Sanitizing human and animal waste (e.g., urine, fecal sludge, or grey water) is a critical step in reducing the spread of disease and ensuring microbially safe reuse of waste materials. Viruses are particularly persistent pathogens and can be transmitted through inadequately sanitized waste. However, adequate storage or digestion of waste can strongly reduce the number of viruses due to increases in pH and uncharged aqueous ammonia (NH3), a known biocide. In this study we investigated the kinetics and mechanisms of inactivation of the single-stranded RNA virus MS2 under temperature, pH and NH3 conditions representative of waste storage. MS2 inactivation was mainly controlled by the activity of NH3 over a pH range of 7.0–9.5 and temperatures lower than 40 °C. Other bases (e.g., hydroxide, carbonate) additionally contributed to the observed reduction of infective MS2. The loss in MS2 infectivity could be rationalized by a loss in genome integrity, which was attributed to genome cleavage via alkaline transesterification. The contribution of each base to genome transesterification, and hence inactivation, could be related to the base pKa by means of a Bronsted relationship. The Bronsted relationship in conjunction with the activity of bases in solution enabled an accurate prediction of MS2 inactivation rates.

Sewage samples have been investigated to study the norovirus concentrations 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 3 years, 2013 to 2016. We performed cross-correlation analysis of the concentrations of norovirus GI or GII in sewage samples collected weekly and the reported number of gastroenteritis cases. Norovirus genotypes in sewage were also analyzed by pyrosequencing and compared with those identified in stool samples. The cross-correlation analysis found the peak coefficient ( = 0.51) at a lag of zero, indicating that the variation in the GII concentration, expressed as the log number of copies per milliliter, was coincident with that in the gastroenteritis cases. A total of 15 norovirus genotypes and up to 8 genotypes per sample were detected in sewage, which included all of the 13 genotypes identified in the stool samples except 2. GII.4 was most frequently detected in both sample types, followed by GII.17. Phylogenetic analysis revealed that a strain belonging to the GII.17 Kawasaki 2014 lineage had been introduced into 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 the presence of GI.3 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. We obtained statistical evidence of the relationship between the variation in the norovirus GII concentration in sewage and that of gastroenteritis cases during the 3-year study period. Sewage sample analysis by a pyrosequencing approach enabled us to understand the temporal variation in the norovirus genotypes circulating in human populations. We found that a strain closely related to the GII.17 Kawasaki 2014 lineage had been introduced into the study area at least 1 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 the 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.

Treatment. DNA and dsRNA viruses were considerably more resistant than ssRNA viruses, resulting in up to 1,000-fold-longer treatment times to reach a 4-log inactivation. The apparently slower inactivation of DNA viruses was rationalized by the higher stability of DNA than that of ssRNA in HEAM. Pushing the system toward harsher pH (>9) and temperature (>35°C) conditions, such as those encountered in thermophilic digestion and alkaline treatments, led to more consistent inactivation kinetics among ssRNA and other viruses. This suggests that the dependence of inactivation on genome type disappeared in favor of protein-mediated inactivation mechanisms common to all viruses. Finally, we recommend the use of MS2 as a conservative indicator to assess the inactivation of ssRNA viruses and the stable ⌽X174 or dsDNA phages as indicators for persistent viruses. IMPORTANCEViruses are among the most environmentally persistent pathogens. They can be present in high concentrations in human excreta and animal manure (HEAM). Therefore, appropriate treatment of HEAM is important prior to its reuse or discharge into the environment. Here, we investigated the factors that determine the persistence of viruses in HEAM, and we determined the main mechanisms that lead to their inactivation. Unlike other organisms, viruses can have four different genome types (double-or single-stranded RNA or DNA), and the viruses studied herein represent all four types. Genome type appeared to be the major determinant for persistence. Single-stranded RNA viruses are the most labile, because this genome type is susceptible to degradation in HEAM. In contrast, the other genome types are more stable; therefore, inactivation is slower and mainly driven by the degradation of viral proteins. Overall, this study allows us to better understand the behavior of viruses in HEAM.

Norovirus is a leading etiological agent of viral gastroenteritis. Because of relatively mild disease symptoms and frequent asymptomatic infections, information on the ecology of this virus is limited. Our objective was to examine the genetic diversity of norovirus circulating in the human population by means of genotyping the virus in municipal wastewater. We investigated norovirus genogroups I and II (GI and GII) in municipal wastewater in Japan by pyrosequencing and quantitative PCR (qPCR) from November 2012 to March 2013. Virological surveillance for gastroenteritis cases was concurrently conducted in the same area. A total of fourteen distinct genotypes in total (GI.1, 3, 4, 6, 7, GII.2, 4, 5, 6, 7, 12, 13, 14, and 17), with up to eight genotypes detected per sample, were observed in wastewater using pyrosequencing; only four genotypes (GI.6, GII.4, 5, and 14) were obtained from clinical samples. Seventy-eight percent of norovirus-positive stool samples contained GII.4, but this genotype was not dominant in wastewater. The norovirus GII.4 Sydney 2012 variant, which appeared and spread during our study period, was detected in both the wastewater and clinical samples. These results suggest that an environmental approach using pyrosequencing yields a more detailed distribution of norovirus genotypes/variants. Thus, wastewater monitoring by pyrosequencing is expected to provide an effective analysis of the distribution of norovirus genotypes causing symptomatic and asymptomatic infections in human populations.