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.     

Simultaneous Concentration of Enteric Viruses and Protozoan Parasites: A Protocol Based on Tangential Flow Filtration and Adapted to Large Volumes of Surface and Drinking Waters

A filtration system, based on tangential flow filtration (TFF) followed by ultracentrifugation was developed in order to concentrate simultaneously viruses and parasites from large volumes of water. For TFF, no pre-treatment of the membrane is performed but a post-rinsing step using high pH-beef extract-based eluant. Applying our protocol to 20 l of surface waters spiked with vaccinal poliovirus-1, ϕX174 and MS2 bacteriophages resulted in an averaged viral recovery of 75% by TFF and 91% by ultracentrifugation (total viral recovery of 70%). Our protocol was further applied to 31 environmental samples including surface, ground and drinking waters from the Grand Duchy of Luxembourg in order to assess the occurrence of protozoan parasites (Cryptosporidium parvum and Giardia lamblia (oo)cysts), pathogenic viruses (enterovirus, norovirus and adenovirus) and infectious bacteriophages (somatic coliphages and F-specific phages) in these samples. High viral recovery rates of > 70% were confirmed concentrating environmental strains of somatic and F-specific coliphages from non-spiked surface waters. Parasites and enteric viruses were detected in 86 and 40% of the surface waters used for drinking water production, respectively. Infectious bacteriophages were isolated from all surface waters and in two out of seven (29%) groundwaters revealing a susceptibility of the corresponding wells to viral pollution. TFF-based method proved to be efficient for surveying the occurrence of non-bacterial pathogens such as enteric viruses and protozoan parasites in large volumes of environmental waters.     

城市地表水中肠道病原微生物与粪便污染指示菌的关系研究

本研究选择肠道病毒、伤寒沙门氏菌、志贺氏菌、大肠埃希氏菌作为城市水体中典型的肠道病原微生物,分别建立起相应的实时荧光定量PCR检测方法.对水源水、景观娱乐用水以及城市河流样品进行长期监测,考察典型肠道病原微生物的分布特征和变化规律.肠道病毒在各地表水体中都有检出,浓度大都在103copy·L-1以下.大肠埃希氏菌的含量在保护良好的水体和接纳城市污水的水体中有明显差别,而伤寒沙门氏菌和志贺氏菌通常出现在受粪便污染较重的水体中.统计学的分析结果显示各种水体中的肠道病原微生物检出浓度均符合对数正态分布规律.粪便污染指示菌与肠道病毒之间不存在显著的相关性,但却能在一定程度上反映典型肠道病原菌的存在情况.在大肠菌群或粪大肠菌群浓度为104CFU·L-1以上的样品中,伤寒沙门氏菌、志贺氏菌具有较高的阳性率.     

Occurrence of Human Enteric Viruses in Water Sources and Shellfish: A Focus on Africa

Enteric viruses are a diverse group of human pathogens which are primarily transmitted by the faecal–oral route and are a major cause of non-bacterial diarrhoeal disease in both developed and developing countries. Because they are shed in high numbers by infected individuals and can persist for a long time in the environment, they pose a serious threat to human health globally. Enteric viruses end up in the environment mainly through discharge or leakage of raw or inadequately treated sewage into water sources such as springs, rivers, dams, or marine estuaries. Human exposure then follows when contaminated water is used for drinking, cooking, or recreation and, importantly, when filter-feeding bivalve shellfish are consumed. The human health hazard posed by enteric viruses is particularly serious in Africa where rapid urbanisation in a relatively short period of time has led to the expansion of informal settlements with poor sanitation and failing or non-existent wastewater treatment infrastructure, and where rural communities with limited or no access to municipal water are dependent on nearby open water sources for their subsistence. The role of sewage-contaminated water and bivalve shellfish as vehicles for transmission of enteric viruses is well documented but, to our knowledge, has not been comprehensively reviewed in the African context. Here we provide an overview of enteric viruses and then review the growing body of research where these viruses have been detected in association with sewage-contaminated water or food in several African countries. These studies highlight the need for more research into the prevalence, molecular epidemiology and circulation of these viruses in Africa, as well as for development and application of innovative wastewater treatment approaches to reduce environmental pollution and its impact on human health on the continent.

     

Rethinking the Significance of Reovirus in Water and Wastewater

The genus Orthoreovirus contains nonenveloped viruses with double-stranded gene segments encased in a double-layered icosahedral capsid shell. These features constitute major determinants of virion stability in the environment and virion resistance against physical and chemical agents. Reovirus (ReoV) is the general term most commonly used for all virus strains that infect humans and nonhuman animals. Several studies have demonstrated the frequent occurrence of ReoV in wastewaters and natural waters, including surface and ground waters from different geographical areas. Most of these studies have reported higher concentrations of ReoV than any other enteric virus analyzed. They are more commonly isolated in chlorine-disinfected wastewaters than other enteric viruses, and appear to survive longer in water. The ability of ReoV to form large aggregates, even with different types of enteric viruses (e.g., poliovirus) and their ability to undergo mechanisms of gene segment reassortment among different serotypes may also explain their greater stability. Different approaches have been applied for concentration of ReoV from water; however, the recovery efficiency of the filtration methods has not been fully evaluated. Recently, molecular methods for identification of ReoV strains and quantification of virus genome have been developed. Studies have shown that the overall detection sensitivity of ReoV RNA is enhanced through initial replication of infectious virions in cell culture. More studies are needed to specifically address unresolved issues about the fate and distribution of ReoV in the environment since this virus is not commonly included in virological investigations.     

