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.     

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.     

Re-emergence of a GII.4 Norovirus Sydney 2012 Variant Equipped with GII.P16 RdRp and Its Predominance over Novel Variants of GII.17 in South Korea in 2016

Noroviruses are major causative pathogen of nonbacterial acute gastroenteritis worldwide. Of the seven genogroups of noroviruses suggested recently, genogroup II genotype 4 (GII.4) had been the most common genotype identified in hospitalized patients in the last few decades. However, since the latter half of 2014, new variants of GII.17 have been reported as the main causes of outbreaks over GII.4 in East Asia and have also occurred in America and Europe. In this study, we monitored norovirus GII in coastal streams at South Gyeongsang province and South Jeolla province of South Korea from March 2015 to May 2016. Norovirus GII.17 capsid sequences were predominantly detected until September 2015 in water samples. However, we found that the number of positive cases of the norovirus GII.4 Sydney 2012 capsid sequence has been increasing since December 2015, overtaking that of GII.17 in 2016. The RdRp genotype of this predominant GII.4 variant in 2016 was identified as GII.P16. The emergence and predominance of the GII.4 pandemic capsid sequence harboring a different RdRp genotype suggested the potential for a future pandemic.     

Norovirus GII.17 Outbreak Linked to an Infected Post-Symptomatic Food Worker in a French Military Unit Located in France

In February 2016, an outbreak of gastroenteritis occurred in a French military unit located in Poitiers, France. Attack rate was of 34% (103/300). A case–control study identified association between illness and cake consumption. Stool samples were tested positive for Norovirus GII.17 for one patient and one post-symptomatic food worker (FW). The FW presented vomiting one day before cake preparation. The NoV strain was probably spread through food worker hand contact. Prevention of Norovirus foodborne outbreaks implies new guidelines for FWs management in France and Europe.     

Pyrosequencing Analysis of Norovirus Genogroup II Distribution in Sewage and Oysters: First Detection of GII.17 Kawasaki 2014 in Oysters

Norovirus GII.3, GII.4, and GII.17 were detected using pyrosequencing in sewage and oysters in January and February 2015, in Japan. The strains in sewage and oyster samples were genetically identical or similar, predominant strains belonging to GII.17 Kawasaki 2014 lineage. This is the first report of GII.17 Kawasaki 2014 in oysters.     

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n?=?6), GII.4 (n?=?1), GII.17 (n?=?3), and GII.unclassified (n?=?3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83–1.85?×?10⁴ genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00?×?10¹ genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

     

Resilience of Norovirus GII.4 to Freezing and Thawing: Implications for Virus Infectivity

Genogroup II.4 norovirus (NoV) remains the predominant NoV strain in food- and water-borne outbreaks. Capsid integrity as well as viral RNA persistence were determined for GII.4 NoV by real-time RT-PCR after 1?C14 freeze/thaw (F/T) cycles (?80?°C/+22?°C) or after ?80?°C storage for up to 120?days. In both cases, capsid integrity and viral RNA titers remained stable. RNase was exogenously added after 1?C14 F/T cycles, but did not alter the amount of genomic NoV RNA detected, indicating that capsids remained intact. Presumptive NoV infectivity was evaluated in functional studies by a porcine gastric mucin binding assay. Viruses frozen and thawed up to 14× bound similarly to porcine mucin, suggesting no reduction in virus infectivity. Overall, this study shows that a) NoV particles retain their integrity for at least 14 F/T cycles, b) long-term (120?day) frozen storage does not decrease NoV RNA titers, and c) capsid binding to receptor-like glycoprotein moieties remains unaltered after 14 F/T cycles. This work indicates that freezing and thawing of foods or beverages would not be a practical processing intervention to reduce NoV contamination. Likewise, repeated freezing and thawing, as might be encountered during winter months, is not expected to inactivate NoV in the environment. Results do show that laboratory samples destined for molecular biological analyses or for use as positive controls may be repeatedly frozen and thawed without any anticipated reduction in NoV RNA titers. This study documents the cryostability of NoV capsids and RNA to freezing and thawing and to the possible retention of virus infectivity.     

