Performance Evaluation of Human-Specific Viral Markers and Application of Pepper Mild Mottle Virus and CrAssphage to Environmental Water Samples as Fecal Pollution Markers in the Kathmandu Valley,Nepal

Monitoring of environmental water is crucial to protecting humans and animals from possible health risks. Although numerous human-specific viral markers have been designed to track the presence of human fecal contamination in water, they lack adequate sensitivity and specificity in different geographical regions. We evaluated the performances of six human-specific viral markers [Aichi virus 1 (AiV-1), human adenoviruses (HAdVs), BK and JC polyomaviruses (BKPyVs and JCPyVs), pepper mild mottle virus (PMMoV), and crAssphage] using 122 fecal-source samples collected from humans and five animal hosts in the Kathmandu Valley, Nepal. PMMoV and crAssphage showed high sensitivity (90–100%) with concentrations of 4.5–9.1 and 6.2–7.0 log₁₀ copies/g wet feces (n = 10), respectively, whereas BKPyVs, JCPyVs, HAdVs, and AiV-1 showed poor performances with sensitivities of 30–40%. PMMoV and crAssphage were detected in 40–100% and 8–90%, respectively, of all types of animal fecal sources and showed no significantly different concentrations among most of the fecal sources (Kruskal–Wallis test, P > 0.05), suggesting their applicability as general fecal pollution markers. Furthermore, a total of 115 environmental water samples were tested for PMMoV and crAssphage to identify fecal pollution. PMMoV and crAssphage were successfully detected in 62% (71/115) and 73% (84/115) of water samples, respectively. The greater abundance and higher mean concentration of crAssphage (4.1 ± 0.9 log₁₀ copies/L) compared with PMMoV (3.3 ± 1.4 log₁₀ copies/L) indicated greater chance of detection of crAssphage in water samples, suggesting that crAssphage could be preferred to PMMoV as a marker of fecal pollution.

     

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 10⁶) > treated sewage (2.9 × 10⁵) > river waters (6.1 × 10²) > estuarine waters (4.8 × 10²) > bathing waters (8.5 × 10¹) > groundwater (5.9 × 10¹). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

     

Comprehensive Study on Enteric Viruses and Indicators in Surface Water in Kyoto,Japan, During 2014–2015 Season

Certain enteric viruses that are present in the water environment are potential risk factors of waterborne infections. To better understand the impact of viruses in water, both enteric viruses and their potential indicators should be comparatively investigated. In this study, occurrences of GI- and GII-noroviruses (NoVs), sapovirus (SaV), rotavirus (RoV), Aichi virus 1 (AiV-1), enterovirus (EV), and pepper mild mottle virus (PMMoV) were quantitatively determined in surface water samples in Japan. Additionally, the genotype distribution of GI- and GII-NoVs was determined using a next-generation amplicon sequencing. PMMoV was the most abundant virus regardless of season and location, indicating its usefulness as an indicator for the viral contamination of water. Other potential indicators, AiV and EV, were less abundant than GII-NoV. Viruses other than PMMoV showed seasonality, i.e., EV and other viruses (NoVs, SaV, RoV, and AiV-1) became prevalent during summer and winter, respectively. SaV showed a relatively high abundance at a location that was affected by untreated wastewater. Regarding NoV genotypes, GI.1, GI.2, GI.4, GI.5, GI.6, GII.3, GII.4, GII.6, and GII.17 were found from the surface water samples. GII.4 and GII.17 seemed to have contributed to the high abundance of GII-NoV in the samples. Interestingly, GII.17 strains became prevalent in the water samples before becoming prevalent among gastroenteritis patients in Japan. These findings provide further insights into the properties of viruses as contaminants in the water environment.     

