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.     

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.     

Distribution of Naturally Occurring Norovirus Genogroups I,II, and IV in Oyster Tissues

This study evaluated different tissues of naturally contaminated oysters (Crassostrea belcheri) for the presence of noroviruses. RNA from digestive tissues, gills, and mantle of the oysters was extracted and tested for norovirus genogroup (G) I, GII, and GIV using RT-nested PCR. In spiking experiments with a known norovirus, GII.4, the detection limits were 2.97 × 10² RNA copies/g of digestive tissues, 2.62 × 10² RNA copies/g of gills, and 1.61 × 10³ RNA copies/g of mantle. A total of 85 oyster samples were collected from a fresh market in Bangkok, Thailand. Noroviruses were found in the oyster samples (40/85, 47%): GI (29/85, 34.1%), GII (9/85, 10.5%), mixed GI and GII (1/85, 1.2%), and GIV (1/85, 1.2%). All three genogroups were found in the digestive tissues of oysters. Norovirus GI was present in all three tissues with the highest frequency in the mantle, and was additionally detected in multiple tissues in some oysters. GII was also detected in all three tissues, but was not detected in multiple tissues in the same oyster. For genogroup I, only GI.2 could be identified and it was found in all tissues. For genogroup II, three different genotypes were identified, namely GII.4 which was detected in the gills and the mantle, GII.17 which was detected in the digestive tissues, and GII.21 which was detected in the mantle. GIV.1 was identified in the digestive tissues of one oyster. This is the first report on the presence of human GIV.1 in oyster in Thailand, and the results indicate oyster as a possible vehicle for transmission of all norovirus genogroups in Thailand.     

Evaluation of a Porcine Gastric Mucin and RNase A Assay for the Discrimination of Infectious and Non-infectious GI.1 and GII.4 Norovirus Following Thermal,Ethanol, or Levulinic Acid Plus Sodium Dodecyl Sulfate Treatments

Human noroviruses (NoVs) are a major source of foodborne illnesses worldwide. Since human NoVs cannot be cultured in vitro, methods that discriminate infectious from non-infectious NoVs are needed. The purpose of this study was to evaluate binding of NoV genotypes GI.1 and GII.4 to histo-blood group antigens expressed in porcine gastric mucin (PGM) as a surrogate for detecting infectious virus following thermal (99 °C/5 min), 70 % ethanol or 0.5 % levulinic acid (LV) plus 0.01 or 0.1 % sodium dodecyl sulfate (SDS) sanitizer treatments and to determine the limit of detection of GI.1 and GII.4 binding to PGM. Treated and control virus samples were applied to 96-well plates coated with 1 µg/ml PGM followed by RNase A (5 ng/µl) treatment for degradation of exposed RNA. Average log genome copies per ml (gc/ml) reductions and relative differences (RD) in quantification cycle (Cq) values after thermal treatment were 1.77/5.62 and 1.71/7.25 (RNase A) and 1.73/5.50 and 1.56/6.58 (no RNase A) for GI.1 and GII.4, respectively. Treatment of NoVs with 70 % EtOH resulted in 0.05/0.16 (GI.1) and 3.54/10.19 (GII.4) log reductions in gc/ml and average RD in Cq value, respectively. LV (0.5 %) combined with 0.1 % SDS provided a greater decrease of GI.1 and GII.4 NoVs with 8.97 and 8.13 average RD in Cq values obtained, respectively than 0.5 % LV/0.01 % SDS. Virus recovery after PGM binding was variable with GII.4 > GI.1. PGM binding is a promising surrogate for identifying infectious and non-infectious NoVs after capsid destruction, however, results vary depending on virus strain and inactivation method.     

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.     

Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log₁₀ (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84–2.5 log₁₀ of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.     