Secretor Status is Associated with Susceptibility to Disease in a Large GII.6 Norovirus Foodborne Outbreak

Food and Environmental Virology - Norovirus is commonly associated with food and waterborne outbreaks. Genetic susceptibility to norovirus is largely dependent on presence of histo-blood group...     

Updating a New Semi-nested PCR Primer Pair for the Specific Detection of GII Norovirus in Oysters

Food and Environmental Virology - Oysters are major transmission vectors of noroviruses (NoVs) in the environment. Outbreaks of NoVs are often associated with the consumption of NoV-contaminated...     

漓江地表水体有机碳来源

科学辨识河流有机碳来源是碳循环研究的关键.本文选取典型岩溶流域漓江流域为研究对象,通过同位素示踪法、相关分析法、端元混合模型,利用碳稳定同位素、C/N对其2016年7~9月有机碳来源进行研究.结果表明:(1)DIC浓度空间分布特征为:岩溶区岩溶区与非岩溶区的混合区非岩溶区;干流区DIC浓度从上游到下游递增,主要受控于流域碳酸盐岩的空间分布比例.(2)DOC是构成漓江水体TOC的主体,TOC来源以内源有机碳为主,内源碳浓度空间分布特征为:岩溶区混合区非岩溶区,可能与岩溶区水生植物丰茂、碳酸酐酶活性较强有关,TOC中内源碳的浓度介于1.02~5.14 mg·L~(-1),平均为2.54 mg·L~(-1);TOC中内源碳的比例空间分布差异不大,平均为73.07%.(3)POC浓度、POC中内源碳的浓度及POC中内源碳的比例空间分布差异不大,POC来源以外源碳为主,POC中内源有机碳浓度介于0.01~0.16 mg·L~(-1),平均为0.05mg·L~(-1),水生生物量对漓江流域POC贡献平均为17.31%.(4)DOC浓度及内源DOC浓度空间分布均为:岩溶区混合区非岩溶区,DOC主要来源于水生生物的初级生产力,DOC中内源碳的浓度介于0.97~5.10 mg·L~(-1),平均为2.48 mg·L~(-1);DOC中内源碳的比例空间分布差异不大,平均为79.51%.研究水生光合生物对流域有机碳的影响,可以为岩溶碳汇稳定性科学问题的解答提供基础.     

九龙江流域地表水中硝酸盐来源辨析

从九龙江流域遴选出2个典型小流域--仙都和五川小流域作为研究区,于2005年春季运用15N同位素示踪法对其地表水中硝酸盐来源进行研究.结果表明,仙都小流域地表水中溶解态总氮、硝氮和氨氮的浓度范围(以N计,下同)分别为1.47～5.31 mg/L、0.83～4.05mg/L和0.21～0.36mg/L,硝酸盐的δ15N值(以样品相对于标准大气N2的15N和14N比值的千分偏差表示)范围在2.5460-7.92%之间;五川小流域地表水中溶解态总氮、硝氮和氨氮的浓度范围分别为1.14～5.56mg/L、0.96～1.46mg/L和0.12～1.28mg/L,硝酸盐的δ15N值范围在-0.19‰～5.89‰之间.对照不同来源的硝酸盐δ15N特征值,结合研究区的农作物种植和施肥状况,得出如下结论:仙都小流域地表水中硝酸盐主要来自无机化肥与土壤有机氮,有机肥有一定的贡献;五川小流域地表水中硝酸盐的来源以无机化肥与土壤有机氮为主,有机肥的贡献很小;2个小流域地表水中硝酸盐的来源随时空变化而有差异,与当地农作物种类及农田时令密切相关.     

