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.     

Molecular Detection of human Noroviruses in Influent and Effluent Samples From Two Biological Sewage Treatment Plants in the Region of Monastir,Tunisia

Noroviruses (NoVs) are responsible for numerous cases of waterborne and foodborne gastroenteritis every year. They are released in the sewage and their detection in this environment can reflect the epidemiology of the viral strains circulating in the community. A three-year (2007–2010) survey was conducted in order to evaluate the presence of human NoVs using RT-PCR in 518 sewage samples collected at the entrance and exit of two biological sewage treatment plants located in Monastir region, Tunisia. In this study, we aimed to genetically characterize the most prevalent GI and GII NoV strains, in order to obtain a rough estimate of the efficacy of disinfection treatments and to compare the results with clinical data documented in the same area during the same period. This work confirms the wide circulation and the genetic diversity of NoVs in Tunisia and the widespread distribution of NoV variants in both raw and treated wastewater. Indeed, NoV was detected in 192 (37.1 %) sewage samples, among them mixed infections with group A rotavirus were detected in 125 (65.1 %) cases. The genotypes of the GI NoVs were GI.1, GI.2, GI.4, GI.5, and GI of unassigned genotype (GI.UA), and the genotypes of the GII NoVs were all GII.12. This study enhances the currently poor environmental virological data gathered in Tunisia, demonstrates the benefit of environmental surveillance as a tool to determine the epidemiology of NoVs circulating in a given community, and underlines the need for the design and support of similar long-term studies in our country, in order to compensate for the absence of a national surveillance system for gastroenteric viruses.     

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.     

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.     

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.     

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.     

独流减河流域绿色基础设施空间格局与景观连通性分析的尺度效应

尺度效应是景观生态学研究的核心问题之一,是正确理解和感知景观格局与生态过程相互作用及其动态变化特征的基础.针对GI（绿色基础设施）进行空间格局与景观连通性分析具有强烈的尺度依赖性的问题,以天津市独流减河流域为例,利用ArcGIS 10.1、ENVI 5.3和eCognition 8.9软件进行面向对象土地利用分类,基于MSPA（形态学空间格局分析）方法和景观连通性分析方法,借助GuidosToolbox 2.6和Conefor 2.6软件,通过设置不同的粒度、边缘宽度和扩散距离,对2016年研究区GI的粒度效应、边缘效应和距离效应进行定量分析和评价.结果表明:①MSP（形态学空间格局）类型具有明显的粒度效应和边缘效应,较小的粒度和边缘宽度会得到更为详细的空间格局信息.②粒度效应改变GI像元的空间分布,会导致GI空间信息的丢失或增加,直接影响MSP类型的空间配置和量化关系;而边缘效应不会改变GI像元的空间分布,使得GI空间信息不发生改变,但对MSP类型的影响更为显著.③景观连通性与扩散距离具有直接关系,扩散距离越大,GI核心区景观连通性越高,当扩散距离增至一定程度时,所有核心区会连接成一个网络整体,景观连通性达到最大,研究区GI核心区的扩散距离关键值在2.5~4.5 km之间.研究显示,独流减河流域GI要素尺度效应明显但机理不同,基于MSPA和景观连通性分析方法能够更加直观地反映各景观类型和网络组分的空间变化特征和数值变化规律.      

Occurrence and Genetic Diversity of Human Cosavirus in Sewage in Italy

Human Cosavirus (HCoSV) is a newly discovered virus whose role in human enteric diseases is still unknown. In Italy, the prevalence and genetic diversity of HCoSV are unexplored. One hundred forty-one raw sewage samples collected throughout Italy were screened for HCoSV by RT-nested PCR. HCoSV was detected in 25.5% of samples. Species A, C, and D, and a potentially new species were detected. Our results show a significant circulation and heterogeneity of HCoSV in Italy.     