Prevalence and Genetic Characterization of Aichivirus in Environmental Waters in Thailand

Aichivirus 1 (AiV-1) is an enteric virus that has been documented to be the causative agent of diarrhea in humans. It is transmitted by fecal–oral route, through person-to-person contact, consumption of contaminated food or water, or recreation of contaminated water. AiV-1 is highly prevalent in water samples and has been proposed as a potential indicator of fecal contamination in water reservoirs. This study aimed to investigate the prevalence and genetic diversity of AiV-1 in environmental water samples in Thailand. A total of 126 samples were collected monthly from November 2016 to July 2018 from various sources of environmental water including irrigation water, reservoir, river, and wastewater. The presence of AiV-1 was detected by RT-nested PCR of the 3CD region and further analyzed by phylogenetic analysis. The AiV-1 was detected in 28 out of 126 (22.2%) of tested samples. A high frequency of AiV-1 detection was in wastewater (52.4%). All 28 AiV-1 strains detected in this study belonged to the genotype B and were closely related to AiV strains detected previously in environmental waters and in humans worldwide. This study demonstrated, for the first time, the contamination of AiV-1 in various sources of water samples in Thailand and provided a better insight into the prevalence of AiV-1 in environmental waters and its potential risk of human health.

     

江苏省某市部分农村河道水体和污水处理厂主要工艺环节病毒污染状况调查

为研究江苏省某市农村河道水体和污水处理厂主要工艺环节病毒的污染状况,分析常见理化因素与病毒污染之间的相关性,对江苏省某市部分农村河道水体和污水处理厂主要工艺环节7个水样、1个污泥和2个沉积物样点进行为期9个月的病毒(包括诺如病毒G Ⅰ、诺如病毒G Ⅱ、轮状病毒、札如病毒、腺病毒、星状病毒、肠道病毒)污染状况监测采样(共90份样本),通过阴离子膜吸附-洗脱法进行富集,荧光定量RT-PCR检测法对病毒进行检测,分析病毒污染状况;同时,监测水温、pH、COD(化学耗氧量)、TP(总磷)、TN(总氮)、EC(电导率)和DO(溶解氧)等7个理化指标的变化情况,探讨这些常见理化因素与病毒污染之间的相关性. 结果表明:90份样本中,诺如病毒G Ⅱ检出率为36.67%,肠道病毒检出率为30%,其他病毒检出率均低于诺如病毒G Ⅱ和肠道病毒. 污水处理后出水中有诺如病毒G Ⅱ、肠道病毒和轮状病毒检出,相关性分析显示,水温与诺如病毒G Ⅱ检出率呈显著负相关(P < 0.05). 研究显示,江苏省某市农村河道水体和污水处理厂主要工艺环节存在病毒污染,诺如病毒G Ⅱ为优势毒株;目前的污水消毒处理工艺并不能完全去除病毒,人群在接触外环境水体时,有潜在病毒暴露风险.      

Persistence of Viruses by qPCR Downstream of Three Effluent-Dominated Rivers in the Western United States

This study was designed to determine the quantitative polymerase chain reaction (qPCR) signal persistence of viruses in three effluent-dominated streams. Samples were collected from the effluent outfall of three wastewater treatment plants in the Western United States and downstream at different locations. All samples were tested for the presence of pepper mild mottle virus (PMMoV), adenoviruses, norovirus GI and GII, Aichi virus, and enteroviruses using qPCR. PMMoV was detected most frequently in 54/57 (94.7%) samples, followed by adenoviruses which was detected in 21/57 (36.8%) samples. PMMoV was detected at all locations downstream and up to 32 km from the discharge point. This study demonstrated that the detection signal of PMMoV was able to persist in wastewater discharges to a greater degree than human enteric viruses in effluent-dominated rivers.     