北京城市生态系统地表水硝酸盐污染空间变化及其来源研究

利用2009～2010年北京城市生态系统地表水10处监测点水环境监测数据,评价了北京城市生态系统地表水硝酸盐污染状况及其空间分布,结合水化学因子相关关系分析了硝酸盐的主要来源.结果表明,北京城市生态系统地表水硝态氮(NO3--N)质量浓度为0.7～7.6 mg.L-1,其中,位于北京市东南部的地表水监测点(东便门和通惠河)水体NO3--N质量浓度为7.0～7.6 mg.L-1,显著高于上游8个监测点NO3--N质量浓度(P<0.01);Cl-质量浓度为14.8～86.0 mg.L-1,东便门、通惠河地表水监测点水体Cl-质量浓度为81.5～85.0 mg.L-1,约为上游其他8个监测点的2.3～5.8倍.东便门、通惠河地表水监测点水体电导率(EC)、SO24-质量浓度也表现出同NO3--N、Cl-相似的变化规律,表明东便门、通惠河两处地表水监测点附近存在明显的污染源.相关分析表明,地表水Cl-/Na+和SO24-/Ca2+呈明显的线性相关,说明地表水NO3--N污染来源比较单一;水体中NO3--N/Cl-及NO3--N的质量浓度状况说明北京城市生态系统地表水NO3--N污染来源主要是城市污水,包括污水处理厂的废水、垃圾沥出液及生活污水.未来北京市地表水治理应重点关注东便门、通惠河等东南部下游水体污染治理.     

Norovirus GII.Pe Genotype: Tracking a Foodborne Outbreak on a Cruise Ship Through Molecular Epidemiology,Brazil, 2014

Norovirus (NoV) is recognized as the most common cause of foodborne outbreaks. In 2014, an outbreak of acute gastroenteritis occurred on a cruise ship in Brazil, and NoV became the suspected etiology. Here we present the molecular identification of the NoV strains and the use of sequence analysis to determine modes of virus transmission. Food (cream cheese, tuna salad, grilled fish, orange mousse, and vegetables soup) and clinical samples were analyzed by ELISA, conventional RT-PCR, qRT-PCR, and sequencing. Genogroup GII NoV was identified by ELISA and conventional RT-PCR in fecal samples from 5 of 12 patients tested (41.7%), and in the orange mousse food sample by conventional RT-PCR and qRT-PCR. Two fecal GII NoV samples and the orange mousse GII NoV sample were successfully genotyped as GII.Pe (ORF 1), revealed 98.0–98.8% identities among them, and shared phylogenetically distinct cluster. Establishing the source of a NoV outbreak can be a challenging task. In this report, the molecular analysis of the partial RdRp NoV gene provided a powerful tool for genotyping (GII.Pe) and tracking of outbreak-related samples. In addition, the same fast and simple extraction methods applied to clinical samples could be successfully used for complex food matrices, and have the potential to be introduced in routine laboratories for screening foods for presence of NoV.     

A Food Handler-Associated, Foodborne Norovirus GII.4 Sydney 2012-Outbreak Following a Wedding Dinner, Austria, October 2012

On October 12, 2012, the provincial public health directorate of Salzburg reported a suspected norovirus (NV) outbreak among guests of a wedding–reception. The investigation aimed to confirm the causative agent, to identify the mode of transmission and to implement appropriate preventive measures. A probable outbreak case was defined as a wedding guest with diarrhoea or vomiting with disease onset from 7 to 10 October 2012 and who consumed food at the wedding dinner prepared by a hotel in the province Salzburg on 6 October 2012. A confirmed outbreak case fulfilled the criteria of a probable outbreak case and had a laboratory-confirmed NV infection. We conducted a cohort-investigation among the wedding guests. The case definitions were fulfilled in 26 wedding guests (25 %) including 2 confirmed cases. Females were 3.2 times more likely to develop disease (95 % CI 1.4–7.2) as compared to males. A mushroom dish was found to be associated with disease risk among females (risk ratio 2.3, 95 % CI 1.2–4.3). Two of 2 tested case-patients and 6 of 14 kitchen workers tested were positive for NV GII.4 Sydney. One kitchen staff-member worked during the wedding dinner despite diarrhoea. No food safety training was documented for the employees and the kitchen staff’s restroom was lacking operational facilities for hand hygiene. We report the first investigated outbreak due to GII.4 Sydney, which was likely due to a symptomatic kitchen worker. Gender-specific eating behaviour may have posed female guests at higher risk of NV infection.     

