., Grabow, W. O. K., & Snozzi, M. (2001). Indicators of microbial water quality. In L. Fewtrell & J. Bartram (Eds.), Water quality: Guidelines, standards and health (pp. 289–316). London, UK: IWA Publishing.Google Scholar
  2. Betancourt, W. Q., Kitajima, M., Wing, A. D., Regnery, J., Drewes, J. E., Pepper, I. L., et al. (2014). Assessment of virus removal by managed aquifer recharge at three full-scale operations. Journal of Environmental Science and Health, Part A, 49(14), 1685–1692.CrossRefGoogle Scholar
  3. Breitbart, M., Hewson, I., Felts, B., Mahaffy, J. M., Nulton, J., Salamon, P., et al. (2003). Metagenomic analyses of an uncultured viral community from human feces. Journal of Bacteriology, 185(20), 6220–6223.PubMedPubMedCentralCrossRefGoogle Scholar
  4. Colson, P., Richet, H., Desnues, C., Balique, F., Moal, V., Grob, J. J., et al. (2010). Pepper mild mottle virus, a plant virus associated with specific immune responses, fever, abdominal pains, and pruritus in humans. PLoS ONE, 5(4), e10041.PubMedPubMedCentralCrossRefGoogle Scholar
  5. Gerba, C. P. (1981). Virus survival in wastewater treatment. In Viruses and wastewater treatment: Proceedings of the international symposium on viruses and wastewater treatment, held at the University of Surrey, Guildford, 1517 September 1980 (Vol. 973, p. 39). London, UK: Pergamon.Google Scholar
  6. Gerba, C. P., Betancourt, W. Q., & Kitajima, M. (2017). How much reduction of virus is needed for recycled water: A continuous changing need for assessment? Water Research, 108, 25–31.PubMedCrossRefGoogle Scholar
  7. Gerba, C. P., Kitajima, M., & Iker, B. C. (2013). Viral presence in waste water and sewage and control methods. Viruses in Food and Water: Risks, Surveillance and Control. Scholar
  8. Gibson, K. E., & Borchardt, M. (2016). Methods for virus recovery in water. In S. M. Goyal & J. L. Cannon (Eds.), Viruses in foods (pp. 277–301). New York: Springer.CrossRefGoogle Scholar
  9. Grabow, W. O. K. (2001). Bacteriophages: Update on application as models for viruses in water. Water SA, 27(2), 251–268.Google Scholar
  10. Gregory, J. B., Litaker, R. W., & Noble, R. T. (2006). Rapid one-step quantitative reverse transcriptase PCR assay with competitive internal positive control for detection of enteroviruses in environmental samples. Applied and Environmental Microbiology, 72(6), 3960–3967.PubMedPubMedCentralCrossRefGoogle Scholar
  11. Hamza, I. A., Jurzik, L., Überla, K., & Wilhelm, M. (2010). Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water. Water Research, 45(3), 1358–1368.PubMedCrossRefGoogle Scholar
  12. Haramoto, E., Kitajima, M., Kishida, N., Konno, Y., Katayama, H., Asami, M., et al. (2013). Occurrence of pepper mild mottle virus in drinking water sources in Japan. Applied and Environmental Microbiology, 79(23), 7413–7418.PubMedPubMedCentralCrossRefGoogle Scholar
  13. Harris, G. D., Adams, V. D., Sorensen, D. L., & Curtis, M. S. (1987). Ultraviolet inactivation of selected bacteria and viruses with photoreactivation of the bacteria. Water Research, 21(6), 687–692.CrossRefGoogle Scholar
  14. Heim, A., Ebnet, C., Harste, G., & Pring-Åkerblom, P. (2003). Rapid and quantitative detection of human adenovirus DNA by real-time PCR. Journal of Medical Virology, 70(2), 228–239.PubMedCrossRefGoogle Scholar
  15. Hijnen, W. A. M., Beerendonk, E. F., & Medema, G. J. (2006). Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo) cysts in water: A review. Water Research, 40(1), 3–22.PubMedCrossRefGoogle Scholar
  16. Hurst, C. J., & Gerba, C. P. (1989). Fate of viruses during wastewater sludge treatment processes. Critical Reviews in Environmental Science and Technology, 18(4), 317–343.Google Scholar
