A systematic review of influenza virus in water environments across human, poultry, and wild bird habitats.

Influenza, a highly contagious acute respiratory disease, remains a major global health concern. This study aimed to comprehensively assess the prevalence of influenza virus in different aquatic environments. Using 43 articles from four databases, we thoroughly examined water matrices from wastewater treatment plants (WTPs) and other human environments, as well as poultry habitats and areas frequented by migratory wild birds. In WTP influents (10 studies), positivity rates for influenza A ranged from 0.0 % to 97.6 %. For influenza B (8 studies), most studies reported no positivity, except for three studies reporting detection in 0.8 %, 5.6 %, and 46.9 % of samples. Within poultry habitats (13 studies), the prevalence of influenza A ranged from 4.3 % to 76.4 %, while in environments frequented by migratory wild birds (11 studies), it ranged from 0.4 % to 69.8 %. Geographically, the studies were distributed as follows: 39.5 % from the Americas, 18.6 % from Europe, 2.3 % from South-East Asia and 39.5 % from the Western Pacific. Several influenza A subtypes were found in water matrices, including avian influenza (H3N6, H3N8, H4N1, H4N2, H4N6, H4N8, H5N1, H5N8, H6N2, H6N6, H7N9, H0N8, and H11N9) and seasonal human influenza (H1N1 and H3N2). The existing literature indicates a crucial requirement for more extensive future research on this topic. Specifically, it emphasizes the need for method harmonization and delves into areas deserving of in-depth research, such as water matrices pertaining to pig farming and prevalence studies in low-income countries.

Detection and full genomic sequencing of rare hepatitis E virus genotype 4d in Italian wastewater, undetected by clinical surveillance.

Hepatitis E is a liver disease caused by the hepatitis E virus (HEV), primarily transmitted through contaminated water or food. There are four different HEV genotypes in humans, with genotypes 1 and 2 being the most widespread. Genotypes 3 and 4 are found in animals and can also infect humans. Genotype 4 is prevalent in Asia, mainly in China. In Italy, only one outbreak of HEV-4 has been documented, which occurred in 2011, involving five patients. In 2013, HEV G4 was also detected in a pig farm. Since then, no further evidence of HEV genotype 4 has been found in the country. This study describes the first detection of HEV genotype 4, subtype d, in wastewater in central Italy, despite a lack of any clinical case reported in the area. By using a multiplex PCR protocol and two sequencing strategies, Illumina and ONT, the virus's complete genome was sequenced and characterized as subtype 4d. These findings shed light on the potential of environmental surveillance for infectious agents to improve our understanding of epidemiology and support public health efforts.

Exploring adenovirus in water environments: a systematic review and meta-analysis.

Adenoviruses (AdVs) have a significant impact in both medical and environmental contexts. The objective of this study was to investigate the prevalence of AdV in different water types, such as untreated and treated wastewater, surface water, groundwater, drinking water, and other water matrices. A total of 239 articles were included in this meta-analysis. Adenoviruses were detected in various waters worldwide. The overall prevalence in water was found to be 59.2%, with the highest prevalence in untreated wastewater (83.1%) and treated wastewater (75.3%), followed by "other water matrices" (53.4%), surface water (49.5%) drinking water (22.7%), and groundwater (18.5%). Most of the studies did not assess the viability of the viruses, leading to weak links between water contamination and risk. Both human and animal AdV were found in water environments. The findings suggest that water, including drinking water, could be a significant route of AdV transmission in both developed and developing economies.

Wastewater surveillance of SARS-CoV-2 variants in October-November 2022 in Italy: detection of XBB.1, BA.2.75 and rapid spread of the BQ.1 lineage.

