Environmental Surveillance of Norovirus Genogroups I and II for Sensitive Detection of Epidemic Variants

Sewage samples have been investigated to study the level of noroviruses concentration in sewage or the genotypes of noroviruses circulating in human populations. However, the statistical relationship between the concentration of the virus and the number of infected individuals, and the clinical importance of genotypes or strains detected in sewage are unclear. In this study, we carried out both environmental and clinical surveillance of noroviruses for three years: 2013-2016. We performed cross-correlation analysis between the concentrations of norovirus GI or GII in sewage samples collected weekly and the number of gastroenteritis cases. Norovirus genotypes in sewage were also analyzed using pyrosequencing and compared with those identified in stool samples. The cross-correlation analysis found the peak coefficient ( R = 0.51) at a lag of zero, indicating that the variation of GII concentration expressed as log 10 copies/mL was coincident with that of the gastroenteritis cases. A total of 15 norovirus genotypes and up to 8 genotypes per sample were detected in sewage, which included all 13 genotypes identified in the stool samples except two. GII.4 was most frequently detected in both samples, followed by GII.17. Phylogenetic analysis revealed that a strain belonging to the GII.17 Kawasaki 2014 lineage had been introduced to the study area in the 2012-2013 season. An increase in GI.3 cases was observed in the 2015-2016 season, and sewage monitoring identified their presence in the previous season (2014-2015). Our results demonstrated that monitoring of noroviruses in sewage is useful for sensitive detection of epidemic variants in human populations. IMPORTANCE Statistical evidence was obtained on the relationship between the variation of norovirus GII concentration in sewage and that of gastroenteritis cases during the 3-year study period. Sewage sample analysis using a pyrosequencing approach enabled us to understand the temporal variation of norovirus genotypes circulating in human populations. We found that a strain closely related to the GII.17 Kawasaki 2014 lineage had been introduced to the study area at least one year before its appearance and identification in clinical cases. A similar pattern was observed for GI.3; cases were reported in the 2015-2016 season, and closely related strains were found in sewage in the previous season. Our observation indicates that monitoring of noroviruses in sewage is useful for rapid detection of an epidemic, and is also sensitive enough to study the molecular epidemiology of noroviruses. Applying this approach to other enteric pathogens in sewage will enhance our understanding of their ecology.