Persistence of Human Noroviruses on Food Preparation Surfaces and Human Hands

The human noroviruses (NoV) are the major cause of acute non-bacterial gastroenteritis and are commonly transmitted by foodborne routes. Epidemiological evidence from propagated outbreaks, as well as environmental sampling, suggest that these viruses are environmentally stable. The purpose of this study was to examine the persistence of representative human NoV on the fingertips of volunteers and on commonly used food preparation surfaces. Human fingerpads and surfaces (stainless steel, Formica^®, and ceramic) were inoculated with 20% fecal suspensions of Norwalk virus (NV) or Snow Mountain virus (SMV). The virus inocula were recovered by elution at serial time points ranging from 0 to 120 min post-inoculation (for fingerpads) and after up to 42 days (for surfaces). The quantity of detectable viral RNA, expressed as genome equivalent particles (GEP) was evaluated using quantitative real-time RT-PCR (RT-qPCR). The amount of NV RNA on the surface decreased gradually over time, with an average reduction ranging from 1.5 to 2.9 log₁₀ GEP after 21–28 days storage under ambient conditions. SMV showed greater environmental persistence, with a 0.4–1.2 log₁₀ GEP reduction on all three surfaces after 42 days of ambient storage. On fingerpads, the amount of human NoV RNA declined slightly (<0.25 log₁₀) after 15 min and remained relatively unchanged thereafter (through 120 min). These results support the epidemiological evidence that food preparation surfaces and human hands can act as vehicles for human NoV transmission long after the initial contamination event has occurred.     

A Review of Known and Hypothetical Transmission Routes for Noroviruses

Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle shedding during asymptomatic infections, and a high environmental persistence, NoVs are easily transmitted pathogens. Norovirus (NoV) outbreaks have often been reported and tend to affect a lot of people. NoV is spread via feces and vomit, but this NoV spread can occur through several transmission routes. While person-to-person transmission is without a doubt the dominant transmission route, human infective NoV outbreaks are often initiated by contaminated food or water. Zoonotic transmission of NoV has been investigated, but has thus far not been demonstrated. The presented review aims to give an overview of these NoV transmission routes. Regarding NoV person-to-person transmission, the NoV GII.4 genotype is discussed in the current review as it has been very successful for several decades but reasons for its success have only recently been suggested. Both pre-harvest and post-harvest contamination of food products can lead to NoV food borne illness. Pre-harvest contamination of food products mainly occurs via contact with polluted irrigation water in case of fresh produce or with contaminated harvesting water in case of bivalve molluscan shellfish. On the other hand, an infected food handler is considered as a major cause of post-harvest contamination of food products. Both transmission routes are reviewed by a summary of described NoV food borne outbreaks between 2000 and 2010. A third NoV transmission route occurs via water and the spread of NoV via river water, ground water, and surface water is reviewed. Finally, although zoonotic transmission remains hypothetical, a summary on the bovine and porcine NoV presence observed in animals is given and the presence of human infective NoV in animals is discussed.     

Follow-Up of Norovirus Contamination in an Oyster Production Area Linked to Repeated Outbreaks

A production area repeatedly implicated in oyster-related gastroenteritis in France was studied for several months over 2 years. Outbreaks and field samples were analyzed by undertaking triplicate extractions, followed by norovirus (NoV) detection using triplicate wells for genomic amplification. This approach allowed us to demonstrate that some variabilities can be observed for samples with a low level of contamination, but most samples analyzed gave reproducible results. At the first outbreak, implicated oysters were collected at the beginning of the contamination event, which was reflected by the higher NoV levels during the first month of the study. During the second year, NoV concentrations in samples implicated in outbreaks and collected from the production area were similar, confirming the failure of the shellfish depuration process. Contamination was detected mainly during winter-spring months, and a high prevalence of NoV GI contamination was observed. A half-life of 18 days was calculated from NoV concentrations detected in oysters during this study, showing a very slow decrease of the contamination in the production area. Preventing the contamination of coastal waters should be a priority.     

