江苏省某市部分农村河道水体和污水处理厂主要工艺环节病毒污染状况调查

为研究江苏省某市农村河道水体和污水处理厂主要工艺环节病毒的污染状况,分析常见理化因素与病毒污染之间的相关性,对江苏省某市部分农村河道水体和污水处理厂主要工艺环节7个水样、1个污泥和2个沉积物样点进行为期9个月的病毒(包括诺如病毒G Ⅰ、诺如病毒G Ⅱ、轮状病毒、札如病毒、腺病毒、星状病毒、肠道病毒)污染状况监测采样(共90份样本),通过阴离子膜吸附-洗脱法进行富集,荧光定量RT-PCR检测法对病毒进行检测,分析病毒污染状况;同时,监测水温、pH、COD(化学耗氧量)、TP(总磷)、TN(总氮)、EC(电导率)和DO(溶解氧)等7个理化指标的变化情况,探讨这些常见理化因素与病毒污染之间的相关性. 结果表明:90份样本中,诺如病毒G Ⅱ检出率为36.67%,肠道病毒检出率为30%,其他病毒检出率均低于诺如病毒G Ⅱ和肠道病毒. 污水处理后出水中有诺如病毒G Ⅱ、肠道病毒和轮状病毒检出,相关性分析显示,水温与诺如病毒G Ⅱ检出率呈显著负相关(P < 0.05). 研究显示,江苏省某市农村河道水体和污水处理厂主要工艺环节存在病毒污染,诺如病毒G Ⅱ为优势毒株;目前的污水消毒处理工艺并不能完全去除病毒,人群在接触外环境水体时,有潜在病毒暴露风险.      

Virucidal Efficacy of a Hydrogen Peroxide Nebulization Against Murine Norovirus and Feline Calicivirus,Two Surrogates of Human Norovirus

Human noroviruses (HuNoV) are amongst the leading causes of acute non-bacterial gastroenteritis in humans and can be transmitted via person-to-person contact, via contact with contaminated surfaces or by consumption of contaminated food. Contaminated surfaces in healthcare settings contribute to the transmission of viruses. No-touch automated room disinfection systems might prevent such a spread of contamination and thus their virucidal effect needs to be evaluated. The aim of this study was to assess the efficacy of a nebulization system spraying hydrogen peroxide on two main surrogates of HuNoV, namely murine norovirus (MNV) and feline calicivirus (FCV). The viruses were dried on cover glasses and on stainless steel discs and exposed to nebulization. The number of infectious viral particles and genomic copies before and after the nebulization was compared. The efficacy in reducing infectivity of both surrogates was demonstrated. For the infectious viral titre of MNV and FCV, a log₁₀ reduction factor ≥4.84 and 4.85 was observed after nebulization, respectively, for tests on cover glasses and ≥3.90 and 5.30, respectively, for tests on stainless steel discs. Only low reductions in genomic copy numbers were observed for both surrogates. The nebulization of hydrogen peroxide showed a clear virucidal effect on both HuNoV surrogates, MNV and FCV, on two different carriers and the use of nebulization should be promoted in complementarity with conventional disinfection methods in healthcare settings and food processing facilities to reduce viral load and spread of contamination.     

Critical Review on the Public Health Impact of Norovirus Contamination in Shellfish and the Environment: A UK Perspective

We review the risk of norovirus (NoV) infection to the human population from consumption of contaminated shellfish. From a UK perspective, risk is apportioned for different vectors of NoV infection within the population. NoV spreads mainly by person-to-person contact or via unsanitary food handling. NoV also enters the coastal zone via wastewater discharges resulting in contamination of shellfish waters. Typically, NoV persists in the marine environment for several days, with its presence strongly linked to human population density, wastewater discharge rate, and efficacy of wastewater treatment. Shellfish bioaccumulate NoV and current post-harvest depuration is inefficient in its removal. While NoV can be inactivated by cooking (e.g. mussels), consumption of contaminated raw shellfish (e.g. oysters) represents a risk to human health. Consumption of contaminated food accounts for 3–11% of NoV cases in the UK (~74,000 cases/year), of which 16% are attributable to oyster consumption (11,800 cases/year). However, environmental and human factors influencing NoV infectivity remain poorly understood. Lack of standard methods for accurate quantification of infective and non-infective (damaged) NoV particles represent a major barrier, hampering identification of an appropriate lower NoV contamination limit for shellfish. Future management strategies may include shellfish quality assessment (at point of harvest or at point of supply) or harvesting controls. However, poor understanding of NoV inactivation in shellfish and the environment currently limits accurate apportionment and risk assessment for NoV and hence the identification of appropriate shellfish or environmental quality standards.     

