Enteric Viruses In Groundwater Research Articles

Wastewater contaminants in a fractured bedrock aquifer and their potential use as enteric virus indicators.

Domestic wastewater is a source of persistent organic pollutants and pathogens to the aquatic environment, including groundwater aquifers. Wastewater contaminants include a variety of personal care products, pharmaceuticals, endocrine disrupters, bacteria, and viruses. Groundwater from 22 wells completed in a semi-confined to confined, fractured Silurian dolostone aquifer in southern Wellington County, Ontario, Canada, was analyzed for 14 organic wastewater contaminants (4 artificial sweeteners, 10 pharmaceuticals) as well as E. coli, total coliforms, and 6 human enteric viruses. Enteric viruses were detected in 8.6% of 116 samples, and at least one organic wastewater contaminant was detected in 82% of the wells (in order of decreasing detection frequency: acesulfame, ibuprofen, sulfamethoxazole, triclosan, carbamazepine, and saccharin). Virus indicator metrics [positive and negative predictive values (PPV, NPV), sensitivity, specificity] were calculated at the sample and well level for the organic wastewater compounds, E. coli, and total coliforms. Fecal bacteria were not good predictors of virus presence (PPV = 0%-8%). Of the potential chemical indicators, triclosan performed the best at the sample level (PPV = 50%, NPV = 100%), and ibuprofen performed the best at the well level (PPV = 60%, NPV = 67%); however, no samples had triclosan or ibuprofen concentrations above their practical quantification limits. Therefore, none of the compounds performed sufficiently well to be considered reliable for assessing the potential threat of enteric viruses in wastewater-impacted groundwater in this bedrock aquifer. Future studies need to evaluate the indicator potential of persistent organic wastewater contaminants in different types of aquifers, especially in fractured rock where heterogeneity is strong.IMPORTANCEAssessing the potential risk that human enteric viruses pose in groundwater aquifers used for potable water supply is complicated by several factors, including: (i) labor-intensive methods for the isolation and quantification of viruses in groundwater, (ii) the temporal variability of these viruses in domestic wastewater, and (iii) their potentially rapid transport in the subsurface, especially in fractured rock aquifers. Therefore, aquifer risk assessment would benefit from the identification of suitable proxy indicators of enteric viruses that are easier to analyze and less variable in wastewater sources. Traditional fecal indicators (e.g., E. coli and coliforms) are generally poor indicators of enteric viruses in groundwater. While many studies have examined the use of pharmaceutical and personal care products as tracers of domestic wastewater and fecal pollution in the environment, there is a paucity of data on the potential use of these chemical tracers as enteric virus indicators, especially in groundwater.

The prevalence and levels of enteric viruses in groundwater of private wells in rural Alberta, Canada

The prevalence and levels of enteric viruses in untreated groundwater of private wells used for drinking and/or agricultural practices in rural Alberta were studied using the qPCR panel assay, integrated cell culture with qPCR and cell culture in the volume of 500 liters per sample through serial sampling. Seven viruses were assessed including adenovirus, rotavirus, norovirus, astrovirus, sapovirus, reovirus and JC virus. Five viruses were detected with an overall positive detection rate of 6.33 % (45 of 711 samples). The most frequently detected virus was adenovirus (48.9%, 22/45) followed by rotavirus (44.4%, 20/45), reovirus (20%, 9/45), JC virus (6.7%, 3/45) and norovirus (6.7%, 3/45). There was no significant difference in the positive detection rates, ranging from 1.1% to 3.4% by various well settings used for broiler farms, cow/calf farms, feedlots and rural acreages. Effects of well characteristics (aquifer type, well depth, static level of water, well seal) and well completion lithology on potential viral contamination of groundwater of private wells were also analyzed upon available data. The findings demonstrate that occurrence of enteric viruses is low and viral contamination is sporadic in groundwater of private wells in rural Alberta. Conventional fecal bacterial indicators (coliform and/or E. coli) were not a representative marker for viral contamination in groundwater wells in rural Alberta.