Norovirus Detection at Oualidia Lagoon,a Moroccan Shellfish Harvesting Area,by Reverse Transcription PCR Analysis

Norovirus (NoV) is the leading cause of acute viral gastroenteritis outbreaks in the world. These outbreaks are frequently associated with bivalve shellfish consumption, particularly because these products are often eaten raw or only slightly cooked. In Morocco, regulations concerning the acceptable levels of enteric bacteria indicator organisms in these products have been put in place. However, these regulations do not take into account the risk of viral contamination, and many gastroenteritis outbreaks have been linked to the ingestion of bivalve shellfish from areas that comply with the current food safety criteria. The aim of this study was to investigate NoV presence in shellfish samples (n = 104) collected at four sites owcff Oualidia lagoon (Moroccan Atlantic coast) from November 2015 to February 2017. Samples were analysed using real-time RT-PCR in accordance with the ISO 15216-2 method. NoVs of the genogroup II were detected in 7% of samples that were all collected during the winter months. Moreover, 71% of NoV-positive samples were harvested at sites upstream of the lagoon. These results highlight the need of regularly monitoring viral contamination in bivalve shellfish to limit the risk of viral gastroenteritis outbreaks.

     

Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR

Several foodborne norovirus gastroenteritis outbreaks have been linked to fresh produce. Rapid and sensitive detection can help prevent the release of contaminated produce items in the market. The objectives of this study were to apply a relatively inexpensive SYBR Green I-based real-time RT-PCR assay for the rapid detection of human norovirus (NoV) GI and GII on the surfaces of lettuce, cherry tomatoes, and green onions. Each washed produce commodity (25 g) was spiked with serial dilutions of NoV GI and GII stool samples. RNA was eluted from the produce surface and extracted using the TRIzol^? method. This was followed by detection using SYBR Green I real-time RT-PCR with primers specific for NoV GI (COG1F-COG1R) and GII (COG2F-COG2R) along with an internal RNA amplification control. End-point detection limits from lettuce and tomatoes were found to be 10 RT-PCR units/25 g for GI and GII and 1 RT-PCR unit/25 g for GI and 10 RT-PCR units/25 g for GII from green onions. These results were confirmed by Tm analysis (showing peaks at 81.5 and 84°C for GI and GII, respectively; and 83°C for the IAC) as well as agarose gel electrophoresis that confirmed products of ~95 bp for GI and GII and ~155 bp for the RNA IAC. Results could be obtained within one working day, showing potential for routine use in diagnostics and monitoring of NoV contamination by the produce industry.     

Detection of Norovirus GII.17 Kawasaki 2014 in Shellfish,Marine Water and Underwater Sewage Discharges in Italy

Norovirus (NoV) is a major cause of non-bacterial acute gastroenteritis worldwide, and the variants of genotype GII.4 are currently the predominant human strains. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been reported as the cause of gastroenteritis outbreaks in Asia, replacing the pandemic strain GII.4 Sydney 2012. The GII.17 Kawasaki 2014 variant has also been reported sporadically in patients with gastroenteritis outside of Asia, including Italy. In this study, 384 shellfish samples were subjected to screening for human NoVs using real-time PCR and 259 (67.4%) tested positive for Genogroup II (GII) NoV. Of these, 52 samples, selected as representative of different areas and sampling dates, were further amplified by conventional PCR targeting the capsid gene, using broad-range primers. Forty shellfish samples were characterized by amplicon sequencing as GII.4 (n = 29), GII.2 (n = 4), GII.6 (n = 2), GII.12 (n = 2), and GII.17 (n = 3). Sixty-eight water samples (39 seawater samples from the corresponding shellfish production areas and 29 water samples from nearby underwater sewage discharge points) were also tested using the above broad-range assay: eight NoV-positive samples were characterized as GII.1 (n = 3), GII.2 (n = 1), GII.4 (n = 2), and GII.6 (n = 2). Based on full genome sequences available in public databases, a novel RT-PCR nested assay specific for GII.17 NoVs was designed and used to re-test the characterized shellfish (40) and water (8) samples. In this second screening, the RNA of GII.17 NoV was identified in 17 additional shellfish samples and in one water sample. Upon phylogenetic analysis, these GII.17 NoV isolates were closely related to the novel GII.17 Kawasaki 2014. Interestingly, our findings chronologically matched the emergence of the Kawasaki 2014 variant in the Italian population (early 2015), as reported by hospital-based NoV surveillance. These results, showing GII.17 NoV strains to be widespread in shellfish samples collected in 2015 in Italy, provide indirect evidence that this strain has started circulating in the Italian population. Notably, using a specific assay, we were able to detect many more samples positive for GII.17 NoV, indicating that, in food and water matrices, broad-range assays for NoV may grossly underestimate the prevalence of some, less common, NoVs. The detection of the GII.17 strain Kawasaki 2014 in clinical, water and food samples in Italy highlights the need for more systematic surveillance for future disease control and prevention.     

