Detection of noroviruses in shellfish in the Netherlands

Shellfish from oyster farms in the Netherlands and imported from other European countries were examined for viral contamination. A method that allows sequence matching between noroviruses from human cases and shellfish was used. The samples of shellfish (n = 42) were analyzed using a semi-nested RT-PCR that had been optimized for detection of norovirus in shellfish (SR primer sets). In addition, a different genome region was targeted using a second primer set which is routinely used for diagnosis of norovirus infection in humans (JV12Y/JV13I). To improve the detection limit for this RT-PCR a semi-nested test format was developed (NV primer sets). One of 21 oyster samples (4.8%) from Dutch farms was norovirus positive, whereas norovirus was detected in 1 out of 8 oyster samples (12.5%) and 5 out of 13 mussel samples (38.5%) collected directly after importation in the Netherlands. RNA from samples associated with an outbreak of gastro-enteritis in the Netherlands in 2001 was re-analyzed using the NV primer sets. At least one identical sequence (142/142 nt) was found in three fecal and in two oyster samples related to this outbreak. Further surveillance of norovirus by detection and typing of viruses from patients with gastroenteritis and shellfish is warranted to clarify the causes of future outbreaks.     

Evaluation of an Extraction Method for the Detection of GI and GII Noroviruses in Fruit and Vegetable Salads

Human norovirus (HuNoV) is a major foodborne virus causing gastroenteritis outbreaks in humans. Salad products can be vectors of transmission for foodborne viruses such as HuNoV when these products are contaminated naturally or through unsanitary food handling. Therefore, development of simple, reliable and sensitive techniques for the detection of HuNoV in salad products is needed to ensure food safety. The purpose of our study was to optimize a method for the detection of HuNoV in artificially contaminated salad products. To this end, 2 different kinds of salads (fruit salads and vegetable salads) were experimentally inoculated with HuNoV GI, HuNoV GII, and MS2 suspensions. The selected method was based on treatment with pectinase followed by Trizol‐chloroform purification, and the recovery efficiencies were 6.07% to 26.52% for HuNoV GI and 5.54% to 37.36% for HuNoV GII. MS2 was used as the process control, and the recovery efficiencies for fruit salad and vegetable salad samples were 38.57% and 41.13%, respectively. The optimized method could be applied in diagnostic laboratories to identify NoV contamination in composite foods, such as salad products, should an event of foodborne outbreak occur.     

Norovirus in retail shellfish

Norovirus is a common cause of gastroenteritis outbreaks associated with consumption of raw shellfish. The majority of norovirus infections worldwide are due to genogroup II noroviruses. Bivalve molluscs (mussels, clams and oysters) at the end of the commercial chain, the points of purchase, were sampled between 2005 and 2008 in several retail points in Apulia, Italy, and screened by a semi-nested RT-PCR specific for genogroup II noroviruses. Noroviral RNA was detected in 12.1% of the samples, with lower frequency being observed in samples obtained from hypermarkets (8.1%) rather than in samples from open-air markets and fish shops (17.6% and 16.2%, respectively). By sequence analysis, the strains were characterized as norovirus variants GII.4/2004 and GII.b/Hilversum, which were both circulating in Italy in the same time-span.     

Norovirus outbreak in a restaurant: investigation of the path of infection by sequence analysis of food and human samples

In September 2014, a small gastroenteritis outbreak occurred in a Greek restaurant. Primary investigations by official food surveillance revealed significant hygienic problems in the premises. Food samples and environmental samples were analyzed for the presence of bacterial and viral food pathogens. Norovirus genogroup I (GI) was detected in 2 environmental samples and in mixed salad. At the same time, stool samples from patients were analyzed and norovirus GI was detected. Further investigations revealed the presence of norovirus GI on some of the restaurant employees. Comparison of nucleic acid sequences revealed full sequence homology between norovirus RNA genotype 1.2 in food, environmental and stool samples, suggesting a common source of contamination and infection. Sequence analysis of food and environmental samples was facilitated by application of a system for total RNA amplification. Despite the fact that original source of contamination could be determined doubtlessly, observed weaknesses in the food production that caused this outbreak were discussed. The mixed salad could have been contaminated either by the lettuce contaminated at primary production or by one of the food-handlers. The investigation of the path of infection is necessary for the kind of legal consequences to be directed by authorities and may contribute to measures to eliminate possible sources of food contamination.     

