Detection of Human Enteric Viruses in Freshwater from European Countries

The transmission of water-borne pathogens typically occurs by a faecal–oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.     

Seasonal Occurrence of Human Enteric Viruses in River Water Samples Collected from Rural Areas of South-East Poland

The study was carried out in 2007, and its main aim was 1 year monitoring of surface water of the River Wieprz in Poland for the presence of human adenoviruses group F and noroviruses (NoVs). In total, 60 water samples were collected from four sampling sites situated along the river. The viruses were concentrated from water samples using glass wool, followed by elution with a glycine buffer containing skimmed milk powder. Subsequently, the viral nucleic acids were extracted and purified from water concentrates using a NucliSENS^® kit and a QIAamp Viral RNA Mini Kit^®. The presence of viral nucleic acids was confirmed by applying traditional PCR-based methods with incorporated internal amplification controls. Human pathogenic viruses were detected in 35% of analysed water samples. Adenoviruses were detected in 28.3% of analysed samples, and were present at all seasons of the year. 11.6% of the samples were positive for NoVs; they were present only during summer, in contrast to conventional findings. Molecular identification of norovirus strains revealed that they belong to genogroup I and II.     

水中粪大肠菌群测定的滤膜法研究

本方法主要研究滤膜法在实际监测工作中的可行性,利用实际水样对滤膜法与发酵法的监测结果进行比对,并对滤膜法的重复性和准确度进行监测研究。     

对硝基苯胺的光化学降解研究

系统研究了氙灯和高压汞灯照射下对硝基苯胺(PNA)在蒸馏水、人工海水和天然海水中的光化学降解情况.结果表明,PNA光降解速率会受到光源、溶液介质、温度、起始浓度、重金属离子、甲醇及过氧化氢浓度的影响.随着光源强度的增强、起始浓度的增大、温度的增高和过氧化氢量的增加,PNA在海水中光降解速率相应加快;然而甲醇的加入可有效...     

环境水体中肠道病毒的膜吸附-洗脱浓缩方法研究

在膜吸附-洗脱和洗脱液浓缩相结合的基础上,建立了一种简便实用的水中肠道病毒浓缩方法.通过实时定量RT-PCR检测,比较了不同材料和不同孔径的微孔滤膜对病毒的吸附效果;对膜洗脱方式进行了改进;研究了在洗脱液浓缩过程中,PEG浓度对于病毒回收率的影响.最后确定了最佳的浓缩方法.选择效果好而且来源广泛的0.22 μm孔径的混合纤维素酯微孔滤膜,采用磁力搅拌来洗脱滤膜上吸附的病毒;洗脱液浓缩步骤中,PEG最佳质量浓度为130　g/L.系统比较了不同病毒接种量下,方法中各步骤的病毒回收率.对接种已知量的肠道病毒的生活污水、二级处理出水和地表水等样品的试验结果表明,该方法效果稳定,适合不同水样中肠道病毒的浓缩分离.     

One-year Surveillance of Human Enteric Viruses in Raw and Treated Wastewaters,Downstream River Waters,and Drinking Waters

Human enteric viruses are a major cause of waterborne diseases, and can be transmitted by contaminated water of all kinds, including drinking and recreational water. The objectives of the present study were to assess the occurrence of enteric viruses (enterovirus, norovirus, adenovirus, hepatitis A and E virus) in raw and treated wastewaters, in rivers receiving wastewater discharges, and in drinking waters. Wastewater treatment plants’ (WWTP) pathogen removal efficiencies by adenovirus quantitative real-time PCR and the presence of infectious enterovirus, by cell culture assays, in treated wastewaters and in surface waters were also evaluated. A total of 90 water samples were collected: raw and treated wastewaters (treated effluents and ultrafiltered water reused for industrial purposes), water from two rivers receiving treated discharges, and drinking water. Nested PCR assays were used for the identification of viral DNA/RNA, followed by direct amplicon sequencing. All raw sewage samples (21/21), 61.9 % of treated wastewater samples (13/21), and 25 % of ultrafiltered water samples (3/12) were contaminated with at least one viral family. Multiple virus families and genera were frequently detected. Mean positive PCRs per sample decreased significantly from raw to treated sewage and to ultrafiltered waters. Moreover, quantitative adenovirus data showed a reduction in excess of 99 % in viral genome copies following wastewater treatment. In surface waters, 78.6 % (22/28) of samples tested positive for one or more viruses by molecular methods, but enterovirus-specific infectivity assays did not reveal infectious particles in these samples. All drinking water samples tested negative for all viruses, demonstrating the effectiveness of treatment in removing viral pathogens from drinking water. Integrated strategies to manage water from all sources are crucial to ensure water quality.     

