Abstract
Human noroviruses are the leading cause of viral gastroenteritis. In the absence of a practical culture technique for routine analysis of infectious noroviruses, several methods have been developed to discriminate between infectious and non-infectious viruses by removing non-viable viruses prior to analysis by RT-qPCR. In this study, two such methods (RNase and porcine gastric mucin) which were designed to remove viruses with compromised capsids (and therefore assumed to be non-viable), were assessed for their ability to quantify viable F-specific RNA bacteriophage (FRNAP) and human norovirus following inactivation by UV-C or heat. It was found that while both methods could remove a proportion of non-viable viruses, a large proportion of non-viable virus remained to be detected by RT-qPCR, leading to overestimations of the viable population. A model was then developed to determine the proportion of RT-qPCR detectable RNA from non-viable viruses that must be removed by such methods to reduce overestimation to acceptable levels. In most cases, nearly all non-viable virus must be removed to reduce the log overestimation of viability to within levels that might be considered acceptable (e.g. below 0.5 log10). This model could be applied when developing alternative pre-treatment methods to determine how well they should perform to be comparable to established infectivity assays.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Araud, E., et al. (2016). Thermal inactivation of enteric viruses and bioaccumulation of enteric foodborne viruses in live oysters (Crassostrea virginica). Applied and Environmental Microbiology, 82(7), 2086–2099. https://doi.org/10.1128/AEM.03573-15.
Bartsch, S. M., et al. (2016). Global economic burden of norovirus gastroenteritis. PLoS ONE, 11(4), e015219. https://doi.org/10.1371/journal.pone.0151219.
Campos, C. J. A., et al. (2016). ‘Human norovirus in untreated sewage and effluents from primary, secondary and tertiary treatment processes. Water Research, 103, 224–232. https://doi.org/10.1016/j.watres.2016.07.045.
Cho, K., et al. (2018). Use of coliphages to investigate norovirus contamination in a shellfish growing area in Republic of Korea. Environmental Science and Pollution Research. https://doi.org/10.1007/s11356-018-2857-6.
Dancho, B. A., Chen, H., & Kingsley, D. H. (2012). Discrimination between infectious and non-infectious human norovirus using porcine gastric mucin. International Journal of Food Microbiology, 155(3), 222–226. https://doi.org/10.1016/j.ijfoodmicro.2012.02.010.
Deshmukh, R. A., et al. (2016). Recent developments in detection and enumeration of waterborne bacteria: A retrospective minireview. MicrobiologyOpen, 5(6), 901–922. https://doi.org/10.1002/mbo3.383.
Dias, E., Ebdon, J., & Taylor, H. (2018). The application of bacteriophages as novel indicators of viral pathogens in wastewater treatment systems. Water Research, 129, 172–179. https://doi.org/10.1016/j.watres.2017.11.022.
Doré, W. J., Henshilwood, K., & Lees, D. N. (2000). Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish. Applied and Environmental Microbiology, 66(4), 1280–1285. https://doi.org/10.1128/AEM.66.4.1280-1285.2000.
Environment Agency (2011) Water discharge permitting: disinfection of wastewater. Operational instruction 347_09. Issued 07/11/2011.
Ettayebi, K., et al. (2017). Replication of human noroviruses in stem cell-derived human enteroids, 353(6306), 1387–1393. https://doi.org/10.1126/science.aaf5211.replication.
Farkas, K., et al. (2018). Seasonal and spatial dynamics of enteric viruses in wastewater and in riverine and estuarine receiving waters. Science of the Total Environment, 634, 1174–1183. https://doi.org/10.1016/j.scitotenv.2018.04.038.
Fraisse, A., et al. (2018). Discrimination of infectious and heat-treated norovirus by combining platinum compounds and real-time RT-PCR. International Journal of Food Microbiology, 269, 64–74. https://doi.org/10.1016/j.ijfoodmicro.2018.01.015.
Hartard, C., et al. (2016). Relevance of F-specific RNA bacteriophages in assessing human norovirus risk in shellfish and environmental waters. Applied and Environmental Microbiology, 82(18), 5709–5719. https://doi.org/10.1128/AEM.01528-16.
Hartard, C., et al. (2018). F-specific RNA bacteriophages, especially members of subgroup II, should be reconsidered as good indicators of viral pollution of oysters. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.01866-17.
Hassard, F., et al. (2017). Critical review on the public health impact of norovirus contamination in shellfish and the environment: A UK perspective. Food and Environmental Virology. https://doi.org/10.1007/s12560-017-9279-3.
ISO (1995) ‘ISO 10705-1:1995 Water quality—detection and enumeration of bacteriophages—part 1: Enumeration of F-specific RNA bacteriophages.