Occurrence of <Emphasis Type="Italic">Pepper Mild Mottle Virus</Emphasis> (PMMoV) in Groundwater from a Karst Aquifer System in the Yucatan Peninsula,Mexico

The Yucatan Peninsula of Mexico hosts a karst aquifer system that is the only source of freshwater for the area; however, it is vulnerable to human-mediated contamination. Pepper mild mottle virus (PMMoV) is one of the most abundant RNA viruses associated with human feces, making it a viable indicator for tracking fecal pollution in aquatic environments, including groundwater. In this study, groundwater samples collected from a karst aquifer from fresh and brackish water locations were analyzed for fecal indicator bacteria, somatic and male F+ specific coliphages, and PMMoV during the rainy and dry seasons. Total coliform bacteria were detected at all sites, whereas Escherichia coli were found at relatively low levels <40 MPN/100 ml. The highest average concentrations of somatic and male F+ specific coliphages were 920 and 330 plaque forming units per 100 ml, respectively, detected in freshwater during the rainy season. PMMoV RNA was detected in 85% of the samples with gene sequences sharing 99–100% of nucleotide identity with PMMoV sequences available in GenBank. Quantification of PMMoV genome copies (GC) by quantitative real-time PCR indicated concentrations ranging from 1.7 × 10¹ to 1.0 × 10⁴ GC/L, with the highest number of GC detected during the rainy season. No significant correlation was observed between PMMoV occurrence by season or water type (p > 0.05). Physicochemical and indicator bacteria were not correlated with PMMoV concentrations. The abundance and prevalence of PMMoV in the karst aquifer may reflect its environmental persistence and its potential as a fecal indicator in this karst aquifer system.     

Impact of Drip Irrigation Method,Soil, and Virus Type on Tomato and Cucumber Contamination

The goal of this study was to better quantify the degree of viral contamination of tomato and cucumber in relationship to virus type, soil type, and irrigation method. Tomatoes and cucumbers were grown in ten-gallon (37.8 L) buckets filled with Pima clay loam or Brazito sandy loam soils. Plants were irrigated with secondary wastewater effluent using surface drip irrigation or subsurface drip irrigation. At specified time intervals irrigation water was seeded with bacteriophages MS-2 and P22, poliovirus type 1 (PV1), enteric adenovirus 40 (Ead 40), and hepatitis A virus. Surface drip irrigation always resulted in viral contamination of both the above and below ground parts of both crops. The roots showed the greatest level of contamination, followed by leaves and fruits. In contrast, with subsurface drip irrigation no viruses were detected in any of the above ground plant surfaces. It was found that under similar soil type and irrigation method, risk of crop contamination was similar for all of the viruses studied. It can be concluded that method of irrigation is the single most critical factor in the contamination trend of different parts of crop plants. Plant parts can be categorized into three groups (root, stem, and leaf/fruit) based on the risk of viral contamination from irrigation water.     

污水稳定塘菌-藻生态系统去除与灭活植物病毒TMV研究

了解污水稳定塘生态系统去除与灭活植物病毒的效率与机理具有重要的理论和实际意义.对模型植物病毒———烟草花叶病毒(tobaccomosaicvirus,TMV)的实验结果显示:稳定塘系统中的悬浮固体(SS)可在短时间内吸附TMV且达到一个饱和值.纯培养的枯草芽孢杆菌及小球藻对TMV的存活无明显不良影响,而光合细菌和稳定塘中的混合细菌群却对TMV具有灭活作用.当它们同藻类形成菌藻共生体系时,对TMV的灭活速率分别从对照的每天0 0404log10(枯斑数·mL-1)升高到0 0783log10(枯斑数·mL-1)和0 075log10(枯斑数·mL-1) 因此,在污水稳定塘生态系统中,由于病毒对悬浮固体的吸附以及菌藻共生体系中微生物的作用可有效地去除与灭活植物病毒,其中悬浮固体吸附病毒并非真正意义上的灭活,稳定塘中存在的细菌特别是光合细菌对植物病毒的灭活才是最主要的因素.     

Quantification and Genotyping of Aichi Virus 1 in Water Samples in the Kathmandu Valley,Nepal

Aichi virus 1 genomes were detected by quantitative PCR in groundwater from shallow dug (10/22) and tube wells (1/15), river water (14/14), and sewage (1/1), with the maximum concentration of 4.0 × 10⁹ copies/l. Nucleotide sequencing analysis demonstrated the prevalence of genotype B in the virus positive samples.     