2020~2021年广州市售牡蛎中GII型诺如病毒污染情况调查

滤食性牡蛎是食源性诺如病毒传播的重要食品媒介。为了解广州市售牡蛎中的诺如病毒污染水平与遗传多样性特点,合理评估消费风险,本研究于2020年6月至2021年5月期间,每月从当地水产市场随机采集牡蛎样本,采用实验室前期建立的蛋白酶K处理偶联聚乙二醇沉淀小体系法,包括荧光定量RT-PCR和巢式RT-PCR技术检测贝类中病毒的污染量以及基因型分布特点。结果共检测牡蛎110只,GII型诺如病毒阳性检出率为52.7%（58/110）,病毒污染含量范围为1.56×103~1.09×106 copies/g（消化腺）。其中,春夏季节（3~8月）牡蛎中诺如病毒的阳性率为35.7%（20/56）,低于在秋冬季节（9~2月）的阳性率70.4%（38/54）;但不同季节中检出的病毒含量无显著差异,分别为春季（2.69±1.46）×105 copies/g（消化腺）,夏季（1.97±2.16）×105 copies/g（消化腺）,秋季（6.91±6.16）×104 copies/g（消化腺）,冬季为（4.83±2.99）×104 copies/g（消化腺）。部分阳性样本测序分析后显示,除1份为GII.17基因型外,其余均为GII.4基因型（n=13）,与当地的临床流行基因型呈现一致性。本研究显示广州市售牡蛎中仍存在较高的诺如病毒污染水平,需要进一步加强病毒防控工作,尤其提醒消费者在食用牡蛎时需加工充分。     

一起疑似食源性诺如病毒胃肠炎事件病原分子生物学特征分析

目的研究厦门市思明区一起聚集性胃肠炎事件的诺如病毒的分子生物学特征。方法将收集到的11份肛拭子标本及1份生蚝样品,采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)方法检测诺如病毒核酸,阳性标本再进行实时荧光定量RT-PCR扩增,扩增产物经过凝胶电泳分析后,进行序列测定,确定基因型,并进行系统发育树分析。结果 11份肛拭子标本中检出5株GⅠ组诺如病毒株,2株测序成功,检出4株GⅡ组毒株,3株测序成功;1份生蚝样品中检出1株GⅠ组毒株,测序成功。对测序成功的6株毒株进行同源性分析,GⅠ.2型毒株所测序列与2014年上海株KP325648等6株参考株高度同源,GⅡ.17型毒株所测序列与2015年韩国株KT384078等8株参考株高度同源,证实这是一起由GⅠ.2和GⅡ.17型诺如病毒混合感染引起的聚集性胃肠炎事件,GⅡ.17型毒株为厦门市首次报道。结论此次聚集性胃肠炎事件是由诺如病毒引起,且为GⅠ.2与GⅡ.17型毒株混合感染引起。     

实时荧光RT-PCR检测冷冻草莓中诺如病毒

目的：建立冷冻草莓中的GI、GII型诺如病毒实时荧光RT-PCR检测方法,并应用于实际样品的检测。方法：对草莓样品进行前处理、病毒富集、病毒RNA的提取和纯化,然后采用实时荧光RT-PCR进行检测。结果：核酸提取方法能够有效地去除抑制因子,同时对104份送检样品进行检测,结果均为阴性。结论：所建立的核酸提取与实时荧光RT-PCR结合的检测体系适合于草莓样品中诺如病毒GI、GII型的检测。     

微滴式数字PCR检测冷冻草莓中GⅠ、GⅡ型诺如病毒

目的:建立一种检测冷冻草莓中诺如病毒(GⅠ和GⅡ)的逆转录微滴数字PCR (RT-ddPCR)方法。方法:根据ISO标准选定检测引物,优化反应体系,退火温度,进行了方法学实验,建立了一种快速检测冷冻草莓中GⅠ和GⅡ亚型诺如病毒的新方法。结果:确定了数字PCR检测GⅠ型诺如病毒退火温度为56.5℃,GⅡ型诺如病毒退火温度为58.1℃。RT-ddPCR检测GⅠ质粒标准品标准曲线的R2=0.9947,RT-ddPCR检测GⅡ质粒标准品标准曲线的R2=0.9950,说明该方法具有良好的线性关系。与RT-qPCR灵敏度对比,RT-ddPCR法的灵敏度比RT-qPCR法高一个数量级。在检测范围内,最低检测限低至个位拷贝数。RSD最小为3.8%,表明该实验重复性良好。浓度较低100 copies/μL左右时,RT-ddPCR的重复性不佳。结论:本研究建立的诺如病毒数字PCR法具有特异性强、灵敏度高、检测限低等优点,可用于冷冻草莓中诺如病毒的定量检测。     