This study adds insight regarding the occurrence and spread of SARS-CoV-2 Variants of Concern (VOCs) and Variants of Interest (VOIs) in Italy in October and November 2022, by testing urban wastewater collected throughout the country. A total of 332 wastewater samples were collected from 20 Italian Regions/Autonomous Provinces (APs) within the framework of national SARS-CoV-2 environmental surveillance. Of these, 164 were collected in the first week of October and 168 in the first week of November. A ∼1600 bp fragment of the spike protein was sequenced by Sanger (for individual samples) and long-read nanopore sequencing (for pooled Region/AP samples). In October, mutations characteristic of Omicron BA.4/BA.5 were detected in the vast majority (91 %) of the samples amplified by Sanger sequencing. A fraction of these sequences (9 %) also displayed the R346T mutation. Despite the low prevalence documented in clinical cases at the time of sampling, amino acid substitutions characteristic of sublineages BQ.1 or BQ.1.1 were detected in 5 % of sequenced samples from four Regions/APs. A significantly higher variability of sequences and variants was documented in November 2022, when the rate of sequences harbouring mutations of lineages BQ.1 and BQ1.1 increased to 43 %, and the number of Regions/APs positive for the new Omicron subvariant more than tripled (n = 13) compared to October. Moreover, an increase in the number of sequences with the mutation package BA.4/BA.5 + R346T (18 %), as well as the detection of variants never observed before in wastewater in Italy, such as BA.2.75 and XBB.1 (the latter in a Region where no clinical cases associated with this variant had ever been documented) was recorded. The results suggest that, as predicted by the ECDC, BQ.1/BQ.1.1 is rapidly becoming dominant in late 2022. Environmental surveillance proves to be a powerful tool for tracking the spread of SARS-CoV-2 variants/subvariants in the population.

Hepatitis E Virus in Water Environments: A Systematic Review and Meta-analysis.

Hepatitis E virus (HEV) is responsible for acute hepatitis in humans, through foodborne, zoonotic, and waterborne transmission routes. This study aimed to assess the prevalence of HEV in water matrices. Six categories were defined: untreated and treated wastewater, surface water (river, lake, and seawater), drinking water, groundwater, and other water environments (irrigation water, grey water, reservoir water, flood water, and effluent of pig slaughterhouse). We searched PubMed, Web of Science, Global Index Medicus, and Excerpta Medica Database. Study selection and data extraction were performed by at least two independent investigators. Heterogeneity (I2) was assessed using the χ2 test on the Cochran Q statistic and H parameter. Sources of heterogeneity were explored by subgroup analysis. This study is registered with PROSPERO, number CRD42021289116. We included 87 prevalence studies from 58 papers, 66.4% of which performed in Europe. The overall prevalence of HEV in water was 9.8% (95% CI 6.4-13.7). The prevalence was higher in untreated wastewater (15.1%) and lower in treated wastewater (3.8%) and in drinking water (4.7%). In surface water, prevalence was 7.4%, and in groundwater, the percentage of positive samples, from only one study available, was 8.3%. Overall, only 36.8% of the studies reported the genotype of HEV, with genotype 3 (HEV-3) prevalent (168 samples), followed by HEV-1 (148 sample), and HEV-4 (2 samples). High-income countries were the most represented with 59/87 studies (67.8%), while only 3/87 (3.5%) of the studies were performed in low-income countries. The overall prevalence obtained of this study was generally higher in industrialized countries. Risk of bias was low in 14.9% of the studies and moderate in 85.1%. The results of this review showed the occurrence of HEV in different waters environments also in industrialized countries with sanitation and safe water supplies. While HEV transmission to humans through water has been widely demonstrated in developing countries, it is an issue still pending in industrialized countries. Better knowledge on the source of pollution, occurrence, survival in water, and removal by water treatment is needed to unravel this transmission path.

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance.

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.

A State-of-the-Art Scoping Review on SARS-CoV-2 in Sewage Focusing on the Potential of Wastewater Surveillance for the Monitoring of the COVID-19 Pandemic.

The outbreak of coronavirus infectious disease-2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has rapidly spread throughout the world. Several studies have shown that detecting SARS-CoV-2 in untreated wastewater can be a useful tool to identify new outbreaks, establish outbreak trends, and assess the prevalence of infections. On 06 May 2021, over a year into the pandemic, we conducted a scoping review aiming to summarize research data on SARS-CoV-2 in sewage. Papers dealing with raw sewage collected at wastewater treatment plants, sewer networks, septic tanks, and sludge treatment facilities were included in this review. We also reviewed studies on sewage collected in community settings such as private or municipal hospitals, healthcare facilities, nursing homes, dormitories, campuses, airports, aircraft, and cruise ships. The literature search was conducted using the electronic databases PubMed, EMBASE, and Web Science Core Collection. This comprehensive research yielded 1090 results, 66 of which met the inclusion criteria and are discussed in this review. Studies from 26 countries worldwide have investigated the occurrence of SARS-CoV-2 in sewage of different origin. The percentage of positive samples in sewage ranged from 11.6 to 100%, with viral concentrations ranging from ˂LOD to 4.6 × 108 genome copies/L. This review outlines the evidence currently available on wastewater surveillance: (i) as an early warning system capable of predicting COVID-19 outbreaks days or weeks before clinical cases; (ii) as a tool capable of establishing trends in current outbreaks; (iii) estimating the prevalence of infections; and (iv) studying SARS-CoV-2 genetic diversity. In conclusion, as a cost-effective, rapid, and reliable source of information on the spread of SARS-CoV-2 and its variants in the population, wastewater surveillance can enhance genomic and epidemiological surveillance with independent and complementary data to inform public health decision-making during the ongoing pandemic.