Confirming the need for virus disinfection in municipal subsurface drinking water supplies

Enteric viruses pose the greatest acute human health risks associated with subsurface drinking water supplies, yet quantitative risk assessment tools have rarely been used to develop health-based targets for virus treatment in drinking water sourced from these supplies. Such efforts have previously been hampered by a lack of consensus concerning a suitable viral reference pathogen and dose-response model as well as difficulties in quantifying pathogenic viruses in water. A reverse quantitative microbial risk assessment (QMRA) framework and quantitative polymerase chain reaction data for norovirus genogroup I in subsurface drinking water supplies were used herein to evaluate treatment needs for such water supplies. Norovirus was not detected in over 90% of samples, which emphasizes the need to consider the spatially and/or temporally intermittent patterns of enteric pathogen contamination in subsurface water supplies. Collectively, this analysis reinforces existing recommendations that a minimum 4-log treatment goal is needed for enteric viruses in groundwater in absence of well-specific monitoring information. This result is sensitive to the virus dose-response model used as there is approximately a 3-log discrepancy among virus dose-response models in the existing literature. This emphasizes the need to address the uncertainties and lack of consensus related to various QMRA modelling approaches and the analytical limitations that preclude more accurate description of virus risks.

Applying Quantitative Molecular Tools for Virus Transport Studies: Opportunities and Challenges.

Bacteriophages have been used in soil column studies for the last several decades as surrogates to study the fate and transport behavior of enteric viruses in groundwater. However, recent studies have shown that the transport behavior of bacteriophages and enteric viruses in porous media can be very different. The next generation of virus transport science must therefore provide more data on mobility of enteric viruses and the relationship between transport behaviors of enteric viruses and bacteriophages. To achieve this new paradigm, labor intensity devoted to enteric virus quantification method must be reduced. Recent studies applied quantitative polymerase chain reaction (qPCR) to column filtration experiments to study the transport behavior of human adenovirus (HAdV) in porous media under a variety of conditions. A similar approach can be used to study the transport of other enteric viruses such as norovirus. Analyzing the column samples with both qPCR and culture assays and applying multiplex qPCR to study cotransport behavior of more than one virus will provide information to under-explored areas in virus transport science. Both nucleic acid extraction kits and one-step lysis protocols have been used in these column studies to extract viral nucleic acid for qPCR quantification. The pros and cons of both methods are compared herein and solutions for overcoming problems are suggested. As better understanding of the transport behavior of enteric viruses is clearly needed, we strongly advocate for application of rapid molecular tools in future studies as well as optimization of protocols to overcome their current limitations.

Occurrence, Survival, and Persistence of Human Adenoviruses and F-Specific RNA Phages in Raw Groundwater

Detection of specific genetic markers can rapidly identify the presence of enteric viruses in groundwater. However, comparison of stability characteristics between genetic and infectivity markers is necessary to better interpret molecular data. Human adenovirus serotype 2 (HAdV2), in conjunction with MS2 phages or GA phages, was spiked into raw groundwater microcosms. Viral stability was periodically assessed by both infectivity and real-time PCR methods. The results of this yearlong study suggest that adenoviruses have the most stable persistence profile and an ability to survive for a long time in groundwater. According to a linear regression model, infectivity reductions of HAdV2 ranged from 0.0076 log(10)/day (4°C) to 0.0279 log(10)/day (20°C) and were significantly lower than those observed for phages. No adenoviral genome degradation was observed at 4°C, and the reduction was estimated at 0.0036 log(10)/day at 20°C. Occurrence study showed that DNA of human adenoviruses could be observed in groundwater from a confined aquifer (7 of the 60 samples were positive by real-time PCR), while no fecal indicators were detected. In agreement with the persistence of genetic markers, the presence of adenoviral DNA in groundwater may be misleading in term of health risk, especially in the absence of information on the infective status.

Human enteric viruses in groundwater indicate offshore transport of human sewage to coral reefs of the Upper Florida Keys

To address the issue of human sewage reaching corals along the main reef of the Florida Keys, samples were collected from surface water, groundwater and coral [surface mucopolysaccharide layers (SML)] along a 10 km transect near Key Largo, FL. Samples were collected semi-annually between July 2003 and September 2005 and processed for faecal indicator bacteria (faecal coliform bacteria, enterococci and Clostridium perfringens) and human-specific enteric viruses (enterovirus RNA and adenovirus DNA) by (RT)-nested polymerase chain reaction. Faecal indicator bacteria concentrations were generally higher nearshore and in the coral SML. Enteric viruses were evenly distributed across the transect stations. Adenoviruses were detected in 37 of 75 samples collected (49.3%) whereas enteroviruses were only found in 8 of 75 samples (10.7%). Both viruses were detected twice as frequently in coral compared with surface water or groundwater. Offshore, viruses were most likely to be found in groundwater, especially during the wet summer season. These data suggest that polluted groundwater may be moving to the outer reef environment in the Florida Keys.