两种水源性诺如病毒富集方法的比较

评估阳离子膜吸附-洗脱法和阴离子膜吸附-洗脱法在富集水源性诺如病毒（NV）过程中的病毒回收效果、消耗时间及使用成本,可以获得更适用于水源性诺如病毒实际监测工作的较优富集方法.以无锡市疾病预防控制中心保存的诺如病毒阳性标准质粒为模板,采用荧光定量RT-PCR（反转录酶-聚合酶链反应）方法绘制病毒定量标准曲线,并应用于病毒的绝对定量.将含诺如病毒的粪便悬液先稀释成3个不同浓度,并加入灭菌水中,分别经过阴离子膜（硝酸纤维素滤膜）或阳离子膜（Nanoceram膜）吸附洗脱,最终相同体积洗脱液提取核酸后用荧光定量RT-PCR方法进行绝对定量,并进一步计算最终病毒回收率.结果表明:①样本病毒浓度为8×101~8×1010 copies/μL时,病毒拷贝数的lg对数值与C_t（荧光定量PCR中达到设定阈值所经过的循环数）呈线性关系.②随着样本病毒浓度的增加,阴离子膜法和阳离子膜法的病毒回收率都相应增加.③当检测样本中病毒浓度分别为8×101、4×102 copies/μL时,阴离子膜法回收率分别为22.2%±4.1%和36.8%±6.2%,阳离子膜法回收率分别为17.4%±1.5%和28.8%±6.1%,说明阴离子膜吸附-洗脱法对病毒的富集回收率显著高于阳离子膜吸附-洗脱法（P < 0.05）.研究显示,综合富集效果、耗费时间和成本等因素,阴离子膜吸附-洗脱法比阳离子膜吸附-洗脱法更适用于日常水源性诺如病毒的富集工作.      

地表水饮用水水源地粪大肠菌群污染现状及趋势分析

本文对衡阳市城区3个集中式生活饮用水水源地近四年(2012-2015年)来,水质中粪大肠菌群的污染状况及污染趋势进行了分析研究。对近四年来各水源地水质中粪大肠菌群的浓度范围、平均值、超标次数、最大超标倍数进行了讨论分析,得出了水源地水质中粪大肠菌群的污染现状及污染趋势,有利于加强饮用水水源地水质监测与监管。     

鄱阳湖地表水硝酸盐时空变异性及其来源研究

利用2011年1月和7月鄱阳湖流域地表水中NO3-的质量浓度实测数据,评价NO3-在不同时期的污染状况及空间分布,运用GIS和统计学方法分析NO3-的时空变异特征,结合水化学因子相关关系分析NO3-的主要污染来源。结果表明,在丰水期,鄱阳湖流域地表水NO3-质量浓度为0~33.33 mg/L,在枯水期NO3-质量浓度为0~51.50 mg/L;空间差值分析显示在丰水期,鄱阳湖南湖区NO3-的质量浓度明显高于北湖区,这主要是来源于生活污水、工业废水、农业及养殖业等产生的氮;而在枯水期恰好相反,北湖区NO3-的质量浓度较高于南湖区,主要是受到沿湖周边工业废水的影响;通过运用水化学因子相关关系分析发现,地表水中各监测点Cl-/Na+和SO42-/Ca2+变化趋势较相似,说明水体中NO3-的污染来源可能比较单一;研究NO3-/Cl-的变化关系曲线发现在枯水期,水体中NO3-的污染来源主要为污水,而丰水期NO3-的污染来源比较复杂,包括农业非点源污染和渔类珍珠养殖业等。     

Prevalence of GII.4 Sydney Norovirus Strains and Associated Factors of Acute Gastroenteritis in Children: 2019/2020 Season in Guangzhou,China

Food and Environmental Virology - Norovirus, the leading cause of non-bacterial acute gastroenteritis (AGE) worldwide, is constantly mutating. Continuous monitoring of the evolution of epidemic...