The presence of Genogroup II Norovirus in Retail Shellfish from Seven Coastal Cities in China

Noroviruses (NoVs) are commonly occurring pathogens that cause gastroenteritis. Outbreaks of viral diseases have often been ascribed to the consumption of contaminated shellfish. Our objective was to evaluate the presence and contamination levels of NoV in shellfish sold at seafood markets in China. We tested 840 shellfish samples (Crassostrea gigas, Mytilus edulis, Azumapecten farreri, SinoNoVacula constricta, Scapharca subcrenata, Ruditapes philippinarum) that were collected from seven cities around the Yellow and Bohai Seas in China between December 2009 and November 2011. We used real-time RT-PCR to detect NoV in purified concentrates from the stomach and digestive diverticula of these shellfish. NoV was detected in 19.35 % (N = 155), 16.67 % (N = 114), 5.70 % (N = 158), 8.82 % (N = 136), 13.74 % (N = 131), and 16.44 % (N = 146) of oyster, mussel, scallop, razor clam, ark shell, and clam samples, respectively. The average detection rate was 13.33 % (112/840). Nucleotide sequencing of the NoV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.12, except two that belonged to GI.3. More than 10² copies of the NoV genome were detected in 69 of 112 positive shellfish samples. Our results suggest that ~13 % of shellfish harbor NoV, and GII.12 NoV is the primary strain in shellfish purchased at markets in seven coastal cities in China.     

GIV Noroviruses in Wastewaters and in Stool Specimens from Hospitalized Patients

Noroviruses (NoVs) are important human pathogens associated with foodborne and waterborne gastroenteritis. These viruses are genetically highly heterogeneous, with more than forty genotypes within three genogroups (GI, GII, and GIV) identified in humans. However, the vast majority of human infections are associated with variants of a unique genotype, GII.4. Aside from these NoV strains of epidemiological relevance, NoV strains of genogroup GIV (Alphatron-like) are reported in a sporadic fashion and their overall prevalence in the community is unknown and this likely reflects the lack of specific diagnostic tools. We analyzed raw sewages collected from 32 wastewater treatment plants distributed throughout Italy (307 samples) and stool specimens collected from hospitalized patients with clinical signs of diarrhea of unknown etiology (285 samples). By using specific qualitative and quantitative RT-PCR assays, 21.8 % of the sewage samples and 3.2 % of the stool specimens tested positive for GIV NoVs. The number of genome copies in fecal samples ranged from 5.08 × 10⁴ to 1.73× 10⁶/g of feces. Sequence analysis showed limited genetic variability in human GIV viruses. The presence of GIV NoV both in sewage and in clinical samples confirms that not only GI and GII NoVs but also GIV strains are circulating in humans. Monitoring of GIV NoV is recommended in order to understand the dynamics of circulation in human populations, environmental contamination, and potential health risks.     

A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia

Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP’s outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75 % and AsV in 67 %. HAV were not detected in wastewater. Overall, 92 % of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92 % of samples and at concentrations up to 7.7 × 10³ genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66 % of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach.     

Semi-Direct Lysis of Swabs and Evaluation of Their Efficiencies to Recover Human Noroviruses GI and GII from Surfaces

Enteric viruses such as noroviruses (NoVs) continue to be the cause of widespread viral outbreaks due to person-to-person transmission, contaminated food, and contaminated surfaces. In order to optimize swabbing methodology for the detection of viruses on (food) contact surfaces, three swab elution/extraction strategies were compared in part one of this study, out of which, one strategy was based on the recently launched ISO protocol (ISO/TS 15216-1) for the determination of hepatitis A virus and NoV in food using real-time RT-PCR (RT-qPCR). These three swab elution/extraction strategies were tested for the detection of GI.4 and GII.4 NoV on high-density polyethylene (HD-PE) surfaces with the use of cotton swabs. For detection of GI.4 and GII.4, the sample recovery efficiency (SRE) obtained with the direct lysis strategy (based on ISO/TS 15216-1) was significantly lower than the SRE obtained with both other strategies. The semi-direct lysis strategy was chosen to assess the SRE of two common swabs (cotton swab and polyester swab) versus the biowipe (Biomérieux, Lyon, France) on three surfaces (HD-PE, neoprene rubber (NR), and nitrile gloves (GL)). For both surfaces, HD-PE and GL, no significant differences in SREs of GI.4 and GII.4 NoVs were detected between the three different swabs. For the coarser NR, biowipes turned out to be the best option for detecting both GI.4 and GII.4 NoV.     