环境水体中肠道病原细菌的定量PCR检测

采用通用引物,通过实时荧光定量PCR方法(QPCR),对西安市5处地表水体中肠道病原细菌的细胞密度进行4个月的连续检测,并将QPCR检测结果与滤膜法测得的大肠菌群CFU值进行比较分析.结果显示, QPCR法可信度为94%,最小检测值为每管未稀释DNA提取物含2.7个大肠杆菌细胞.5个水体(N=60)检测结果表明, QPCR检测结果是大肠菌群CPU的2.2～5倍.病原细菌的几何平均值范围, QPCR法在25～67 000 CCE/100 mL之间, MF法大肠菌群为3～45 000 CFU/100 mL之间.2种方法的离散和回归分析表明, QPCR检测结果与大肠菌群CFU显著正相关,秩相关系数为r=0.983.     

入境船舶压载水潜在致病菌组成及影响因素研究

船舶压载水直接促进了地理性隔离海域之间的水体交换,可作为潜在致病菌迁移扩散的媒介。本文以采集到的25艘入境船舶的压载水样品为研究对象,采用高通量测序技术对潜在致病菌群落组成和影响因素进行了初步解析。结果表明:变形菌（Proteobacteria）、拟杆菌（Bacteroidetes）和放线菌（Actinobacteria）为压载水中的优势菌门。船舶压载水中共检出36个潜在致病菌属和19个潜在致病菌种。痤疮丙酸杆菌（Cutibacterium acnes）、大肠埃希氏杆菌（Escherichia coli）和表皮葡萄球菌（Staphylococcus epidermidis）为压载水中最普遍的潜在致病菌,检出率超过50%。置换和未置换压载水中潜在致病菌的种类数量和丰度并无显著差异。此外,压载水中的潜在致病菌与磷酸盐及大肠埃希氏杆菌呈显著正相关性,表明其可能与近岸的人类活动有关。综上所述,相关部门应当对船舶压载水加强管理,以降低潜在致病菌的入侵扩散风险。     

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.     

Human Enteric Viruses in Groundwater

Waterborne outbreaks of enteric viruses are a major public health concern. The present study has been carried out to assess the presence of enteric viruses responsible for human acute gastroenteritis (AGE) in groundwater intended for drinking and produce washing. In total, 62 samples from groundwater for drinking and produce washing collected from Dec 2007 to Dec 2008 in Seoul were tested for enteric viruses using conventional RT–PCR, ELISA, and real-time RT–PCR. Our results showed that enteric viruses were detected in 7 (8.8%) groundwater samples. Rotaviruses were detected in 3 (4.8%) of the samples by ELISA; human adenoviruses were detected in 2 (3.2%) of the samples by ELISA; and nested RT–PCR detected noroviruses in 2 (3.2%) of the samples. In one of the groundwater sample, the norovirus RNA was detected by conventional RT–PCR which was confirmed positive by real-time RT–PCR. Additionally, real-time RT–PCR successfully detected norovirus RNA in five out of 62 water samples (8.1%). The data demonstrate that real-time RT–PCR will be useful as a rapid and sensitive method for detecting norovirus in water samples. Phylogenetic analysis revealed that the noroviruses detected in two of the groundwater samples belonged to GII-4. These studies can provide important information for the prevalence of enteric viruses in Korean groundwater.     

Solar Disinfection of Water for Inactivation of Enteric Viruses and its Enhancement by Riboflavin

Solar disinfection (SODIS) has been described as a cheap and effective method of treating contaminated water to inactivate pathogenic microorganisms. In this study, SODIS was assessed for its efficacy in inactivating three enteric viruses (coxsackievirus B3, coxsackievirus B5 and poliovirus), either on its own or in the presence of riboflavin as a disinfection enhancer. On its own, SODIS produced a reduction of virus infectivity of 4–6 log₁₀ in 6 h. In the presence of riboflavin, inactivation was more rapid in all viruses studied, and with coxsackievirus B5 and poliovirus an extra 1–2 log₁₀ increase in reduction of infectivity was observed after 6 h exposure. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances.     

The Detection Rate of Enteric Viruses and <Emphasis Type="Italic">Clostridium difficile</Emphasis> in a Waste Water Treatment Plant Effluent

Waste water treatment plant (WWTP) is considered as an important source of surface water contamination by enteric pathogens. In this study, we describe the occurrence of enteric viruses (group A rotaviruses, noroviruses, astroviruses, sapoviruses, hepatitis A virus, and hepatitis E virus) and Clostridium difficile in the effluent of a wastewater treatment plant during a 1-year period. Enteric viruses were simultaneously and efficiently concentrated in a single step using methacrylate monolithic chromatographic support. Rotaviruses, noroviruses (genogroup I and II), and sapoviruses were detected in all 12 concentrated samples, whereas astroviruses were not detected in August and September and hepatitis A and E viruses were not detected at all. Clostridium difficile was detected in all samples and altogether 121 strains were isolated and grouped into 32 different ribotypes of which 014/020 and 010 were most prevalent. Pathogens detected in WWTP effluent partially reflect the epidemiological situation of enteric viruses and C. difficile in human population and open the discussion on implementation of possible techniques for virus and bacteria removal from WWTP effluent prior to release into the surface water system.     