太湖地区稻作系统不同水体硝态氮同位素特征及污染源

为辨别稻作系统不同水体硝态氮来源,选择太湖地区典型稻作区域,应用硝态氮δ15N同位素技术,结合水化学方法(如NO3-, NH4+, TP, Cl-, SO42-),研究水稻施肥之前(4~5月),施肥期(6月),及施肥之后(7~8月)地表水和地下水硝态氮来源.结果表明,地表水和地下水硝态氮含量普遍较高.在施肥期,各水体硝态氮中δ15N均较低,表明该时期农业化肥是水体硝态氮的主要来源.在施氮前期,池塘水δ15N较低,其可能原因是受雨水的影响;而地下水δ15N较高,可能是水体发生了强烈的反硝化.在施肥后期,池塘水δ15N较高可能受养殖废水影响;地下水δ15N较低,可能受农田渗漏水的影响.河水和灌溉水硝态氮δ15N在各时期波动不大,其中河水硝态氮主要来源是生活污水和动物粪肥,但灌溉水硝态氮主要来源于雨水.本研究提出新的Cl-浓度和NO3-/Cl-物质的量比区间以辨别太湖地区水体硝态氮来源.     

Neural stem cell-based in vitro bioassay for the assessment of neurotoxic potential of water samples

Intensive agriculture activities, industrialization and growing numbers of wastewater treatment plants along river banks collectively contribute to the elevated levels of neurotoxic pollutants in natural water reservoirs across Europe. We established an in vitro bioassay based upon neural stem cells isolated from the subventricular zone of the postnatal mouse to evaluate the neurotoxic potential of raw wastewater, treated sewage effluent, groundwater and drinking water. The toxic potential of water samples was evaluated employing viability, proliferation, differentiation and migration assays. We found that raw wastewater could reduce the viability and proliferation of neural stem cells, and decreased the neuronal and astrocyte differentiation, neuronal neurite growth, astrocyte growth and cell migration. Treated sewage water also showed inhibitory effects on cell proliferation and migration. Our results indicated that relatively high concentrations of nitrogenous substances, pesticides, mercuric compounds, bisphenol-A, and phthalates, along with some other pollutants in raw wastewater and treated sewage water, might be the reason for the neuroinhibitory effects of these water samples. Our model successfully predicted the neurotoxicity of water samples collected from different sources and also revealed that the incomplete removal of contaminants from wastewater can be problematic for the developing nervous system. The presented data also provides strong evidence that more effective treatments should be used to minimize the contamination of water before release into major water bodies which may be considered as water reservoirs for human usage in the future.     

病毒对地下水的入侵风险与防控对策浅析

病毒可能会成为人类未来的“头号公敌”,全球地下水病毒污染事件频繁发生,因此,病毒对地下水的影响和防控逐渐受到国际重视.为进一步了解地下水病毒入侵,通过文献检索调研,分析了病毒进入含水层的途径、在地下水中迁移的限制性因素、在多孔介质中的转化过程,总结了地下水病毒入侵防控相关方法、技术、政策,探讨了风险防范意识有待加强、地下水病毒科学研究存在短板、应急防控与预警能力不足等问题及挑战.参考国内外相关研究成果和实践经验,初步提出地下水病毒入侵风险防控对策建议:①源头排查风险,防患于未然.在地下水饮用水水源地保护区及补给区,排查防疫类污染源渗漏情况,经过化学或高温消毒后再使用农村地下水饮用水、生活用水.②加强地下水饮用水水源地病毒调查研究.研究病毒在土壤及地下水中的存活能力、迁移转化机制、环境行为特征、模拟预测技术、健康风险评价方法,制定相关规范、指南.③筹划应急防控对策.做好地下水应急监测预警、饮用水消毒处理、环境质量信息公开等相关准备工作,制定地下水饮用水水源地病毒应急防控预案.      

