Deciphering the Diversities of Astroviruses and Noroviruses in Wastewater Treatment Plant Effluents by a High-Throughput Sequencing Method

Abstract

cAlthough clinical epidemiology lists human enteric viruses to be among the primary causes of acute gastroenteritis in the human population, their circulation in the environment remains poorly investigated. These viruses are excreted by the human population into sewers and may be released into rivers through the effluents of wastewater treatment plants (WWTPs). In order to evaluate the viral diversity and loads in WWTP effluents of the Paris, France, urban area, which includes about 9 million inhabitants (approximately 15% of the French population), the seasonal occurrence of astroviruses and noroviruses in 100 WWTP effluent samples was investigated over 1 year. The coupling of these measurements with a high-throughput sequencing approach allowed the specific estimation of the diversity of human astroviruses (human astrovirus genotype 1 [HAstV-1], HAstV-2, HAstV-5, and HAstV-6), 7 genotypes of noroviruses (NoVs) of genogroup I (NoV GI.1 to NoV GI.6 and NoV GI.8), and 16 genotypes of NoVs of genogroup II (NoV GII.1 to NoV GII.7, NoV GII.9, NoV GII.12 to NoV GII.17, NoV GII.20, and NoV GII.21) in effluent samples. Comparison of the viral diversity in WWTP effluents to the viral diversity found by analysis of clinical data obtained throughout France underlined the consistency between the identified genotypes. However, some genotypes were locally present in effluents and were not found in the analysis of the clinical data. These findings could highlight an underestimation of the diversity of enteric viruses circulating in the human population. Consequently, analysis of WWTP effluents could allow the exploration of viral diversity not only in environmental waters but also in a human population linked to a sewerage network in order to better comprehend viral epidemiology and to forecast seasonal outbreaks.T he epidemiology of viral gastroenteritis is mainly evaluated through the use of clinical data. However, clinical data provide only a partial vision of the gastroenteritis viruses circulating in the human population. Indeed, many viral infections do not require specialized consultations or hospitalizations and so are not directly included in clinical statistics. As a consequence, these data sets offer a limited view of the diversity and occurrence of enteric viruses in the human population.Human astroviruses (HAstVs) and noroviruses (NoVs) are among the main causes of human acute gastroenteritis worldwide (1, 2). HAstVs and NoVs are nonenveloped viruses and have a positive-sense single-stranded RNA. The genome of HAstV is composed of three open reading frames (ORFs), and ORF2, encoding the capsid protein precursor, allows discrimination of eight genotypes of HAstV, HAstV-1 to HAstV-8 (3). The genome of NoV is also composed of three ORFs, and genotyping based on the genetic similarity of ORF2 and ORF3 allows discrimination of 9 genotypes of NoV genogroup I (NoV GI) and 22 genotypes of NoV genogroup II (NoV GII), which are the most common genogroups associated with human infections.HAstVs and NoVs are able to infect i...

Emerging recombinant noroviruses identified by clinical and waste water screening

“…To date, only a handful of studies have examined the genotypic diversity of viruses in wastewater, in which most used traditional Sanger sequencing/cloning methods 54 - 56 , with only two employing NGS technologies 57 , 58 . Studies by Kazama et al 57 and Prevost et al 58 applied second-generation sequencing technologies (pyrosequencing) to characterise norovirus diversity in waste water, which was then compared with the diversity found in clinical samples. A French study also identified a total of 16 norovirus GII genotypes of which GII.4 (86% Sydney 2012 variant) was the most prevalent in both sample types between May 2013 and May 2014 58 .…”

“…Studies by Kazama et al 57 and Prevost et al 58 applied second-generation sequencing technologies (pyrosequencing) to characterise norovirus diversity in waste water, which was then compared with the diversity found in clinical samples. A French study also identified a total of 16 norovirus GII genotypes of which GII.4 (86% Sydney 2012 variant) was the most prevalent in both sample types between May 2013 and May 2014 58 . In contrast to our study, Prevost et al 58 found a strikingly different distribution of norovirus diversity between waste water and clinical samples.…”

Retrospective Surveillance of Wastewater To Examine Seasonal Dynamics of Enterovirus Infections

“…NGS has also increased the discovery rate of human-pathogenic viruses in feces (39) and has revealed that culture-based enrichment can indeed alter the taxonomic profiles of environmental samples (40). Recently, NGS approaches have been described that examine the diversity of adenovirus (42), norovirus (43), and astrovirus (43) in sewage. Those studies highlighted the utility NGS approaches for community-wide enteric virus surveillance and offered advantages over Sanger sequencing, which provides information only on the dominant circulating strain (42), and can facilitate identification of virulent recombinant virus strains that may negatively impact public health (43).…”

Molecular characterization of classic human astrovirus in eastern China, as revealed by environmental sewage surveillance

“…Due to the differences in viral concentration method, primer pair and sample volume, the detection rate of classic HAstVs in sewage varies greatly (Prevost et al . ). In this study, all detected sewage samples were positive for classic HAstVs, suggesting a high HAstVs prevalence and a large number of infected individuals in the study area.…”