Molecular Study of the Persistence of Infectious Human Norovirus on Food-Contact Surfaces

The aim of this study was to investigate the effect of relative humidity (RH) and temperature on norovirus (NoV) persistence as infectious particles on food-contact surfaces such as stainless steel and polyvinyl chloride (PVC). For this purpose, a new method combining enzymatic digestion with molecular beacon-based NASBA targeting the ORF1-ORF2 domain was developed to discriminate between infectious and noninfectious NoV. Stainless steel and PVC disks were contaminated with known amounts of human NoV and kept for 56 days at 7 and 20°C at high (86% ± 4%) and low (30% ± 10%) RH. NoV retained its putative infectivity for 56 days on PVC and for 49 days on stainless steel at 7°C and for 7 and 28 days, respectively, at low and high RH at 20°C on both tested surfaces. These results confirm that NoV persists in an infective state on inert surfaces for long periods of time and consequently may cause illness. The new molecular approach to detecting infectious NoV on inert surfaces may provide valuable information for evaluating environmental surface decontamination strategies.     

Nucleic Acid Amplification-Based Methods for Detection of Enteric Viruses: Definition of Controls and Interpretation of Results

The most effective methods for virus detection in food and environmental samples are those based on nucleic acid amplification. Complex methods must be applied by the analyst in order to control for false negative results of virus detection assays in those samples that may be contaminated by virus concentrations above the detection level. The verification of analytical results is a necessity and this depends on using an appropriate suite of controls to monitor the efficacy of the critical steps in the method and allow correct result interpretations to be made. We describe the suite of controls necessary for analysing food and environmental samples for the presence of enteric viruses by nucleic acid amplification-based methods. To exclude false negative and positive interpretations of results, the inclusion of this suite of controls will be essential when considering incorporating monitoring of viruses in food or environmental safety management plans.     

Comparative Virucidal Efficacy of Seven Disinfectants Against Murine Norovirus and Feline Calicivirus,Surrogates of Human Norovirus

Human noroviruses (HuNoV) are the leading cause of acute non-bacterial gastroenteritis in humans and can be transmitted either by person-to-person contact or by consumption of contaminated food. A knowledge of an efficient disinfection for both hands and food-contact surfaces is helpful for the food sector and provides precious information for public health. The aim of this study was to evaluate the effect of seven disinfectants belonging to different groups of biocides (alcohol, halogen, oxidizing agents, quaternary ammonium compounds, aldehyde and biguanide) on infectious viral titre and on genomic copy number. Due to the absence of a cell culture system for HuNoV, two HuNoV surrogates, such as murine norovirus and feline calicivirus, were used and the tests were performed in suspension, on gloves and on stainless steel discs. When, as criteria of efficacy, a log reduction >3 of the infectious viral titre on both surrogates and in the three tests is used, the most efficacious disinfectants in this study appear to be biocidal products B, C and D, representing the halogens, the oxidizing agents group and a mix of QAC, alcohol and aldehyde, respectively. In addition, these three disinfectants also elicited a significant effect on genomic copy number for both surrogate viruses and in all three tests. The results of this study demonstrate that a halogen compound, oxidizing agents and a mix of QAC, alcohol and aldehyde are advisable for HuNoV disinfection of either potentially contaminated surfaces or materials in contact with foodstuffs.     

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.     

Development of a Practical Method to Detect Noroviruses Contamination in Composite Meals

Various methods to detect foodborne viruses including norovirus (NoV) in contaminated food have been developed. However, a practical method suitable for routine examination that can be applied for the detection of NoVs in oily, fatty, or emulsive food has not been established. In this study, we developed a new extraction and concentration method for detecting NoVs in contaminated composite meals. We spiked NoV-GI.4 or -GII.4 stool suspension into potato salad and stir-fried noodles. The food samples were suspended in homogenizing buffer and centrifuged to obtain a food emulsion. Then, anti-NoV-GI.4 or anti-NoV-GII.4 rabbit serum raised against recombinant virus-like particles or commercially available human gamma globulin and Staphylococcus aureus fixed with formalin as a source of protein A were added to the food emulsion. NoV-IgG-protein A-containing bacterial complexes were collected by centrifugation, and viral RNA was extracted. The detection limits of NoV RNA were 10–35 copies/g food for spiked NoVs in potato salad and stir-fried noodles. Human gamma globulin could also concentrate other NoV genotypes as well as other foodborne viruses, including sapovirus, hepatitis A virus, and adenovirus. This newly developed method can be used as to identify NoV contamination in composite foods and is also possibly applicable to other foodborne viruses.     