Kozel, T. R., & Burnham-marusich, A. R. (2017). Point-of-care testing for infectious diseases: Past, present, and future. Journal of Clinical Microbiology, 55(8), 2313–2320.
Langlet, J., Kaas, L., & Greening, G. (2015). Binding-based RT-qPCR assay to assess binding patterns of noroviruses to shellfish. Food and Environmental Virology, 7(2), 88–95. https://doi.org/10.1007/s12560-015-9180-x.
Li, D., et al. (2012). Evaluation of methods measuring the capsid integrity and/or functions of noroviruses by heat inactivation. Journal of Virological Methods, 181(1), 1–5. https://doi.org/10.1016/j.jviromet.2012.01.001.
Lowther, J. A., et al. (2017). Validation of ISO method 15216 part 1—quantification of hepatitis A virus and norovirus in food matrices. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2017.11.014.
Lowther, J. A., et al. (2018). A one-year survey of norovirus in UK oysters collected at the point of sale. Food and Environmental Virology. https://doi.org/10.1007/s12560-018-9338-4.
Nuanualsuwan, S., & Cliver, D. O. (2002). Pretreatment to avoid positive RT-PCR results with inactivated viruses. Journal of Virological Methods, 104(2), 217–225. https://doi.org/10.1016/S0166-0934(02)00089-7.
Park, G. W., Linden, K. G., & Sobsey, M. D. (2011). Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F +) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation. Letters in Applied Microbiology, 52(2), 162–167. https://doi.org/10.1111/j.1472-765X.2010.02982.x.
Pecson, B. M., Martin, L. V., & Kohn, T. (2009). Quantitative PCR for determining the infectivity of bacteriophage MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: Advantages and limitations of an enzymatic treatment to reduce false-positive results. Applied and Environmental Microbiology, 75(17), 5544–5554. https://doi.org/10.1128/AEM.00425-09.
Randazzo, W., et al. (2016). Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2016.04.010.
Randazzo, W., et al. (2018). Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples. International Journal of Food Microbiology. Elsevier, 266, 1–7. https://doi.org/10.1016/j.ijfoodmicro.2017.11.011.
Rönnqvist, M., et al. (2014). Ultraviolet light inactivation of murine norovirus and human norovirus GII: PCR may overestimate the persistence of noroviruses even when combined with pre-PCR treatments. Food and Environmental Virology, 6(1), 48–57. https://doi.org/10.1007/s12560-013-9128-y.
Topping, J. R., et al. (2009). Temperature inactivation of Feline calicivirus vaccine strain FCV F-9 in comparison with human noroviruses using an RNA exposure assay and reverse transcribed quantitative real-time polymerase chain reaction-A novel method for predicting virus infectivity. Journal of Virological Methods, 156(1–2), 89–95. https://doi.org/10.1016/j.jviromet.2008.10.024.
Weng, S. C., et al. (2018). Infectivity reduction efficacy of UV irradiation and peracetic acid-UV combined treatment on MS2 bacteriophage and murine norovirus in secondary wastewater effluent. Journal of Environmental Management, 221, 1–9. https://doi.org/10.1016/j.jenvman.2018.04.064.
Wigginton, K. R., et al. (2012). Virus inactivation mechanisms: Impact of disinfectants on virus function and structural integrity. Environmental Science and Technology, 46(21), 12069–12078. https://doi.org/10.1021/es3029473.
Wolf, S., et al. (2008). Detection and characterization of F + RNA bacteriophages in water and shellfish: Application of a multiplex real-time reverse transcription PCR. Journal of Virological Methods, 149(1), 123–128. https://doi.org/10.1016/j.jviromet.2007.12.012.
Yang, Y., & Griffiths, M. W. (2014). Enzyme treatment reverse transcription-PCR to differentiate infectious and inactivated F-specific RNA phages. Applied and Environmental Microbiology, 80(11), 3334–3340. https://doi.org/10.1128/AEM.03964-13.
Zou, W. Y., et al. (2017). Human intestinal enteroids: New models to study gastrointestinal virus infections. Methods in Molecular Biology, 45, 89. https://doi.org/10.1007/7651_2017_1.
Acknowledgments
This work was jointly supported by the Natural Environment Research Council (NERC) and the Food Standards Agency (FSA) under the Environmental Microbiology and Human Health (EMHH) Programme (Grant No. NE/M010996/1). Additional funds were provided by Cefas Seedcorn project code DM008. The authors would like to thank the Enteric Virus Unit at Public Health England, Colindale for providing faecal samples used in this study.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Walker, D.I., Cross, L.J., Stapleton, T.A. et al. Assessment of the Applicability of Capsid-Integrity Assays for Detecting Infectious Norovirus Inactivated by Heat or UV Irradiation. Food Environ Virol 11, 229–237 (2019). https://doi.org/10.1007/s12560-019-09390-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12560-019-09390-4