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Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
Department of Clinical Microbiology, County Hospital Ryhov, 55185 Jönköping, Sweden.
Gryaab AB, Gothenburg, Sweden.
Linköping University, Department of Clinical and Experimental Medicine, Molecular Virology . Linköping University, Faculty of Health Sciences.
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2009 (English)In: Water Research, ISSN 0043-1354, Vol. 43, no 4, 1117-1125 p.Article in journal (Refereed) Published
Abstract [en]

Norovirs (NoV) is a leading cause of acute gastroenteritis and is often spread via wastewater contamination. Little is known about how the wastewater treatment process affects norovirus, and which factors influence virus concentrations. To investigate this, we collected wastewater samples monthly during one year at eight different key sites at the municipal wastewater treatment plant in Gothenburg, Sweden. Virus particles were concentrated using ultracentrifugation, viral RNA was subsequently extracted, and transformed into cDNA by reverse transcription. The quantification was performed with real-time PCR assays for NoV genogroups I (GGI) and II (GGII), respectively. We found seasonal changes of NoV genogroups, with the highest concentration of NoV GGII during the winter months, and the highest concentration of NoV GGI during the summer months. Virus transmission in wastewater was more stable for NoV GGI, with NoV GGII demonstrating larger seasonal peaks. Virus reduction took place at similar rates in the primary settling, and in the activated sludge in combination with the secondary settling. Different physicochemical parameters and incoming virus concentrations were correlated to reduction of NoV between different treatment sites. This study gives new information about NoV transmission and virus reduction in a wastewater treatment plant.

Place, publisher, year, edition, pages
2009. Vol. 43, no 4, 1117-1125 p.
Keyword [en]
Wastewater treatment, Norovirus, Removal, Physicochemical parameters, real-time PCR
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-17654DOI: 10.1016/j.watres.2008.11.053OAI: diva2:210971
Original Publication: Johan Nordgren, Andreas Matussek, Ann Mattsson, Lennart Svensson and Per-Eric Lindgren, Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant, 2009, Water Research, (43), 4, 1117-1125. Copyright: Elsevier Science B.V., Amsterdam. Available from: 2009-05-09 Created: 2009-04-07 Last updated: 2009-05-11Bibliographically approved
In thesis
1. Norovirus Epidemiology: Prevalence, transmission, and determinants of disease susceptibility
Open this publication in new window or tab >>Norovirus Epidemiology: Prevalence, transmission, and determinants of disease susceptibility
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Norovirus (NoV) is today recognized as the most important agent of acute human gastroenteritis, causing a high number of diarrheal episodes in both adults and children. Outbreaks in hospitals, nursing homes, day-care centers, and from consumption of contaminated food and drinking water are common. Wastewater can be a source of NoV dissemination, e.g. when used for irrigation of crops, or due to shellfish cultivation near the outlet of wastewater treatment plants. Today, at least 25 different genotypes of NoV belonging to two major genogroups (GG) have been observed in humans. These genotypes are associated with different transmission patterns and disease severity in humans. Also host genetic factors, such as presence of ABO antigens and mutations in the FUT2 gene affect susceptibility, and can even render complete resistance to symptomatic infections, but only the most common NoV genotypes have been studied regarding this.

In this thesis, we wanted to find prevention strategies for NoV disease through four studies of NoV epidemiology: Development of a sensitive real-time PCR assay for detection and quantification of human NoVs, characterization of NoV in children with diarrhea in Nicaragua, investigation of the prevalence and parameters influencing NoV concentration in a wastewater treatment plant in Gothenburg, Sweden, and studying host susceptibility factors in a foodborne NoV outbreak in Jönköping, Sweden.

First we developed a real-time PCR assay which can detect and quantify NoV in various settings, both in stool samples of patients, and in wastewater samples from which virus was first concentrated using ultracentrifugation. This assay was found to be more sensitive than commercial immunological assays and conventional PCR methods. The assay is furthermore able to differentiate between the two major human genogroups of NoV using melting curve analysis, which provides valuable information about the circulating NoV strains.

The survey of NoV in pediatric diarrhea in Nicaragua revealed a large impact of NoV, both in community and hospital based settings, with 15% of the severe diarrhea cases attributed to NoV. Peaks of clinically diagnosed NoV gastroenteritis were associated with emerging variants of genotype GGII.4, largely replacing the many different NoV genotypes circulating before the peak of diarrheal cases. Children infected with the GGII.4 genotype were found to shed more virus compared to children infected with other genotypes, which could partly explain the high transmission of GGII.4.

At the wastewater treatment plant in Gothenburg, both NoV GGI and GGII were detected during a whole year, not only during the winter season when clinical cases are common. This indicates that NoV infections are frequently occurring at clinical and/or sub-clinical levels in the community. During winter, GGII was present in high concentrations, whereas GGI concentration increased to higher levels than GGII in summer, possibly due to the emergence of new genotypes following the winter outbreaks. The levels of NoV GGI were stable during the year, and hence incoming concentrations were affected by dilution factors such as rain. Primary treatment and treatment in a conventional, non-nitrifying activated sludge system reduced the NoV concentration by a factor of about 30. The detection of NoV in outgoing water, together with the low reduction and lack of correlation to indicator bacteria, suggest that better monitoring tools for virus in wastewater are warranted to reduce environmental contamination.

A foodborne NoV outbreak in Jönköping in October 2007, by a NoV GGI.3 strain, revealed a surprising pattern of host susceptibility. In contrast to previous findings, this strain infected individuals irrespective of secretor status and Lewis (Le) phenotype, with non-secretors and Lea+bindividuals having a higher risk of disease. Individuals with blood group B had a partial protection to symptomatic infection, but none of the host factors investigated mediated complete resistance. Furthermore, we observed differences in susceptibility regarding homozygosity and heterozygosity in the FUT2 gene, with heterozygous secretor-positive individuals being more susceptible to symptomatic NoV infection than homozygous secretors.

In summary, the developed LUX real-time PCR assay was successfully used in all studies in this thesis, which yielded important information about the prevalence and transmission of NoV. We observed the emergence of GGII.4 variants, causing the majority of diarrheal cases in children, largely replacing the other circulating genotypes, possibly due to better replication leading to a higher viral shedding. After the peak of NoV-induced diarrheal episodes, the incidence of GGII.4 decrease and other strains emerge which can infect people not previously exposed. This was observed in the foodborne outbreak in Jönköping, where individuals expected to be resistant to NoV were infected, and indeed had a higher risk of developing disease. A similar seasonal pattern was also indirectly observed in wastewater, with high levels of GGII in winter, which subsequently declined, followed by an increase of GGI in summer. Taken together, these results provide a better insight into the epidemiology of the virus, which hopefully can lead to better preventive measures for NoV gastroenteritis.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 45 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1111
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-17687 (URN)978-91-7393-670-5 (ISBN)
Public defence
2009-04-23, Berzeliussalen, Campus US, Linköpings Universitet , Linköping, 13:00 (English)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2009-04-29Bibliographically approved

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