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.

Enteric viruses in raw vegetables and groundwater used for irrigation in South Korea.

Raw vegetables irrigated with groundwater that may contain enteric viruses can be associated with food-borne viral disease outbreaks. In this study, we performed reverse transcription-PCR (RT-PCR) and cell culture-PCR to monitor the occurrence of enteric viruses in groundwater samples and in raw vegetables that were cultivated using that groundwater in South Korea. Samples were collected 10 times from three farms located in Gyeonggi Province, South Korea. RT-PCR and cell culture-PCR were performed to detect adenoviruses (AdVs), enteroviruses (EVs), noroviruses (NoVs), and rotaviruses, followed by sequence analyses of the detected strains. Of the 29 groundwater samples and the 30 vegetable samples, five (17%) and three (10%) were positive for enteric viruses, respectively. AdVs were the most frequently detected viruses in four groundwater and three vegetable samples. EVs and NoVs were detected in only one groundwater sample and one spinach sample, respectively. The occurrence of enteric viruses in groundwater and vegetable samples was not correlated with the water temperature and the levels of indicator bacteria, respectively. Phylogenetic analysis indicated that most of the detected AdVs were temporally distributed, irrespective of sample type. Our results indicate that raw vegetables may be contaminated with a broad range of enteric viruses, which may originate from virus-infected farmers and virus-contaminated irrigation water, and these vegetables may act as a potential vector of food-borne viral transmission.

Human Enteric Viruses in Groundwater from a Confined Bedrock Aquifer

Confined aquifers are overlain by low-permeability aquitards that are commonly assumed to protect underlying aquifers from microbial contaminants. However, empirical data on microbial contamination beneath aquitards is limited. This study determined the occurrence of human pathogenic viruses in well water from a deep sandstone aquifer confined by a regionally extensive shale aquitard. Three public water-supply wells were each sampled 10 times over 15 months. Samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for several virus groups and by cell culture for infectious enteroviruses. Seven of 30 samples were positive by RT-PCR for enteroviruses; one of these was positive for infectious echovirus 18. The virus-positive samples were collected from two wells cased through the aquitard, indicating the viruses were present in the confined aquifer. Samples from the same wells showed atmospheric tritium, indicating water recharged within the pastfew decades. Hydrogeologic conditions support rapid porous media transport of viruses through the upper sandstone aquifer to the top of the aquitard 61 m below ground surface. Natural fractures in the shale aquitard are one possible virus transport pathway through the aquitard; however, windows, cross-connecting well bores, or imperfect grout seals along well casings also may be involved. Deep confined aquifers can be more vulnerable to contamination by human viruses than commonly believed.

Virus occurrence in municipal groundwater sources in Quebec, Canada

A 1 year study was undertaken on groundwater that was a source of drinking water in the province of Quebec, Canada. Twelve municipal wells (raw water) were sampled monthly during a 1 year period, for a total of 160 samples. Using historic data, the 12 sites were categorized into 3 groups: group A (no known contamination), group B (sporadically contaminated by total coliforms), and group C (historic and continuous contamination by total coliforms and (or) fecal coliforms). Bacterial indicators (total coliform, Escherichia coli, enteroccoci), viral indicators (somatic and male-specific coliphages), total culturable human enteric viruses, and noroviruses were analyzed at every sampling site. Total coliforms were the best indicator of microbial degradation, and coliform bacteria were always present at the same time as human enteric viruses. Two samples contained human enteric viruses but no fecal pollution indicators (E. coli, enterococci, or coliphages), suggesting the limited value of these microorganisms in predicting the presence of human enteric viruses in groundwater. Our results underline the value of historic data in assessing the vulnerability of a well on the basis of raw water quality and in detecting degradation of the source. This project allowed us to characterize the microbiologic and virologic quality of groundwater used as municipal drinking water sources in Quebec.