浅谈我国海洋环境中人类肠病毒的污染监管

介水传染病是当今世界主要环境问题之一,其中人类肠病毒通过污染水体导致人类感染的胃肠道等疾病是主要传染病之一。本文以人类肠病毒经过污水处理厂排放为起点,分析其进入海洋环境后通过海水或海产品等途径给人体健康带来的风险;综述了国内外关于海洋环境中病原微生物的监测与评价技术体系的最新进展,指出目前我国海洋环境微生物监测指标和评价方法还不能满足指示人类肠病毒污染的问题。基于此,本文提出为更好地保护公众健康,我国迫切需要制定切实可行的海洋环境中人类肠病毒的风险监管措施,包括构建科学的海洋环境中人类肠病毒监测技术体系,构建全国相关的监测网络体系,构建海洋环境中人类肠病毒风险评价和预警体系,制定加强我国海洋环境中人类肠病毒的污染监管办法等。     

Development and Evaluation of a Multiplexed Real-Time TaqMan RT-PCR Assay with a Sample Process Control for Detection of F-specific RNA Coliphage Genogroups I and IV

There are increasing concerns of zoonotic transmission of some animal enteric viruses, such as calicivirus, hepatitis E virus, and rotavirus, which are closely related to human pathogenic strains. Most enteric viruses are detected by molecular techniques because they cannot be cultured. Surrogates such as F-RNA coliphages are cultivable but few molecular methods exist. Individual real-time TaqMan RT-PCR assays for the replicase gene of F-RNA coliphage genogroups I and IV were developed and multiplexed with a real-time TaqMan RT-PCR assay for feline calicivirus as a sample process control for the simultaneous detection and enumeration of genogroup I and IV F-RNA coliphages. Genogroup IV were successfully detected with the multiplexed assay in 80% of fecal samples that contained F-RNA coliphage levels ≥3.2 log plaque forming units (pfu). F-RNA coliphage were at or below the limit of detection in most fecal samples when levels were ≤4 log pfu/g.     

Detection of Viruses in Coastal Seawater Using <Emphasis Type="Italic">Mytilus Galloprovincialis</Emphasis> as an Accumulation Matrix

The purpose of this study was to demonstrate that shellfish can be used to detect enteric viruses in marine waters where they are present at very low concentrations. Aqua-cultured mussels were placed in the sea just off the mouth of a drainage channel affected by human and animal faecal contamination. Samples were taken from the channel, the sea and the mussels at intervals over two 4-week periods. The samples were tested to verify the presence of both rotaviruses and E. coli. Rotaviruses were detected by Real Time-PCR, typed by multiplex PCR and subsequently sequenced. E. coli was enumerated in water matrices by a filtering method and in mussels by the MPN method. The presence of E. coli in the examined matrices demonstrates contamination of faecal origin throughout the studied environments. Rotaviruses were recorded in channel waters, but not in sea water. In both experiments, rotaviruses were detected in mussels 21 and 28 days after being placed in the sea water off the channel mouth. The use of mussels thus enabled the detection of rotaviruses in waters where the high dilution rendered direct investigation impossible. This study indicates that mussels can be used in marine virological surveillance programs.     

Identification of Enteric Viruses in Foods from Mexico City

Foodborne viruses are a common and, probably, the most under-recognized cause of outbreaks of gastroenteritis. Among the main foods involved in the transmission of human enteric viruses are mollusks, and fruits and vegetables irrigated with wastewater and/or washed with non-potable water or contaminated by contact with surfaces or hands of the infected personnel during its preparation. In this study, 134 food samples were analyzed for the detection of Norovirus, Rotavirus, and Hepatitis A virus (HAV) by amplification of conserved regions of these viruses. From the 134 analyzed samples, 14 were positive for HAV, 6 for Norovirus, and 11 for Rotavirus. This is the first report in Mexico where emphasis is given to the presence of HAV and Norovirus on perishable foods and food from fisheries, as well as Rotavirus on frozen vegetables, confirming the role of vegetables and bivalve mollusks as transmitting vehicles of enteric viruses.     

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.     

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.     

病原微生物基准对再生水检测的指导意义

精确高效地定量检测再生水中病原微生物含量对于保障水资源安全循环利用有重要意义.基于微生物定量风险评价(QMRA)方法,以年感染风险10-4作为公众最大可容忍风险,计算了我国再生水中4种常见病原微生物在4种主要使用用途下的基准浓度.然后,讨论了培养法和分子生物学法2种检测方法中对取样体积的科学要求,提出了基于水质健康基准...