Detection and Molecular Characterization of Human Noroviruses in Korean Groundwater Between 2008 and 2010

RT-PCR, nucleotide sequencing, and phylogenetic analysis were performed for genotyping and molecular characterization of noroviruses isolated from Korean groundwater. Among 160 samples collected from 80 sites between 2008 and 2010, 14 samples (8.7?%) from 12 sites were positive for noroviruses (NoVs). The percentages of NoV-positive samples in 2008, 2009, and 2010 were 22.2, 3.2, and 0?%, respectively, representing a yearly decrease. GII-positive samples (n?=?9, 5.6?%) outnumbered GI-positive samples (n?=?5, 3.1?%). The genotypes of the GI NoVs were GI.2, GI.5, and GI.6, and the genotypes of the GII NoVs were all GII.4. One sample, HM623465, was very similar to CUK-3 and CBNU2 and two GII.4 sequences isolated from the stool of Korean gastroenteritis patients. A BLASTN search revealed several nucleotide sequences highly similar to those of NoVs isolated in this study. The original isolation sources for these similar NoVs were mostly stool (n?=?731, 80.0?%) and groundwater (n?=?135, 14.8?%), and all the countries from which they were isolated were almost in Asia (96.0?%); specifically, China (n?=?192, 21.0?%), Japan (n?=?383, 41.9?%), Korea (n?=?296, 32.4?%), and other Asian countries (n?=?6, 0.7?%). These results suggest that Korean groundwater might be contaminated with NoVs from the stool of infected patients and that these NoVs in turn cause new cases of gastroenteritis through a typical fecal-oral route with region-specific circulation. Therefore, it is important to properly treat sewage, which may include waterborne viruses and manage point sources in groundwater for national health and sanitation. In addition, continuous molecular surveillance remains important for understanding circulating NoVs.     

Occurrence of Norovirus and Hepatitis A Virus in Wild Mussels Collected from the Baltic Sea

The aim of the study was to define the occurrence of human noroviruses of genogroup I and II (NoV GI and NoV GII) and hepatitis A virus (HAV) in the Baltic Sea mussels. The shellfish samples were taken at the sampling sites located on the Polish coast. In total, 120 shellfish were tested as pooled samples using RT-PCR and hybridisation with virus specific probes. NoV GI was detected in 22 (18.3 %), NoV GII in 28 (23.3 %), and HAV in 9 (7.5 %) of the shellfish. The nucleotide sequence analysis of the detected NoV GII strains showed a 97.3–99.3 % similarity to GII.4 virus strain. This is the first report describing the NoV and HAV occurrence in wild Baltic mussels and their possible role as bioindicators of seawater contamination with human enteric viruses.     

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.     

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.     

Norovirus GII.17 Associated with a Foodborne Acute Gastroenteritis Outbreak in Brazil, 2016

Foodborne transmission gastroenteritis (AGE) outbreak occurred during a celebration lunch in July, 2016, Brazil. All stool samples tested were positive for noroviruses (NoV) and phylogenetic analysis revealed that strains were genetically close to GII.17 Kawasaki_2014. These findings indicated circulation of NoV GII.17 Kawasaki_2014 in the Brazilian population, associated with AGE outbreak.     

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.     

Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish

Shellfish complying with European Regulations based on quantification of fecal bacterial indicators (FIB) are introduced into markets; however, information on viruses, more stable than FIB, is not available in the literature. To assess the presence of noroviruses (NoVs) GI and GII and human adenoviruses (HAdV) in domestic and imported mussels and clams (n = 151) their presence was analyzed during winter seasons (2004–2008) in north-west Spanish markets through a routine surveillance system. All samples tested negative for NoV GI and 13 % were positive for NoV GII. The role of HAdV as viral indicator was evaluated in 20 negative and 10 positive NoV GII samples showing an estimated sensitivity and specificity of HAdV to predict the presence of NoV GII of 100 and 74 % (cut-off 0.5). The levels of HAdV and NoVs and the efficiency of decontamination in shellfish depuration plants (SDP) were evaluated analyzing pre- and post-depurated mussels collected in May–June 2010 from three different SDP. There were no statistically significant differences in the prevalence and quantification of HAdV between pre- and post-depurated shellfish and between seawater entering and leaving the depuration systems. Moreover, infectious HAdV were detected in depurated mussels. These results confirm previous studies showing that current controls and depuration treatments limiting the number of FIB do not guarantee the absence of viruses in shellfish.