Detection and Molecular Characterization of Aichivirus 1 in Wastewater Samples from Uruguay

Aichivirus 1 (AiV-1) is an enteric virus with 30 nm in diameter, belonging to the genus Kobuvirus in the Picornaviridae family being a causative agent of gastroenteritis in humans. The transmission is via the fecal-oral route, through person to person contact, recreation in contaminated waters, or through the consumption of contaminated food or water. The aim of this study was to determine the frequency and the molecular characterization of AiV-1 in wastewater from Uruguay. Biweekly collections from March 2011 to February 2012 were performed in the cities of Bella Unión, Salto, Paysandú, and Fray Bentos, northwestern region of Uruguay. A total of 96 samples were collected; viruses were concentrated by ultracentrifugation, and AiV-1 was detected by using a nested PCR with primers directed to a conserved region (3CD junction) of the viral genome. A high frequency of AiV-1 (n = 54) was observed at all the cities analyzed mainly in the colder months of the year. AiV-1 was not evidenced as an appropriate viral fecal indicator since when compared with other previously detected enteric viruses, no correlation was observed. All 13 characterized AiV-1 belonged to the genotype B after the phylogenetic analysis performed with the sequences obtained from the first round PCR amplicon. This study demonstrates that AiV-1 is a frequently detected enteric viruses present in wastewater and excreted by infected persons in the northwestern region of Uruguay.     

多环芳烃在不同灌区土壤剖面的分布特征研究

文章在不同类型灌溉用水的三个典型灌区进行土壤精细剖面钻探采样分析,研究16种优控多环芳烃在土壤剖面的分布特征和不同灌溉条件对土壤质量的影响。结果表明:表土是多环芳烃的主要累积层位,污灌区、再生水灌区、清灌区表土的多环芳烃总量分别为726、200、34μg/kg,说明长期进行污水和再生水灌溉均会造成不同程度的土壤污染;受多环芳烃自身理化性质的影响,低环的多环芳烃容易向土壤剖面的深部迁移,在表土以下的层位占绝对优势分布,高环的多环芳烃迁移性很弱,基本只在表层有检出;通过对各灌区剖面的典型多环芳烃含量和土壤理化指标进行相关分析和回归分析,得出TOC是多环芳烃在土壤剖面垂向迁移的主要影响因素。     

Pre-harvest Viral Contamination of Crops Originating from Fecal Matter

Pre-harvest contamination of fresh produce and fruits is a possible route for viral transmission which should not be ignored. The contamination originates from viruses shed in human or animal fecal materials which eventually reach crops through many steps and hurdles, including spread of viruses into the agricultural environment through leakage of septic tanks/pipes or runoff from animal lagoons, virus survival during biosolids and manure treatment, virus survival and transport in soil and subsequent contamination of irrigation water, and virus transmission to crops through irrigation water, etc. Initially, large quantities of virus particles may be released from infected humans or animals, and then in the environment viruses are gradually inactivated under various natural conditions (e.g., temperature, water activity, microbial activities, etc.) and only a tiny portion of viruses may reach crops and cause the contamination. However, the fact that the infectious dose of some foodborne viruses is as low as 10–100 particles makes the pre-harvest contamination still a threat to human health. In the USA, the Environmental Protection Agency (USEPA) and United States Department of Agriculture (USDA) regulations and guidances on proper treatment and usage of manure and biosolids for agricultural purpose may largely reduce the release of viruses to the environment; however, pre-harvest viral contamination due to fecal matter is not completely avoidable. This review focuses on the current knowledge of pre-harvest viral contamination and describes the complex transmission process regarding the presence and survival of virus in soil and fecal material, the effect of different biosolids/manure treatment on virus inactivation, the transmission of virus from soil to water, the contamination of crops by irrigation water and survival of virus on crops.     

Assessment of the environmental impact of artificial effluent lagoon in Jiayuguan City of China

An artificial effluent lagoon for storing wastewater were excavated in Jiayuguan City since 1994. As a part of a demonstration project of Sino-Austmlia cooperation, an assessment of the environmental impact of the lagoon was carried out. The assessment was based on field and laboratory tests and predictive model. The main impacts from the lagoon site are likely to be on the groundwater system, and, to a lesser extent,on ambient air quality in the vicinity. Currently it is expected that groundwater is being pollutod with effluent from the effluent Ingcen. Air pollution(odor nuisance) is mainly caused by untreated effluent in the irrigation channel. The impact of high total dissolved salt(TDS) on groundwater is likely to be significant in the long run if the lagoon is continuously used. There is, consequently, no likelihood of contamination of surface water system, particularly of the city water supply system, from infiltration of effluent at the lagoon.     