Determination of norovirus contamination in oysters from two commercial harvesting areas over an extended period, using semiquantitative real-time reverse transcription PCR

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses, which cause gastroenteritis, are the principal agents of shellfish-related illness. Fecal-indicator quality standards based on Escherichia coli are well established in Europe and elsewhere. However, norovirus outbreaks after consumption of shellfish meeting these standards still occur, and the need to improve consumer health protection is well recognized. Alternative approaches proposed include direct monitoring of viral pathogens and the use of alternative indicator organisms capable of providing a better indication of virus risk. This study applies a recently developed TaqMan PCR assay to assess norovirus contamination in shellfish. Comparison was made with E. coli as the existing sanitary standard and a male-specific RNA bacteriophage as a possible alternative. Two commercial pacific oyster (Crassostrea gigas) harvesting areas were monitored over a 31-month period. The results show peaks of norovirus contamination in both areas during winter months, with average levels approximately 17 times higher in oysters sampled October to March than during the remainder of the year, consistent with epidemiological data for the United Kingdom showing oyster-associated illness is confined to winter months. While there was no apparent association with E. coli, an association between levels of norovirus contamination and the male-specific RNA bacteriophage was noted, with average norovirus levels over 40 times higher in samples with male-specific RNA bacteriophage counts of >1,000 PFU/100 g than in samples with <100 PFU/100 g. Overall, these results suggest that norovirus monitoring in shellfish production areas could be an effective strategy for reduction of virus risk.     

草莓中诺如病毒和甲肝病毒的多重实时荧光逆转录PCR检测方法的建立

目的建立草莓中诺如病毒GI、诺如病毒GII和甲肝病毒等3种食源性病毒的多重实时荧光RT-PCR检测方法,并应用于实际样品检测。方法对草莓样品进行前处理、病毒富集、病毒RNA提取和纯化后,先采用单重实时荧光RT-PCR进行检测,随后进行多重实时荧光RT-PCR反应条件优化,建立多重实时荧光RT-PCR检测方法并分析其特异性和灵敏度。结果所采用的病毒富集和核酸提取方法可以实现病毒的有效富集和抑制剂的去除,建立的多重实时荧光RT-PCR方法特异性强(100%),对草莓样品中诺如病毒GI、诺如病毒GII和甲肝病毒的检测灵敏度分别为56.2 RT-PCR50/20 g、31.6 RT-PCR50/20 g和31.4 CCID50/20 g。同时对50份样品进行检测,结果均为阴性。结论所建立的检测方法快速、灵敏、特异性强,适用于草莓产品中诺如病毒GI、诺如病毒GII和甲肝病毒的同时检测。     

Surveillance of Enteric Viruses and Microbial Indicators in the Eastern Oysters (Crassostrea virginica) and Harvest Waters along Louisiana Gulf Coast

Noroviruses are the most common causative agent of viral gastroenteritis in humans, and are responsible for major foodborne illnesses in the United States. Filter‐feeding molluscan shellfish exposed to sewage‐contaminated waters bioaccumulate viruses, and if consumed raw, transmit the viruses to humans and cause illness. We investigated the occurrence of norovirus GI and GII and microbial indicators of fecal contamination in the eastern oysters (Crassostrea virginica) and water from commercial harvesting areas along the Louisiana Gulf Coast (January to November of 2013). Microbial indicators (aerobic plate count, enterococci, fecal coliforms, Escherichia coli, male‐specific coliphages, and somatic coliphages) were detected at the densities lower than public health concerns. Only one oyster sample was positive for norovirus GII at 3.5 ± 0.2 log₁₀ genomic equivalent copies/g digestive tissues. A stool specimen obtained from an infected individual associated with a norovirus outbreak and the suspected oysters (Cameron Parish, La., area 30, January 2013) were also analyzed. The norovirus strain in the stool belonged to GII.4 Sydney; however, the oysters were negative and could not be linked. In general, no temporal trend was observed in the microbial indicators. Low correlation among bacterial indicators was observed in oysters. Strongest correlations among microbial indicators were observed between enterococci and fecal coliforms (r = 0.63) and between enterococci and E. coli (r = 0.64) in water (P < 0.05); however, weak correlations were found in oysters (r < 0.45) and between oysters and harvest water (r ≤ 0.36, P > 0.05). Our results emphasize the need for regular monitoring of pathogenic viruses in commercial oyster harvesting areas to reduce the risks of viral gastroenteritis incidences.     