Morphological and Biochemical Characteristics of Avian Faecal Droppings and Their Impact on Survival of Avian Influenza Virus

Environmental specimens such as faecal droppings are considered important for the detection of avian influenza viruses (AIV). In view of lower rates of AIV isolation from avian faecal droppings, characterization of droppings is imperative to elucidate contributing factors. However, there are no reports on morphological and biochemical characteristics of droppings. The objective of the present study was the characterization of droppings from different avian species and their impact on the AIV detection and isolation. A total of 373 droppings belonging to 61 avian species from 22 families of apparently healthy wild migratory, resident, domestic birds and poultry were studied during five winter migratory bird seasons between 2007 to 2012 and 2016–2017. The colour, morphology and size of the droppings varied from species-to-species. These data could be useful for the identification of avian species. Droppings from 67% of the avian species showed acidic pH (4.5–6.5); nine species showed neutral pH (7.0), and 11 species showed alkaline pH (7.5). The infectious titers of AIV in droppings with acidic pH were significantly lower (p < 0.05) than those of the droppings with neutral and alkaline pH. However, acidic pH did not hamper AIV detection by real-time RT-PCR. In order to avoid the impact of acidic pH, collecting fresh droppings into viral transport medium (pH 7.0–7.5) would help improve the rate of AIV isolation.     

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.     

Effectiveness of Poliovirus Concentration and Recovery from Treated Wastewater by Two Electropositive Filter Methods

Enteric viruses are often present in low numbers in various water matrices. Virus sampling therefore involves multiple concentration steps to condense large samples down to small volumes for detection by cell culture or molecular assays. The NanoCeram^® Virus Sampler has been demonstrated to be effective for the recovery of viruses from tap water, surface waters, and seawater. The goal of this study was to evaluate a new method using NanoCeram^® filters for the recovery of poliovirus 1 (PV-1) from treated wastewater. Activated sludge effluent samples were spiked with PV-1 and concentrated in side-by-side tests by two methods: (1) NanoCeram^® filtration, elution with sodium polyphosphate buffer, secondary concentration via centrifugal ultrafiltration; and (2) 1MDS filtration, elution with beef extract, secondary concentration via organic flocculation. The virus retention and elution efficiencies did not differ significantly between the two methods. In contrast, the secondary concentrate volume was smaller for the NanoCeram^® method (8.4 vs. 30 mL) and the secondary concentration efficiencies were different between the two methods with 98 % for centrifugal ultrafiltration (NanoCeram^®) and 45 % for organic flocculation (1MDS). The overall method efficiencies were significantly different (P ≤ 0.05) with the NanoCeram^® method yielding a 57 % and the 1MDS a 23 % virus recovery. In addition, there appeared to be less interference with viral detection via polymerase chain reaction with the NanoCeram^® concentrates. This NanoCeram^® method therefore is able to efficiently recover PV-1 from large volumes of wastewater and may serve as an inexpensive alternative to the standard 1MDS filter method for such applications.     