  17. Katayama, H., Shimasaki, A., & Ohgaki, S. (2002). Development of a virus concentration method and its application to detection of enterovirus and Norwalk virus from coastal seawater. Applied and Environmental Microbiology, 68(3), 1033–1039.PubMedPubMedCentralCrossRefGoogle Scholar
  18. King, A. M., Lefkowitz, E., Adams, M. J., & Carstens, E. B. (Eds.). (2011). Virus taxonomy: Ninth report of the International Committee on Taxonomy of Viruses. San Diego, CA: Elsevier Academic Press.Google Scholar
  19. Kitajima, M., Hata, A., Yamashita, T., Haramoto, E., Minagawa, H., & Katayama, H. (2013). Development of a reverse transcription-quantitative PCR system for detection and genotyping of Aichi viruses in clinical and environmental samples. Applied and Environmental Microbiology, 79(13), 3952–3958.PubMedPubMedCentralCrossRefGoogle Scholar
  20. Kitajima, M., Iker, B. C., Pepper, I. L., & Gerba, C. P. (2014). Relative abundance and treatment reduction of viruses during wastewater treatment processes—identification of potential viral indicators. Science of the Total Environment, 488, 290–296.PubMedCrossRefGoogle Scholar
  21. Kitajima, M., Oka, T., Takagi, H., Tohya, Y., Katayama, H., Takeda, N., et al. (2010). Development and application of a broadly reactive real-time reverse transcription-PCR assay for detection of murine noroviruses. Journal of Virological Methods, 169(2), 269–273.PubMedCrossRefGoogle Scholar
  22. Kuroda, K., Nakada, N., Hanamoto, S., Inaba, M., Katayama, H., Do, A. T., et al. (2015). Pepper mild mottle virus as an indicator and a tracer of fecal pollution in water environments: Comparative evaluation with wastewater-tracer pharmaceuticals in Hanoi, Vietnam. Science of the Total Environment, 506, 287–298.PubMedCrossRefGoogle Scholar
  23. La Rosa, G., Pourshaban, M., Iaconelli, M., & Muscillo, M. (2010). Quantitative real-time PCR of enteric viruses in influent and effluent samples from wastewater treatment plants in Italy. Annali dell’Istituto superiore di sanita, 46(3), 266–273.PubMedGoogle Scholar
  24. Leong, L. Y. C. (1983). Removal and inactivation of viruses by treatment processes for potable water and wastewater—a review. Water Science and Technology, 15(5), 91–114.CrossRefGoogle Scholar
  25. Oneby, M. A., Bromley, C. O., Borchardt, J. H., & Harrison, D. S. (2010). Ozone treatment of secondary effluent at US municipal wastewater treatment plants. Ozone Science and Engineering, 32(1), 43–55.CrossRefGoogle Scholar
  26. Pavlov, D. N., Van Zyl, W. B., Van Heerden, J., Grabow, W. O. K., & Ehlers, M. M. (2005). Prevalence of vaccine-derived polioviruses in sewage and river water in South Africa. Water Research, 39(14), 3309–3319.PubMedCrossRefGoogle Scholar
  27. Rachmadi, A. T., Kitajima, M., Pepper, I. L., & Gerba, C. P. (2016). Enteric and indicator virus removal by surface flow wetlands. Science of the Total Environment, 542, 976–982.PubMedCrossRefGoogle Scholar
  28. Rosario, K., Symonds, E. M., Sinigalliano, C., Stewart, J., & Breitbart, M. (2009). Pepper mild mottle virus as an indicator of fecal pollution. Applied and Environmental Microbiology, 75(22), 7261–7267.PubMedPubMedCentralCrossRefGoogle Scholar
  29. Rosiles-González, G., Ávila-Torres, G., Moreno-Valenzuela, O. A., Acosta-González, G., Leal-Bautista, R. M., Grimaldo-Hernández, C. D., et al. (2017). Occurrence of pepper mild mottle virus (PMMoV) in groundwater from a karst aquifer system in the Yucatan Peninsula, Mexico. Food and Environmental Virology, 9(4), 487–497.PubMedCrossRefGoogle Scholar