南宁市中水回用问题与对策研究

中水水量稳定,处理达标后的中水可回用于工业冷却、农业灌溉、城市杂用及河流景观补水等方面。中水回用对缓解城市干旱缺水具有一定作用。以近年来南宁市遭遇的干旱为依据,阐述了中水回用的必要性。介绍了南宁市水资源及中水回用状况,分析中水回用存在问题,提出了南宁市中水回用对策。根据南宁市污水处理厂及潜在中水用户分布,结合南宁市各行业用水实际情况,南宁市中水宜就近回用作工业用水及生态环境用水,也可由罐车输送用于道路冲洗及城市绿化灌溉。     

Virological Quality of Irrigation Water in Leafy Green Vegetables and Berry Fruits Production Chains

This study condenses data acquired during investigations of the virological quality of irrigation water used in production of fresh produce. One hundred and eight samples of irrigation water were collected from five berry fruit farms in Finland (1), the Czech Republic (1), Serbia (2), and Poland (1), and sixty-one samples were collected from three leafy green vegetable farms in Poland, Serbia, and Greece. Samples were analyzed for index viruses of human or animal fecal contamination (human and porcine adenoviruses, and bovine polyoma viruses), and human pathogenic viruses (hepatitis A virus, hepatitis E virus, and noroviruses GI/GII). Both index and pathogenic viruses were found in irrigation water samples from the leafy green vegetables production chain. The data on the presence of index viruses indicated that the highest percentage of fecal contamination was of human origin (28.1 %, 18/64), followed by that of porcine (15.4 %, 6/39) and bovine (5.1 %, 2/39) origins. Hepatitis E virus (5 %, 1/20) and noroviruses GII (14.3 %, 4/28) were also detected. Samples from berry fruit production were also positive for both index and pathogenic viruses. The highest percentage of fecal contamination was of human origin (8.3 %, 9/108), followed by that of porcine, 4.5 % (4/89) and bovine, 1.1 % (1/89) origins. Norovirus GII (3.6 %, 2/56) was also detected. These data demonstrate that irrigation water used in primary production is an important vehicle of viral contamination for fresh produce, and thus is a critical control point which should be integrated into food safety management systems for viruses. The recommendations of Codex Alimentarius, as well as regulations on the use of water of appropriate quality for irrigation purposes, should be followed.     

Estimation of Contamination Sources of Human Enteroviruses in a Wastewater Treatment and Reclamation System by PCR–DGGE

A polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) method was employed to estimate the contamination sources of human enteroviruses and understand how their dominant strains vary in a wastewater treatment and reclamation system consisting of sewage collection, wastewater treatment with membrane bioreactor and open lakes for reclaimed water storage and reuse. After PCR–DGGE using a selected primer set targeting enteroviruses, phylogenetic analysis of acquired enterovirus gene sequences was performed. Enteroviruses identified from the septic tank were much more diverse than those from grey water and kitchen wastewater. Several unique types of enterovirus different from those in wastewater samples were dominant in a biological wastewater treatment unit. Membrane filtration followed by chlorination was proved effective for physically eliminating enteroviruses; however, secondary contamination likely occurred as the reclaimed water was stored in artificial lakes. Enterovirus 71 (EV71), a hand-foot-and-mouth disease (HFMD) viral pathogen, was detected mainly from the artificial lakes, implying that wastewater effluent was not the contamination source of EV71 and that there were unidentified non-point sources of the contamination with the HFMD viral pathogen in the reclaimed water stored in the artificial lakes. The PCR–DGGE targeting enteroviruses provided robust evidence about viral contamination sources in the wastewater treatment and reclamation system.