Detection and Characterization of Hepatitis A Virus and Norovirus in Mussels from Galicia (NW Spain)

Shellfish are recognized as a potential vehicle of viral disease and despite the control measures for shellfish safety there is periodic emergence of viral outbreaks associated with shellfish consumption. In this study a total of 81 mussel samples from Ría do Burgo, A Coruña (NW Spain) were analysed. Samples were collected in seven different harvesting areas with the aim to establish a correlation between the prevalence of norovirus (NoV) and hepatitis A virus (HAV) in mussel samples and the water quality. In addition, the genogroup of the detected HAV and NoV strains was also determined. The HAV presence was detected in 18.5 % of the samples. Contamination levels for this virus ranged from 1.1 × 10² to 4.1 × 10⁶ RNA copies/g digestive tissue. NoV were detected in 49.4 % of the cases reaching contamination levels from 5.9 × 10³ to 1.6 × 10⁹ RNA copies/g digestive tissue for NoV GI and from 6.1 × 10³ to 5.4 × 10⁶ RNA copies/g digestive tissue for NoV GII. The χ²-test showed no statistical correlation between the number of positive samples and the classification of molluscan harvesting area based on the E. coli number. All the detected HAV strains belong to genogroup IB. NoV strains were assigned to genotype I.4, II.4 and II.6.     

Environmental Surveillance for Noroviruses in Selected South African Wastewaters 2015–2016: Emergence of the Novel GII.17

Norovirus (NoV) GII.4 is the predominant genotype associated with gastroenteritis pandemics and new strains emerge every 2–3 years. Between 2008 and 2011, environmental studies in South Africa (SA) reported NoVs in 63% of the sewage-polluted river water samples. The aim of this study was to assess whether wastewater samples could be used for routine surveillance of NoVs, including GII.4 variants. From April 2015 to March 2016, raw sewage and effluent water samples were collected monthly from five wastewater treatment plants in SA. A total of 108 samples were screened for NoV GI and GII using real-time RT-qPCR. Overall 72.2% (78/108) of samples tested positive for NoVs with 4.6% (5/108) GI, 31.5% (34/108) GII and 36.1% (39/108) GI + GII strains being detected. Norovirus concentrations ranged from 1.02 × 10² to 3.41 × 10⁶ genome copies/litre for GI and 5.00 × 10³ to 1.31 × 10⁶ genome copies/litre for GII. Sixteen NoV genotypes (GI.2, GI.3, GI.4, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GII.9, GII.10, GII.14, GII.16, GII.17, GII.20, and GII.21) were identified. Norovirus GII.2 and GII.17 co-dominated and the majority of GII.17 strains clustered with the novel Kawasaki 2014 variant. Sewage surveillance facilitated detection of Kawasaki 2014 in SA, which to date has not been detected with surveillance in children with gastroenteritis <5 years of age. Combined surveillance in the clinical setting and environment appears to be a valuable strategy to monitor emergence of NoV strains in countries that lack NoV outbreak surveillance.     

New Proof-of-Concept in Viral Inactivation: Virucidal Efficacy of 405 nm Light Against Feline Calicivirus as a Model for Norovirus Decontamination

The requirement for novel decontamination technologies for use in hospitals is ever present. One such system uses 405 nm visible light to inactivate microorganisms via ROS-generated oxidative damage. Although effective for bacterial and fungal inactivation, little is known about the virucidal effects of 405 nm light. Norovirus (NoV) gastroenteritis outbreaks often occur in the clinical setting, and this study was designed to investigate potential inactivation effects of 405 nm light on the NoV surrogate, feline calicivirus (FCV). FCV was exposed to 405 nm light whilst suspended in minimal and organically-rich media to establish the virucidal efficacy and the effect biologically-relevant material may play in viral susceptibility. Antiviral activity was successfully demonstrated with a 4 Log₁₀ (99.99%) reduction in infectivity when suspended in minimal media evident after a dose of 2.8 kJ cm^?2. FCV exposed in artificial faeces, artificial saliva, blood plasma and other organically rich media exhibited an equivalent level of inactivation using between 50–85% less dose of the light, indicating enhanced inactivation when the virus is present in organically-rich biologically-relevant media. Further research in this area could aid in the development of 405 nm light technology for effective NoV decontamination within the hospital environment.     