铜锌及其复合污染对西瓜种子萌发的影响

通过室内培养实验,研究了铜(Cu)和锌(Zn)单一及其复合污染对西瓜种子萌发的影响.结果表明,低浓度(20μmol/L)铜、锌单一及其复合处理对西瓜种子的发芽率没有影响,但增加了发芽指数、活力指数;促进r幼芽和幼根的伸长生长;提高了生物量的积累.高浓度(200 μmol/L)铜、锌单一及复合处理对西瓜种子的发芽率、发芽...     

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.     

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.     

Genetic Analysis of Norovirus Strains that Caused Gastroenteritis Outbreaks Among River Rafters in the Grand Canyon,Arizona

Toilet solid waste samples collected from five outbreaks among rafters in the Grand Canyon were subjected to sequencing analysis of norovirus partial capsid gene. The results revealed that a GI.3 strain was associated with one outbreak, whereas the other outbreaks were caused by GII.5 whose sequences shared >98.9% homology.     

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.     

景观规划导向的绿色基础设施研究进展——基于“格局—过程—服务—可持续性”研究范式

中国目前处于新型城镇化与生态文明建设的重要时期,引入绿色基础设施（Green Infrastructure,GI）规划框架对协调城市发展与生态保护具有重要意义。基于景观生态学中“格局—过程—服务—可持续性”研究范式,对近年来中国内外GI规划进行针对性综述,利用该范式对相关研究的理论来源与进展进行梳理。研究表明：GI格局研究中,景观格局指数是应用最为广泛的方法。针对于景观连接度,形态学空间格局分析等结构连接度评价方法具有计算简单、适用性广的优势,但缺乏明确的生态学含义;图论等功能连接度则针对特定的生态过程,是GI格局与生态过程的重要联系,将是下一步研究重点。GI生态过程研究借鉴生态网络的理论与方法,其规划步骤可分为核心区选取、阻力面构建、潜在廊道识别三部分。GI生态系统服务研究中,生物物理量模型与经济计量模型广泛应用于调节、供给与支持服务评价中,而文化服务评价则将问卷调查作为基础数据获取的重要途径。另外,生态系统服务需求研究涉及到生态系统服务的实际需求与使用偏好,相关定量评价方法以及成果的实际应用有待进一步探索。GI景观可持续性研究中,在明晰GI提供的生态系统服务总量的基础上,服务间权衡与协同效应已成为GI规划中的重要考量,而情景规划可以有效模拟GI生态系统服务的动态变化。最后,GI规划应将提升景观可持续性作为规划目标,据此研究提出了重视格局与过程相互影响机制的基础研究、加深GI生态系统服务需求端研究、明晰生态系统服务间协同与权衡效应、将GI与实际规划结合四项研究展望。     

不同物料堆肥腐熟度评价指标的变化特性

为探讨工厂化好氧堆肥的堆肥周期及腐熟度评价体系,提高工厂堆肥效率,选取上海地区不同来源典型的9种物料,采用工厂化工艺进行堆肥试验,对堆体的温度、含水率、pH、C/N、w(OM)、ρ(NH₄+-N)、ρ(NO₃--N)、ρ(DOC)、ρ(DOC)/ρ(DON)及GI(种子发芽指数)腐熟度评价指标变化规律进行研究. 结果表明:堆肥腐熟度受多方面因素影响,T〔(C/N)终点/(C/N)起点)〕与w(OM)、ρ(NH₄+-N)、GI、ρ(DOC)/ρ(DON)之间相关性显著,T、ρ(NH₄+-N)、GI 3个指标能准确有效地判断堆肥腐熟情况,堆肥结束后T在0.50~0.59之间,ρ(NH₄+-N)为301~346 mg/L,GI为81.31%~91.03%. 不同物料堆肥腐熟难易程度不同,通过聚类分析将9种物料分为5类:第1类,厨余、杂草、生活垃圾、园林垃圾;第2类,果蔬、污泥;第3类,秸秆;第4类,鸡粪;第5类,猪粪. 厨余、杂草、生活垃圾、园林垃圾、污泥、秸秆堆肥成分复杂较难腐熟,需要35 d达到腐熟标准;鸡粪及猪粪堆肥结构简单较易腐熟,29 d即可达到腐熟标准. 据此可适当将工厂化堆肥周期缩短为35 d.      

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.