The Impact of Winter Relocation and Depuration on Norovirus Concentrations in Pacific Oysters Harvested from a Commercial Production Site

Oysters contaminated with norovirus present a significant public health risk when consumed raw. In this study, norovirus genome copy concentrations were determined in Pacific oysters (Magallana gigas) harvested from a sewage-impacted production site and then subjected to site-specific management procedures. These procedures consisted of relocation of oysters to an alternative production area during the norovirus high-risk winter periods (November to March) followed by an extended depuration (self-purification) under controlled temperature conditions. Significant differences in norovirus RNA concentrations were demonstrated at each point in the management process. Thirty-one percent of oyster samples from the main harvest area (Site 1) contained norovirus concentrations >?500 genome copies/g and 29% contained norovirus concentrations <?100 genome copies/g. By contrast, no oyster sample from the alternative harvest area (Site 2) or following depuration contained norovirus concentrations >?500 genome copies/g. In addition, 60 and 88% of oysters samples contained norovirus concentrations <?100 genome copies/g in oysters sampled from Site 2 and following depuration, respectively. These data demonstrate that site-specific management processes, supported by norovirus monitoring, can be an effective strategy to reduce, but not eliminate, consumer exposure to norovirus genome copies.     

The Fate of Murine Norovirus and Hepatitis A Virus During Preparation of Fresh Produce by Cutting and Grating

Human noroviruses and hepatitis A virus (HAV) are commonly associated with outbreaks occurring in restaurant establishments and catered events. Food handlers are major contributing factors to foodborne illnesses initiated in the kitchen setting. In this study, transfer of HAV and murine norovirus (MNV-1), a human norovirus surrogate, between produce (cucumbers, strawberries, tomatoes, cantaloupes, carrots, and honeydew melons) and common kitchen utensils (graters and knives) was investigated. The extent of virus transfer to produce during utensil application, in the presence and the absence of food residue, and the impact of knife surface properties (sharp, dull, serrated) was also investigated. Transfer of MNV-1 and HAV from produce items, initially contaminated with ~5.5 log PFU, to knives and graters during application ranged from 0.9 to 5.1 log PFU. MNV-1 transfer to knives was the greatest for cucumbers, strawberries, and tomatoes, and the least for honeydew melons, while transfer of HAV to knives was greater for tomatoes and honeydew melons than strawberries, cantaloupes, and cucumbers. After preparation of a contaminated produce item, knife cross-contamination easily occurred as viruses were detected on almost all of the seven produce items successively prepared. Produce residues on utensils often resulted in less virus transfer when compared to utensils without residue accumulation. Knife surface properties did not impact virus transfer. The ease of virus transfer between produce and utensils demonstrated by the current study highlights the importance of efforts aimed toward preventing cross-contamination in the kitchen environment.     

Effect of the Shellfish Proteinase K Digestion Method on Norovirus Capsid Integrity

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log₁₀ was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log₁₀ loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log₁₀, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.     

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.     

Assessment of Water Quality in a Border Region Between the Atlantic Forest and an Urbanised Area in Rio de Janeiro,Brazil

The preservation of water resources is one of the goals of the designation of parks that act as natural reservoirs. In order to assess the impact of the presence of humans in an environmental preservation area bordering urban areas, the presence of four pathogenic enteric viruses [group A rotavirus (RV-A), norovirus (NoV), human adenoviruses (HAdV), and hepatitis A virus (HAV)], as well as the physico-chemical parameters, and Escherichia coli levels were assessed in riverine water samples. From June 2008 to May 2009, monthly monitoring was performed along the Engenho Novo River. RV-A, NoV, and HAdV were observed in 29 % (31/108) of the water samples, with concentrations of up to 10³ genome copies/liter. The natural occurrence of infectious HAdV was demonstrated by Integrated Cell Culture-PCR (ICC-PCR). This study confirms the suitability of using the detection of fecal-oral transmitted viruses as a marker of human fecal contamination in water matrices and indicates the spread of pathogenic viruses occurring in an alleged area of environmental protection.     

Detection and Typing of Norovirus from Frozen Strawberries Involved in a Large-Scale Gastroenteritis Outbreak in Germany

During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.     

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.