Evaluation of a norovirus detection methodology for ready-to-eat foods

Despite recent norovirus (NoV) foodborne outbreaks related to consumption of ready-to-eat (RTE) foods, a standardized assay to detect NoV in these foods is not available yet. Therefore, the robustness of a methodology for NoV detection in RTE foods was evaluated. The NoV detection methodology consisted of direct RNA extraction with an eventual concentration step, followed by RNA purification and a multiplex RT-qPCR assay for the detection of GI and GII NoV and the murine norovirus-1 (MNV-1), the latter used as process control. The direct RNA extraction method made use of the guanidine-isothiocyanate containing reagent (Tri-reagent?, Ambion) to extract viral RNA from the food sample (basic protocol called TriShort), followed by an eventual concentration step using organic solvents (extended protocol called TriConc). To evaluate the robustness of the NoV detection method, the influence of (1) the NoV inoculum level and (2) different food types on the recovery of NoV from RTE foods was investigated. Simultaneously, the effect of two RNA purification methods (manual RNeasy minikit (Qiagen) and automated NucliSens EasyMAG (BioMérieux)) on the recovery of NoV from these foods was examined. Finally, MNV-1 was evaluated as process control. First of all, high level GI and GII NoV inocula (~10? NoV genomic copies/10 g) could be recovered from penne salad samples (10 g) in at least 4 out of 6 PCRs, while low level GI and GII NoV inocula (~10? NoV genomic copies/10 g) could be recovered from this food product in maximally 3 out 6 PCRs, showing a significant influence of the NoV inoculum level on its recovery. Secondly, low level GI and GII NoV inocula (10? NoV genomic copies/10 g) were spiked onto 22 ready-to-eat food samples (10 g) classified in three categories (soups, deli sandwiches and composite meals). The GI and GII NoV inocula could be recovered from 20 of the 22 samples. The TriConc protocol provided better recoveries of GI and GII NoV for soups while the TriShort protocol yielded better results for the recovery of GII NoV from composite meals. NoV recovery from deli sandwiches was problematic using either protocol. Thirdly, the simultaneous comparison of two RNA purification protocols demonstrated that automated RNA purification performed equally or better compared to manual RNA extraction. Finally, MNV-1 was successfully evaluated as process control when detecting NoV in RTE foods using this detection methodology. In conclusion, the evaluated NoV detection method was capable of detecting NoV in RTE foods, although recoveries were influenced by the inoculum level and by the food type.     

Genetic variation in the conservative gene region of Norovirus genogroup II strains in environmental and stool samples

Noroviruses (NoVs) have been one of leading etiological agents for infectious gastroenteritis over the world. Gastroenteritis caused by NoVs is prevalent in winter season, and the contamination of the water environment with NoVs in the epidemic cold season is frequently reported. In contrast, the number of gastroenteritis patients and NoVs in the water environment are reduced during the nonepidemic summer season, and the year-round fate of NoVs has remained to be elucidated. In this study, we collected nucleotide sequences of NoV genogroup II (GII) from domestic sewage, sewage sludge, treated wastewater, river water, and stool samples of gastroenteritis patients in geographically close areas. Phylogenetic analysis of the obtained NoV gene revealed that six out of seven isolates from environmental samples and 10 out of 11 isolates from stool samples belong to genotype 3 (NoV GII.3) or 4 (NoV GII.4), which have been prevalent throughout the world. Genetic distances between the conservative gene region of NoV GII.4 variants implied that genetically diverse strains are likelyto occur in environmental samples. The evaluation of the evolutionary change of NoV gene obtained from environmental samples would make it possible to elucidate the year-round fate of NoVs.     

实时荧光RT-PCR检测贝类中的札幌病毒

建立检测贝类中GⅠ、GⅡ、GⅣ和GⅤ型札幌病毒的实时荧光RT-PCR新方法。首先使用PEG 8000对贝类中的札幌病毒进行富集,然后采用Tri-reagent提取材料中的总RNA,针对札幌病毒RNA 3'端含Poly A尾的特点,使用带有Poly(dT)25的磁珠对病毒RNA进行纯化,用所获的高纯度RNA进行四种型别札幌病毒的实时荧光RT-PCR检测。该方法高效、灵敏,检测下限为101数量级拷贝,能够用于日常检验。