废铁屑预处理被4-硝基苯胺污染的土壤

利用机械厂废弃铁屑和分析纯级铁粉对受4-硝基苯胺(PNA)污染土壤的修复进行了对比研究,探讨了土壤酸度和反应温度对还原效果的影响,并对反应时间、铁屑用量和土壤含水量3个影响因素进行了优化选择.实验结果表明,在1.5g含PNA浓度约为1.3×10-5mol/g的模拟污染土样中加入100mg铁屑或铁粉和0.5mL蒸馏水,在25℃下反应4h,PNA的还原率分别为99%和76%,产物均为苯二胺(PDA);铁粉仅在偏酸性土壤中对PNA有较高还原率,铁屑则在中性或弱碱性土壤中仍能保持较好的还原效果;反应温度的变化对还原效果影响不大;正交实验结果显示反应时间对PNA的还原效果影响最大,土壤含水量次之,铁屑用量影响最小。     

四种人肠道病毒在大连金石滩浴场中的分布

于2007年5～10月,每月一次采集大连金石滩浴场各站位10 L表层海水,用超滤的方法进行病毒浓缩,采用PCR方法分析了该浴场表层海水中的四种主要肠道病毒-轮状病毒、星状病毒、脊髓灰质炎病毒和腺病毒.调查结果为:在所采集的24个样品中轮状病毒检出率为12.5%、星状病毒0.9%、脊髓灰质炎病毒和腺病毒分别达29.2%;每个月份均有病毒检出,依据病毒的阳性检出率,8月份该浴场受到的污染最严重,10月份和9月份次之.     

A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia

Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP’s outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75 % and AsV in 67 %. HAV were not detected in wastewater. Overall, 92 % of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92 % of samples and at concentrations up to 7.7 × 10³ genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66 % of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach.     

Concentration and Recovery of Viruses from Water: A Comprehensive Review

Enteric viruses are a cause of waterborne disease worldwide, and low numbers in drinking water can present a significant risk of infection. Because the numbers are often quite low, large volumes (100–1,000 L) of water are usually processed. The VIRADEL method using microporous filters is most commonly used today for this purpose. Negatively charged filters require the addition of multivalent salts and acidification of the water sample to effect virus adsorption, which can make large-volume sampling difficult. Positively charged filters require no preconditioning of samples, and are able to concentrate viruses from water over a greater pH range than electronegative filters. The most widely used electropositive filter is the Virosorb 1MDS; however, the Environmental Protection Agency has added the positively charged NanoCeram filters to their proposed Method 1615. Ultrafilters concentrate viruses based on size exclusion rather than electrokinetics, but are impractical for field sampling or processing of turbid water. Elution (recovery) of viruses from filters following concentration is performed with organic (e.g., beef extract) or inorganic solutions (e.g., sodium polyphosphates). Eluates are then reconcentrated to decrease the sample volume to enhance detection methods (e.g., cell culture infectivity assays and molecular detection techniques). While the majority of available filters have demonstrated high virus retention efficiencies, the methods to elute and reconcentrate viruses have met with varying degrees of success due to the biological variability of viruses present in water.     

种植绿肥对作物产量和细菌群落稳定性的影响

为深入研究青海高原地区长期种植绿肥,减施化肥条件下小麦/油菜产量及土壤理化性状及微生物群落的变化,通过2011年建立的定位试验,设GF0（毛苕子作为绿肥,不施用化肥）、GF60（毛叶苕子配施60%化肥）、GF70（毛叶苕子配施70%化肥）、GF80（毛叶苕子配施80%化肥）、GF90（毛叶苕子配施90%化肥）、GF100（毛叶苕子配施100%化肥）、F0（休耕并且不施化肥）、F100（休耕并且施用100%化肥）共8个处理,利用高通量测序等技术,研究了种植利用绿肥减施化肥条件下小麦和油菜的产量、土壤性质和微生物群落结构的变化特征.结果表明,在保证小麦、油菜不减产的基础上,种植绿肥后茬作物减施化肥达到30%左右.绿肥配施减量化肥对土壤肥力有显著的提升作用,特别是土壤有机碳和全氮含量有明显的提高,分别提高1.34%~7.46%、2.16%~7.48%.化肥与绿肥配施增强了土壤微生物群落多样性,其中酸杆菌门和变形菌门是本研究土壤中的优势菌群.共生网络分析表明,绿肥处理提高了细菌群落的稳定性和抗干扰能力,同时绿肥应用增加了细菌群落中的关键物种.本研究表明,不同土壤微生物丰度受种植模式、施肥量影响很大,长期种植绿肥、减施化肥提高了土壤微生物丰度和多样性,增强微生物群落的稳定性.种植绿肥条件下化肥减肥量30%左右保证作物稳产可为当地的施肥管理提供参考.     