  30. Sánchez, G., & Bosch, A. (2016). Survival of enteric viruses in the environment and food. In S. M. Goyal & J. L. Cannon (Eds.), Viruses in foods (pp. 367–392). New York: Spinger.CrossRefGoogle Scholar
  31. Schmitz, B. W., Kitajima, M., Campillo, M. E., Gerba, C. P., & Pepper, I. L. (2016). Virus reduction during advanced Bardenpho and conventional wastewater treatment processes. Environmental Science and Technology, 50(17), 9524–9532.PubMedCrossRefGoogle Scholar
  32. Shrestha, S., Shrestha, S., Shindo, J., Sherchand, J. B., & Haramoto, E. (2018). Virological quality of irrigation water sources and pepper mild mottle virus and tobacco mosaic virus as index of pathogenic virus contamination level. Food and Environmental Virology, 10(1), 107–120.PubMedCrossRefGoogle Scholar
  33. Sobsey, M. D., & Meschke, J. S. (2003). Virus survival in the environment with special attention to survival in sewage droplets and other environmental media of fecal or respiratory origin (p. 70). Geneva: Report for the World Health Organization.Google Scholar
  34. Symonds, E. M., Griffin, D. W., & Breitbart, M. (2009). Eukaryotic viruses in wastewater samples from the United States. Applied and Environmental Microbiology, 75(5), 1402–1409.PubMedPubMedCentralCrossRefGoogle Scholar
  35. Symonds, E. M., Sinigalliano, C., Gidley, M., Ahmed, A., McQuaig-Ulrich, S. M., & Breitbart, M. (2016). Faecal pollution along the southeastern coast of Florida and insight into the use of pepper mild mottle virus as an indicator. Journal of Applied Microbiology, 121, 1469–1481.PubMedCrossRefGoogle Scholar
  36. Tam, L. S., Tang, T. W., Lau, G. N., Sharma, K. R., & Chen, G. H. (2007). A pilot study for wastewater reclamation and reuse with MBR/RO and MF/RO systems. Desalination, 202(1–3), 106–113.CrossRefGoogle Scholar
  37. Thompson, S. S., Jackson, J. L., Suva-Castillo, M., Yanko, W. A., El Jack, Z., Kuo, J., et al. (2003). Detection of infectious human adenoviruses in tertiary-treated and ultraviolet-disinfected wastewater. Water Environment Research, 75(2), 163–170.PubMedCrossRefGoogle Scholar
  38. van den Berg, H., Lodder, W., van der Poel, W., Vennema, H., & de Roda Husman, A. M. (2005). Genetic diversity of noroviruses in raw and treated sewage water. Research in Microbiology, 156(4), 532–540.PubMedCrossRefGoogle Scholar
  39. Wetter, C., Conti, M., Altschuh, D., Tabillion, R., & van Regenmortel, M. H. V. (1984). Pepper mild mottle virus, a tobamovirus infecting pepper cultivars in Sicily. Phytopathology, 74, 405–410.CrossRefGoogle Scholar
  40. Wintgens, T., Melin, T., Schäfer, A., Khan, S., Muston, M., Bixio, D., et al. (2005). The role of membrane processes in municipal wastewater reclamation and reuse. Desalination, 178(1–3), 1–11.CrossRefGoogle Scholar
  41. Xagoraraki, I., Yin, Z., & Svambayev, Z. (2014). Fate of viruses in water systems. Journal of Environmental Engineering, 140(7), 04014020.CrossRefGoogle Scholar
  42. Xu, P., Janex, M. L., Savoye, P., Cockx, A., & Lazarova, V. (2002). Wastewater disinfection by ozone: main parameters for process design. Water Research, 36(4), 1043–1055.PubMedCrossRefGoogle Scholar
  43. Zhang, T., Breitbart, M., Lee, W. H., Run, J. Q., Wei, C. L., Soh, S. W. L., et al. (2005). RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biology, 4(1), e3.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of Science, School of Life and Environmental ScienceThe University of SydneySydneyAustralia
  2. 2.Department of Soil, Water and Environmental ScienceThe University of ArizonaTucsonUSA
  3. 3.Department of Environmental Engineering, Faculty of EngineeringHokkaido UniversitySapporoJapan

Personalised recommendations