An innovative approach for the non-invasive surveillance of communities and early detection of SARS-CoV-2 via solid waste analysis.

The diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection requires the detection of viral RNA by reverse transcription-polymerase chain reaction (RT-qPCR) performed mainly using nasopharyngeal swabs. However, this procedure requires separate analysis per each individual, performed in advanced centralized laboratory facilities with specialized medical personnel. In this study, an alternative approach termed "solid waste-based surveillance (SWBS)" was explored, in order to investigate SARS-CoV-2 infection in small communities through the indirect sampling of saliva left on waste. Sampling was performed at 20 different sites in Italy during the second peak of COVID-19. Three swabs were positive for SARS-CoV-2 using a published RT-qPCR protocol targeting the non-structural protein 14 region, and the viral load ranged 4.8 × 103-4.0 × 106 genome copies/swab. Amino acid substitutions already reported in SARS-CoV-2 sequences circulating in Italy (A222V and P521S) were detected in two positive samples. These findings confirmed the effectiveness of SWBS for non-invasive and dynamic SARS-CoV-2 surveillance.

Rapid screening for SARS-CoV-2 variants of concern in clinical and environmental samples using nested RT-PCR assays targeting key mutations of the spike protein.

New SARS-CoV-2 mutations are constantly emerging, raising concerns of increased transmissibility, virulence or escape from host immune response. We describe a nested RT-PCR assay (~1500 bps) to detect multiple nucleotide changes resulting in key spike protein mutations distinctive of the major known circulating SARS-CoV-2 variants, including the three Variants of Concern (VOCs) 20I/501Y.V1 (United Kingdom), 20H/501Y.V2 (South Africa), and 20 J/501Y.V3 (Brazil), as well as the 20E.EU1 variant (Spain), the CAL.20C recently identified in California, and the mink-associated variant (GR, lineage B.1.1.298). Prior to application to field samples, the discriminatory potential of this PCR assay was explored using GISAID and Nextclade. To extend variant detection to challenging matrices such as sewage, where the amplification of long fragments is problematic, two short nested RT-PCR assays (~300 bps) were also designed, targeting portions of the region spanned by the long nested assay. The three newly-designed assays were then tested on field samples, including 31 clinical samples (7 fully-sequenced swab samples, and 24 uncharacterized ones) and 34 urban wastewater samples, some of which collected in areas where circulation of VOCs had been reported. The long assay successfully amplified 29 of the 31 swabs (93%), allowing the correct identification of variants 20I/501Y.V1 and 20E.EU1 present in the panel of previously characterized samples. The Spanish variant was detected in 14/24 of the uncharacterized samples as well. The sequences obtained using the short assays were consistent with those obtained with the long assay. Mutations characteristic of VOCs (UK and Brazilian variant) and of other variant (Spanish) were detected in sewage samples. To our knowledge, this is the first evidence of the presence of sequences harboring key mutations of 20I/501Y.V1 and 20 J/501Y.V3 in urban wastewaters, highlighting the potential contribution of wastewater surveillance to explore SARS-CoV-2 diversity. The developed nested RT-PCR assays can be used as an initial rapid screening test to select clinical samples containing mutations of interest. This can speed up diagnosis and optimize resources since it allows full genome sequencing to be done only on clinically relevant specimens. The assays can be also employed for a rapid and cost-effective detection of VOCs or other variants in sewage for the purposes of wastewater-based epidemiology. The approach proposed here can be used to better understand SARS-CoV-2 variant diversity, geographic distribution and impact worldwide.

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy.

