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.     

Prevalence of Human Parainfluenza Viruses and Noroviruses Genomes on Office Fomites

The aim of this study was to evaluate the potential role of office fomites in respiratory (human parainfluenza virus 1—HPIV1, human parainfluenza virus 3—HPIV3) and enteric (norovirus GI—NoV GI, norovirus GII—NoV GII) viruses transmission by assessing the occurrence of these viruses on surfaces in office buildings. Between 2016 and 2017, a total of 130 surfaces from open-space and non-open-space rooms in office buildings located in one city were evaluated for HPIV1, HPIV3, NoV GI, and NoV GII viral RNA presence. Detection of viruses was performed by RT-qPCR method. Study revealed 27 positive samples, among them 59.3% were HPIV3-positive, 25.9% HPIV1-positive, and 14.8% NoV GII-positive. All tested surfaces were NoV GI-negative. Statistical analysis of obtained data showed that the surfaces of office equipment including computer keyboards and mice, telephones, and desktops were significantly more contaminated with respiratory viruses than the surfaces of building equipment elements such as door handles, light switches, or ventilation tracts (χ ² p = 0.006; Fisher’s Exact p = 0.004). All examined surfaces were significantly more contaminated with HPIVs than NoVs (χ ² p = 0.002; Fisher’s Exact p = 0.003). Office fomites in open-space rooms were more often contaminated with HPIVs than with NoVs (χ ² p = 0.016; Fisher’s Exact p = 0.013). The highest average concentration of HPIVs RNA copies was observed on telephones (1.66 × 10² copies/100 cm²), while NoVs on the light switches (1.40 × 10² copies/100 cm²). However, the Kruskal–Wallis test did not show statistically significant differences in concentration levels of viral RNA copies on surfaces between the all tested samples. This study unequivocally showed that individuals in office environment may have contact with both respiratory and enteric viral particles present on frequently touched surfaces.     

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.     

Prevalence and Molecular Genotyping of Noroviruses in Market Oysters,Mussels, and Cockles in Bangkok,Thailand

Noroviruses are the most common cause of acute gastroenteritis associated with bivalve shellfish consumption. This study aimed to detect and characterize noroviruses in three bivalve shellfish species: oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The virus concentration procedure (adsorption-twice elution-extraction) and a molecular method were employed to identify noroviruses in shellfish. RT-nested PCR was able to detect known norovirus GII.4 of 8.8 × 10^?2 genome copies/g of digestive tissues from oyster and cockle concentrates, whereas in mussel concentrates, the positive result was seen at 8.8 × 10² copies/g of digestive tissues. From August 2011 to July 2012, a total of 300 shellfish samples, including each of 100 samples from oysters, cockles, and mussels were collected and tested for noroviruses. Norovirus RNA was detected in 12.3 % of shellfish samples. Of the noroviruses, 7.7 % were of the genogroup (G) I, 2.6 % GII, and 2.0 % were mixed GI and GII. The detection rate of norovirus GI was 2.1 times higher than GII. With regards to the different shellfish species, 17 % of the oyster samples were positive, while 14.0 and 6.0 % were positive for noroviruses found in mussels and cockles, respectively. Norovirus contamination in the shellfish occurred throughout the year with the highest peak in September. Seventeen norovirus-positive PCR products were characterized upon a partial sequence analysis of the capsid gene. Based on phylogenetic analysis, five different genotypes of norovirus GI (GI.2, GI.3, GI.4, GI.5, and GI.9) and four different genotypes of GII (GII.1, GII.2, GII.3, and GII.4) were identified. These findings indicate the prevalence and distribution of noroviruses in three shellfish species. The high prevalence of noroviruses in oysters contributes to the optimization of monitoring plans to improve the preventive strategies of acute gastroenteritis.     

Evaluation of Virus Reduction by Ultrafiltration with Coagulation–Sedimentation in Water Reclamation

The evaluation of virus reduction in water reclamation processes is essential for proper assessment and management of the risk of infection by enteric viruses. Ultrafiltration (UF) with coagulation–sedimentation (CS) is potentially effective for efficient virus removal. However, its performance at removing indigenous viruses has not been evaluated. In this study, we evaluated the reduction of indigenous viruses by UF with and without CS in a pilot-scale water reclamation plant in Okinawa, Japan, by measuring the concentration of viruses using the real-time polymerase chain reaction (qPCR). Aichi virus (AiV) and pepper mild mottle virus (PMMoV) were targeted in addition to the main enteric viruses of concern for risk management, namely, norovirus (NoV) genogroups I and II (GI and GII) and rotavirus (RoV). PMMoV, which is a plant pathogenic virus and is present at high concentrations in water contaminated by human feces, has been suggested as a useful viral indicator. We also investigated the reduction of a spiked model virus (F-specific RNA bacteriophage MS2) to measure the effect of viral inactivation by both qPCR and plaque assay. Efficiencies of removal of NoV GI, NoV GII, RoV, and AiV by UF with and without CS were >0.5 to 3.7 log₁₀, although concentrations were below the detection limit in permeate water. PMMoV was the most prevalent virus in both feed and permeate water following UF, but CS pretreatment could not significantly improve its removal efficiency (mean removal efficiency: UF, 3.1 log₁₀; CS + UF, 3.4 log₁₀; t test, P > 0.05). CS increased the mean removal efficiency of spiked MS2 by only 0.3 log₁₀ by qPCR (t-test, P > 0.05), but by 2.8 log₁₀ by plaque assay (t-test, P < 0.01). This difference indicates that the virus was inactivated during CS + UF. Our results suggest that PMMoV could be used as an indicator of removal efficiency in water reclamation processes, but cultural assay is essential to understanding viral fate.     