污染场地环境调查的土壤监测点位布设方法初探

结合我国污染场地环境调查中土壤监测面临的现状,通过分析国外土壤监测采样的点位布设方法及国内相关技术标准的相应要求,对我国污染场地环境调查土壤监测的点位布设方法进行了探讨。提出土壤样品采集的平面点位、纵向分层布设应考虑的影响因素、基本原则和布点要求,分别对具体的平面、纵向采样点位布设方法进行了分析阐述,为开展污染场地环境调查的土壤监测采样提供借鉴。     

三唑酮在水体系中的光化学降解研究

系统研究了在天然日光和高压汞灯照射下三唑酮在蒸馏水、人工海水和天然海水中的光降解情况.结果表明,天然日光照射下,三唑酮在三种介质中降解较慢.高压汞灯较天然日光能够更有效地激发三唑酮的降解,且降解符合一级动力学规律.在300 W高压汞灯照射下,三唑酮在蒸馏水、人工海水和天然海水中的半寿期分别为54.14 min、144.38 min、177.69 min.三唑酮在蒸馏水中的光降解速率大于其在天然海水中的,这主要是由于天然海水中存在的阴、阳离子和其它特殊的物质所引起的.同蒸馏水中的光反应相比,人工海水中的离子抑制了三唑酮的光降解反应.在500 W高压汞灯照射下,三唑酮的反应速率明显提高.实验发现溶液中加入重金属离子(Cu2+、Pb2+、Cd2+)能够改变三唑酮的光降解速率.     

几种粪大肠菌群检测方法的比较

粪大肠菌群作为水体粪便污染的指示菌,其在地表水水质监测评价中具有重要作用。采取精确、快速的粪大肠菌群检测方法,对控制流行疾病的发生和传播有重要的科学意义。为此对应用多管发酵法、滤膜法、快速检测法(纸片法)、酶底物法检测粪大肠菌群的优缺点作了对比研究。得出了快速检测法(纸片法)、酶底物法有较准确、快速等方法特性,有较好的实用价值。     

水体系中氧氟沙星的光化学降解研究

研究了高压汞灯和氙灯照射下氧氟沙星(OFLX)在蒸馏水、人工海水和天然海水中的光降解过程,探讨了光源、起始浓度、丙酮、表面活性剂等因素对OFLX光化学降解速率的影响.结果表明,OFLX在高压汞灯下的光反应速率比在氙灯下快得多,均符合一级反应动力学过程;在相同光源下,OFLX在海水中降解最快,其次是人工海水;当OFLX初始浓度为2、4、6mg.L-1时,其光解速率常数分别为0.163、0.140和0.132 min-1,随着OFLX初始浓度增大,其光解率降低;不同浓度的丙酮均能促进OFLX光降解的反应,其反应速率常数为0.084 2～0.102 min-1,且光敏效率与丙酮浓度呈正相关关系;然而当十六烷基三甲基溴化铵(CTAB)、十二烷基苯磺酸钠(SDBS)、吐温-20(TW-20)等表面活性剂的浓度为5 mg.L-1,它们均抑制了水体中OFLX光降解反应的进程.另外,利用金藻8701单种培养进行了OFLX光化学降解前后的毒性试验,表明OFLX光解过程产生风险较高的中间产物,随着光解进行,产物毒性降低.