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 106) > treated sewage (2.9 × 105) > river waters (6.1 × 102) > estuarine waters (4.8 × 102) > bathing waters (8.5 × 101) > groundwater (5.9 × 101). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

Evidence of Saffold virus circulation in Italy provided through environmental surveillance.

Saffold virus (SAFV) is an emerging human cardiovirus associated with respiratory and gastrointestinal infection, and, more recently, to symptoms related to the endocrine, cardiovascular, and neurological systems. Information about SAFV circulation in Italy is scarce. In order to provide insights into the epidemiology of SAFV in Italy, 141 raw sewage samples collected throughout Italy were tested using broad-range nested RT-PCR primers targeting the 5'-NC region. Seven samples (5·0%) were confirmed as SAFV in samples collected in North, Centre and Southern Italy. Typing was attempted through amplification of the VP1 coding region, using both published and newly designed primers, and one sample was characterized as SAFV-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence, genetic diversity and geographic distribution of SAFV in Italy is currently unknown. This study represents the first detection of SAFV in sewage samples in Italy, suggesting that it is circulating in the population despite lack of clinical reporting. Whether the virus is associated with asymptomatic cases or with undetected gastroenteritis or respiratory illness is unknown. Further studies are needed to investigate on the occurrence and persistence of SAFV in water environments and its waterborne transmission potential.

Enteric viruses, somatic coliphages and Vibrio species in marine bathing and non-bathing waters in Italy.

Microbial safety of recreational waters is a significant public health issue. In this study we assessed the occurrence and quantity of enteric viruses in bathing and non-bathing waters in Italy, in parallel with microbial faecal indicators, somatic coliphages and Vibrio spp. Enteric viruses (aichivirus, norovirus and enterovirus) were detected in 55% of bathing water samples, including samples with bacterial indicator concentrations compliant with the European bathing water Directive. Aichivirus was the most frequent and abundant virus. Adenovirus was detected only in non-bathing waters. Somatic coliphages were identified in 50% bathing water samples, 80% of which showed simultaneous presence of viruses. Vibrio species were ubiquitous, with 9 species identified, including potential pathogens (V. cholerae, V. parahaemoylticus and V. vulnificus). This is the first study showing the occurrence and high concentration of Aichivirus in bathing waters and provides original information, useful in view of a future revision of the European Directive.

Molecular characterization of human Sapovirus in untreated sewage in Italy by amplicon-based Sanger and next-generation sequencing.

AIMS: Human Sapoviruses (HSaVs) are etiological agents of sporadic cases and outbreaks of acute gastroenteritis in humans of all ages. Evidence of worldwide distribution of HSaV has been documented; however, little is known about HSaV circulation in Italy. To study their occurrence and genetic diversity a nation-wide environmental surveillance was undertaken. METHODS AND RESULTS: One hundred and sixty-six raw sewage samples, collected from 16 wastewater treatment plants throughout Italy, were processed and analysed by a RT-nested PCR targeting the capsid region, followed by amplicon sequencing. HSaV was detected in 56 of 166 (33·7%) samples, characterized as genotypes GI.1 (n = 30 samples), GI.2 (n = 3), GI.3 (n = 2) and GII.1 (n = 1). Mixed electropherograms were detected in 20 samples. Next-generation sequencing (NGS)-based amplicon sequencing was performed on pools of PCR amplicons to detect viruses in mixed samples and less prevalent genotypes undetectable by conventional Sanger sequencing. NGS revealed three additional genotypes (GI.6, GII.6 and GV.1) beyond the four detected by Sanger sequencing. CONCLUSIONS: Our results show a significant circulation of HSaV in Italy with three genogroups (GI, GII and GV) and seven genotypes detected. The high detection rate in sewage samples suggests that HSaV infection in Italy could be underestimated or associated with asymptomatic or mild cases. SIGNIFICANCE AND IMPACT OF THE STUDY: The study detected HSaV in a relevant proportion of raw sewage samples, reflecting a considerable circulation of these viruses in the Italian population, pointing to the usefulness of including HSaV in testing patients with gastroenteritis. Furthermore, our results confirm that wastewater surveillance coupled with NGS is a powerful tool to study the molecular epidemiology of enteric viruses.

Occurrence and Genetic Diversity of Human Cosavirus in Sewage in Italy.