Hepatitis A Virus,Hepatitis E Virus,and Rotavirus in Foods of Animal Origin Traded at the Borders of Brazil,Argentina, and Uruguay

The aim of this study was to investigate hepatitis A virus (HAV), hepatitis E (HEV), and rotavirus (RV) in fresh and processed meat traded on the border of Brazil with Argentina and Uruguay. In total, 159 samples of raw and processed foods of animal origin were collected in Paso de los Libres, Argentina (n?=?53 raw meat, n?=?24 processed meat) and Rivera, Uruguay (n?=?55 raw meat, n?=?18 processed meat), or were seized by the Brazilian International Agricultural Surveillance System—VIGIAGRO (Brazil–Argentina border) (n?=?8 raw meat, n?=?1 bush meat). All samples were tested for the presence of HAV, HEV, and RV genomes. HAV genes were detected in 18.23% of samples and RV genes in 23.89%. No HEV-positive samples were detected. HAV was also detected in two of the VIGIAGRO samples. Processed meats from Argentina and Uruguay had a higher rate of HAV and RV than raw meat (P?>?0.05). The median HAV in the Argentinian and Uruguayan samples was 6.9?×?10⁴ and 3.5?×?10³ copies/g, respectively. The presence of RV viral genes in raw meats from Argentina was significant, and this was not observed in processed meats. The presence of HAV and RV genes in a significant portion of products from Argentina and Uruguay is a potential source of human infection. This also indicates precarious conditions of acquisition, processing, and manipulation, which could be improved by improved regulation of food across borders.     

Temperature-Dependent Persistence of Human Norovirus Within Oysters (<Emphasis Type="Italic">Crassostrea virginica</Emphasis>)

This study characterizes the persistence of human norovirus in Eastern oysters (Crassostrea virginica) held at different seawater temperatures. Oysters were contaminated with human norovirus GI.1 (Norwalk strain 8FIIa) by exposing them to virus-contaminated water at 15 °C, and subsequently holding them at 7, 15, and 25 °C for up to 6 weeks. Viral RNA was extracted from oyster tissue and hemocytes and quantitated by RT-qPCR. Norovirus was detected in hemocytes and oysters held at 7 and 15 °C for 6 weeks and in hemocytes and oysters held at 25 °C for up to 2 and 4 weeks, respectively. Results confirm that NoV is quite persistent within oysters and demonstrate that cooler water temperatures extend norovirus clearance times. This study suggests a need for substantial relay times to remove norovirus from contaminated shellfish and suggests that regulatory authorities should consider the effects of water temperature after a suspected episodic norovirus-contamination event.     

Oyster Contamination with Human Noroviruses Impacted by Urban Drainage and Seasonal Flooding in Vietnam

This study investigated the level of norovirus contamination in oysters collected at a lagoon receiving urban drainage from Hue City for 17 months (August 2015–December 2016). We also investigated the genetic diversity of norovirus GI and GII in oyster and wastewater samples by using pyrosequencing to evaluate the effect of urban drainage on norovirus contamination of oysters. A total of 34 oyster samples were collected at two sampling sites (stations A and B) in a lagoon. Norovirus GI was more frequently detected than GII (positive rate 79 vs. 41%). Maximum concentrations of GI and GII were 2.4 × 10⁵ and 2.3 × 10⁴ copies/g, respectively. Co-contamination with GI and GII was observed in 35% of samples. Norovirus GII concentration was higher at station A in the flood season than in the dry season (P = 0.04, Wilcoxon signed-rank test). Six genotypes (GI.2, GI.3, GI.5, GII.2, GII.3, and GII.4) were identified in both wastewater and oyster samples, and genetically similar or identical sequences were obtained from the two types of samples. These observations suggest that urban drainage and seasonal flooding contribute to norovirus contamination of oysters in the study area.     