Persistent Norovirus Contamination of Groundwater Supplies in Two Waterborne Outbreaks

Microbiological contamination of groundwater supplies causes waterborne outbreaks worldwide. In this study, two waterborne outbreaks related to microbiological contamination of groundwater supplies are described. Analyses of pathogenic human enteric viruses (noroviruses and adenoviruses), fecal bacteria (Campylobacter spp. and Salmonella spp.), and indicator microbes (E. coli, coliform bacteria, intestinal enterococci, Clostridium perfringens, heterotrophic plate count, somatic and F-specific coliphages) were conducted in order to reveal the cause of the outbreaks and to examine the effectiveness of the implemented management measures. Moreover, the long-term persistence of noro- and adenovirus genomes was investigated. Noroviruses were detected in water samples from both outbreaks after the intrusion of wastewater into the drinking water sources. In the outbreak I, the removal efficiency of norovirus genome (3.0 log₁₀ removal) in the sand filter of onsite wastewater treatment system (OWTS) and during the transport through the soil into the groundwater well was lower than the removal efficiencies of E. coli, coliform bacteria, intestinal enterococci, and spores of C. perfringens (6.2, 6.0, > 5.9, and > 4.8 log₁₀ removals, respectively). In the outbreak II, cleaning of massively contaminated groundwater well and drinking water distribution network proved challenging, and noro- and adenovirus genomes were detected up to 3 months (108 days). The long-term persistence study showed that noro- and adenovirus genomes can remain detectable in the contaminated water samples up to 1277 and 1343 days, respectively. This study highlights the transport and survival properties of enteric viruses in the environment explaining their potency to cause waterborne outbreaks.     

Prevalence of Human Noroviruses in Frozen Marketed Shellfish,Red Fruits and Fresh Vegetables

Noroviruses (NoVs), currently recognised as the most common human food-borne pathogens, are ubiquitous in the environment and can be transmitted to humans through multiple foodstuffs. In this study, we evaluated the prevalence of human NoV genogroups I (GI) and II (GII) in 493 food samples including soft red fruits (n = 200), salad vegetables (n = 210) and bivalve mollusc shellfish (n = 83), using the Bovine Enterovirus type 1 as process extraction control for the first time. Viral extractions were performed by elution concentration and genome detection by TaqMan Real-Time RT-PCR (RT-qPCR). Experimental contamination using hepatitis A virus (HAV) was used to determine the limit of detection (LOD) of the extraction methods. Positive detections were obtained from 2 g of digestive tissues of oysters or mussels kept for 16 h in seawater containing 2.0–2.7 log₁₀ plaque-forming units (PFU)/L of HAV. For lettuces and raspberries, the LOD was, respectively, estimated at 2.2 and 2.9 log₁₀ PFU per 25 g. Of the molluscs tested, 8.4 and 14.4 % were, respectively, positive for the presence of GI NoV and GII NoV RNA. Prevalence in GI NoVs varied from 11.9 % for the salad vegetables samples to 15.5 % for the red soft fruits. Only 0.5 % of the salad and red soft fruits samples were positive for GII NoVs. These results highlight the high occurrence of human NoVs in foodstuffs that can be eaten raw or after a moderate technological processing or treatment. The determination of the risk of infection associated with an RT-qPCR positive sample remains an important challenge for the future.     

Virus Genome Quantification Does not Predict Norovirus Infectivity After Application of Food Inactivation Processing Technologies

When determining the effect of food processing on the infectivity of any contaminating virus, it is necessary to distinguish unambiguously between infectious and non-infectious viruses present. However, this can be difficult in the particular case of noroviruses (NoVs) because no reliable cell culture model is available. The aim of this study was to assess the use of molecular methods—RT real-time PCR (RT-qPCR) and enzymatic treatment (ET) coupled to RT-qPCR—to quantify the infectivity of NoV after application of various inactivating food-processing technologies. RT-qPCR and ET-RT-qPCR gave significantly different (P < 0.01) results concerning the reduction in viral genome counts by all inactivation procedures and conditions used, except for HHP treatment at 600 MPa for 5 min. These findings indicate that the ET prior to RT-qPCR has an effect on the estimation of the reduction of virus genome counts, and may eliminate genomes of affected virus particles. However, no correlation was found between the results obtained by ET-RT-qPCR and those obtained by cell culture. Therefore, the effect is presumably only partial, and not adequate to allow accurate estimation of virus inactivation. Consequently, our results indicate that the quantification of virus genomes by PCR, regardless of prior ET, is not adequate for establishing virus inactivation and/or infectivity. In addition, our results also illustrate that the general effect of virus inactivation is not directly correlated to effects on the integrity of virus genome and protein capsid. Presumably, inactivation by food processing is the consequence of effects on proteins involved in adhesion and invasion stages.     

Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011–2016) Revealed by Next-Generation and Sanger Sequencing

Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013–2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.