Human Cosavirus (HCoSV) is a newly discovered virus whose role in human enteric diseases is still unknown. In Italy, the prevalence and genetic diversity of HCoSV are unexplored. One hundred forty-one raw sewage samples collected throughout Italy were screened for HCoSV by RT-nested PCR. HCoSV was detected in 25.5% of samples. Species A, C, and D, and a potentially new species were detected. Our results show a significant circulation and heterogeneity of HCoSV in Italy.

Development of a method for direct extraction of viral RNA from bivalve molluscs.

The detection of foodborne viruses in bivalve molluscs is a challenging procedure in relation to low virus concentration and to the presence of significant RT-PCR inhibitors. The aim of this study was the development of an efficient direct extraction method for foodborne viral RNA from bivalve molluscs. Using Mengovirus as a surrogate for foodborne viruses, five extraction methods based on RNA release by Trizol were compared on clams and oysters. A procedure consisting of Trizol, PureLink RNA Mini Kit, followed by Cetyltrimethylammonium bromide (CTAB) treatment and LiCl precipitation was found to provide RNA with the highest extraction efficiency and negligible inhibitory effect on real-time RT-PCR. This procedure was further compared to standard extraction method (ISO 15216) using clam, mussel and oyster samples spiked with Hepatitis A virus, Norovirus (NoV) GI and GII as well as bivalve samples naturally contaminated with NoV GI or GII. Results clearly demonstrated that the developed method provided, on average, a recovery 4·3 times higher than the standard reference protocol as well as good repeatability. SIGNIFICANCE AND IMPACT OF THE STUDY: A direct extraction procedure was developed to recover viral RNA from shellfish with improved efficiency in comparison to reference extraction method (ISO 15216). Without the need for specific equipment, this procedure offers an alternative for performing food safety controls and for risk assessment studies. Given the inclusion in this extraction method of several steps for the efficient removal of food components inhibiting PCR reaction, this approach could serve as a general scheme for the extraction of nucleic acids of other enteric viruses and/or from other food categories.

Correction to: 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.

The original version of this article unfortunately contained a mistake. The presentation of Table 1 was incorrect. The corrected table is given below. The original article has been corrected.

Detection of Human Bocavirus Species 2 and 3 in Bivalve Shellfish in Italy.

Human bocavirus (HBoV) has been shown to be a common cause of respiratory infections and gastroenteritis in children. Recently, HBoVs have been detected in sewage and river waters in Italy and worldwide. However, studies on their presence in other water environments and in bivalve mollusks are not yet available. In this study, 316 bivalve shellfish samples collected in three Italian regions over a 6-year period (2012 to 2017) were analyzed by nested PCR and sequencing using broad-range primer pairs targeting the capsid proteins VP1 and VP2 of HBoV. The virus was detected in 27 samples (8.5% of the total samples), and a statistically significant difference was found within the three regions. A further 13 samples, collected in geographic and temporal proximity to positive samples, were included in the study to assess the spread of HBoV in shellfish production areas at the time of contamination. Twelve of these additional samples were found to be positive for HBoV. All positive samples in this study were characterized as HBoV species 2 (17 samples; 8 different sequences) or species 3 (22 samples; 4 different sequences). This study reports the occurrence of HBoV in bivalve shellfish and shows evidence of considerable spatial spread of the virus throughout shellfish production areas. Further studies are needed to elucidate both the role of HBoV as an agent of gastroenteritis and the risk for foodborne transmission of this virus.IMPORTANCE Human bocavirus is recognized as an important cause of acute respiratory tract infections and has recently been considered an etiological agent of gastroenteritis in the pediatric population. Our findings document that HBoVs are detected in bivalve shellfish with a relevant prevalence and suggest that an assessment of the risk for foodborne transmission of these viruses should be undertaken.

First Detection of Hepatitis E Virus in Shellfish and in Seawater from Production Areas in Southern Italy.

Shellfish samples (n = 384) from production areas, water samples from the same areas (n = 39) and from nearby sewage discharge points (n = 29) were analyzed for hepatitis E virus (HEV) by real-time and nested RT-PCR. Ten shellfish samples (2.6%) and five seawater samples (12.8%) tested positive for HEV; all characterized strains were G3 and showed high degree of sequence identity. An integrated surveillance in seafood and waters is relevant to reduce the risk of shellfish-associated illnesses.

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.

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.