The Presence of Norovirus and Adenovirus on Environmental Surfaces in Relation to the Hygienic Level in Food Service Operations Associated with a Suspected Gastroenteritis Outbreak

Norovirus (NoV) gastroenteritis outbreaks appear frequently in food service operations (FSOs), such as in restaurants and canteens. In this study the presence of NoV and adenovirus (AdV) genomes was investigated on the surfaces of premises, especially in kitchens, of 30 FSOs where foodborne gastroenteritis outbreaks were suspected. The objective was to establish a possible association between the presence of virus genomes on surfaces and a visual hygienic status of the FSOs. NoV genome was found in 11 and AdV genome in 8 out of 30 FSOs. In total, 291 swabs were taken, of which 8.9% contained NoV and 5.8% AdV genome. The presence of NoV genomes on the surfaces was not found to associate with lower hygiene level of the premises when based on visual inspection; most (7/9) of the FSOs with NoV contamination on surfaces and a completed evaluation form had a good hygiene level (the best category). Restaurants had a significantly lower proportion of NoV-positive swabs compared to other FSOs (canteens, cafeteria, schools etc.) taken together (p = 0.00014). The presence of a designated break room for the workers was found to be significantly more common in AdV-negative kitchens (p = 0.046). Our findings suggest that swabbing is necessary for revealing viral contamination of surfaces and emphasis of hygiene inspections should be on the food handling procedures, and the education of food workers on virus transmission.     

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.     

Inhibition of Murine Norovirus and Feline Calicivirus by Edible Herbal Extracts

Human noroviruses (HuNoVs) cause foodborne and waterborne viral gastroenteritis worldwide. Because HuNoV culture systems have not been developed thus far, no available medicines or vaccines preventing infection with HuNoVs exist. Some herbal extracts were considered as phytomedicines because of their bioactive components. In this study, the inhibitory effects of 29 edible herbal extracts against the norovirus surrogates murine norovirus (MNV) and feline calicivirus (FCV) were examined. FCV was significantly inhibited to 86.89 ± 2.01 and 48.71 ± 7.38% by 100 μg/mL of Camellia sinensis and Ficus carica, respectively. Similarly, ribavirin at a concentration of 100 μM significantly reduced the titer of FCV by 77.69 ± 10.40%. Pleuropterus multiflorus (20 μg/mL) showed antiviral activity of 53.33 ± 5.77, and 50.00 ± 16.67% inhibition was observed after treatment with 20 μg/mL of Alnus japonica. MNV was inhibited with ribavirin by 59.22 ± 16.28% at a concentration of 100 μM. Interestingly, MNV was significantly inhibited with 150 µg/mL Inonotus obliquus and 50 μg/mL Crataegus pinnatifida by 91.67 ± 5.05 and 57.66 ± 3.36%, respectively. Treatment with 20 µg/mL Coriandrum sativum slightly reduced MNV by 45.24 ± 4.12%. The seven herbal extracts of C. sinensis, F. carica, P. multiflorus, A. japonica, I. obliquus, C. pinnatifida, and C. sativum may have the potential to control noroviruses without cytotoxicity.     

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.     

Occurrence and Trend of Hepatitis A Virus in Bivalve Molluscs Production Areas Following a Contamination Event

The aim of this study was to assess the trend of hepatitis A virus (HAV) in a coastal zone impacted by a contamination event, providing data for the development of management strategies. A total of 352 samples, including four bivalve mollusc species (Mytilus galloprovincialis, Solen vagina, Venus gallina and Donax trunculus), were taken over a period of 6 months from 27 production areas of the coast and analysis were performed according to ISO/TS 15216-1:2013. HAV presence was detected in 77 samples from 11 production areas and all positive results were related to samples collected in the first 3 months of the surveillance, during which HAV prevalence was 39.9% and values as high as 5096 genome copies/g were detected. A progressive reduction of viral contamination was evident during the first trimester of the monitoring, with prevalence decreasing from 78.8% in the first month, to 37.8% in the second and 3.9% in the third and quantitative levels reduced from an average value of 672 genome copies/g to 255 genome copies/g over a period of 4 weeks (virus half-life: 21.5 days). A regression analysis showed that, during the decreasing phase of the contamination, the data fitted a reciprocal quadratic model (Ra² = 0.921) and, based on the model, a residual presence of HAV could be estimated after negativization of the production areas. The statistical analysis of the results per shellfish species and per production area showed that there were limited differences in contamination prevalence and levels among diverse bivalve species, while a statistically significant difference was present in quantitative levels of one production area. These data could be useful for the development of both risk assessment models and code of practice for the management of viral contamination in primary production.     

Virus Type-Specific Removal in a Full-Scale Membrane Bioreactor Treatment Process

We investigated removal of noroviruses, sapoviruses, and rotaviruses in a full-scale membrane bioreactor (MBR) plant by monitoring virus concentrations in wastewater samples during two gastroenteritis seasons and evaluating the adsorption of viruses to mixed liquor suspended solids (MLSS). Sapoviruses and rotaviruses were detected in 25% of MBR effluent samples with log reduction values of 3- and 2-logs in geometric mean concentrations, respectively, while noroviruses were detected in only 6% of the samples. We found that norovirus and sapovirus concentrations in the solid phase of mixed liquor samples were significantly higher than in the liquid phase (P < 0.01, t test), while the concentration of rotaviruses was similar in both phases. The efficiency of adsorption of the rotavirus G1P[8] strain to MLSS was significantly less than norovirus GI.1 and GII.4 and sapovirus GI.2 strains (P < 0.01, t test). Differences in the adsorption of viruses to MLSS may cause virus type-specific removal during the MBR treatment process as shown by this study.     

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.