Virus Transport Experiments in a Sandy Aquifer

The occurrence of human enteric viruses in ground water has been well documented in the literature. Bacteriophages such as MS-2 and PRD1 have properties similar to pathogenic human viruses suggesting that bacteriophages can be used as proxies for virus transport. The objective of this study is to investigate a “worst case scenario” for virus transport in a ground water aquifer, i.e., sand and gravel aquifer under a forced-gradient, by using bacteriophages. Field studies have been conducted to trace large-scale (34 m) and small-scale (10 m) virus transport under natural- and forced-gradients through a sand and gravel aquifer at a ground water research site at the Texas A&M University. Virus transport was monitored under natural and forced-gradient conditions using MS-2 and PRD-1 as virus tracers and bromide as a conservative tracer. Results indicate virus and bromide transport to down-gradient monitoring wells in both the large- and small-scale field tests. During the tests conducted, MS-2 transport appears to exhibit little longitudinal dispersion, showing a narrow peak at the well nest 34 m down-gradient in 13 days which is 20 days before the bromide breakthroughs, indicating that bacteriophage transport through the aquifer was mainly by advective flow. Differences in tracer transport can be attributed to the heterogeneity of the sand and gravel aquifer at the site, different injection methods, different sampling methods, and different tracer properties. Heterogeneity of the aquifer would cause virus transport through preferential flow paths.

Incidence of enteric viruses in groundwater from household wells in Wisconsin.

Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.

Bacteriophages as indicators of enteric viruses and public health risk in groundwaters.

Low concentrations of all types of bacteriophages in groundwater limit their power to predict the presence of enteric viruses. There is little concordance in the literature regarding phage detection methods, thus making comparisons extremely difficult. Different authors have used different hosts, phage concentration methods, and end-point determinations. Also, markedly different volumes of sample have been employed, varying from 1 litre to 400 l. Bacteriophage concentration methods are not reproducible. There has been marked variability among groups in the natural substrates used (for example, beef extract), the type of adsorbing filter used, centrifugation instruments and conditions, and the delivery of the concentrate to the host cells. There is no consensus on the best bacterial host strain. Currently, several are employed with each showing differential sensitivities and specificities. In particular, host stability must be considered. Host stability has two components: the ability of the host to continue to be receptive to the bacteriophage after continued sub-culture, and the lack of lysogenic or temperate bacteriophage in the host cell line which may be randomly and unpredictably activated. There is a lack of consistent recovery of bacteriophages from individual faecal specimens. In particular, only approximately 3% of individual humans carry the FRNA phages. While there is some evidence to indicate that the phages multiply in sewage, it is not clear how they do so since the host pili should not be produced at lower temperatures. These ecological factors need to be understood. Of all the phages thus far studied, Bacteroides fragilis HSP40 has the highest recovery rate from individual people. However, Bacteroides, being an anaerobe, is a difficult host for routine laboratory analysis. Methods for the enumeration of F(+)-specific phages and Bacteroides phages are complex, time-consuming, costly and not reproducible. Conversely, somatic coliphage methods are simpler and results can be available in 4-6 h. The occurrence of phages and viruses in groundwater depends on physicochemical characteristics that control their fate and transport in the groundwater/aquifer environment. There are very little actual data taken from the field that allow an understanding of the ecology and life span of phages in their natural environment. Moreover, the ability of phages to serve as a source of food for other microbes needs to be understood. There has been a lack of association of bacteriophage recovery with gastroenteritis outbreaks due to enteric viruses. There is only a small epidemiological database concerning the occurrence of enteric viruses in groundwater.

A Strategy for Detection of Viruses in Groundwater by PCR

We evaluated the use of the PCR for detection of enteric viruses in groundwater. To do this, we used an improved sample-processing technique and a large-volume amplification protocol. The objective of this study was to use advanced molecular techniques to develop a rapid and simple method which can be used by the water industry for detection of viral contamination in a variety of water samples. The strategy described here fulfills the water industry's need for a rapid, reliable, easily performed method for analyzing groundwater for virus contamination. Viruses were detected after concentration from at least 400 gallons (1,512 liters) of water by a filter adsorption and elution method, which resulted in a concentrate containing viruses. A total of 150 samples were analyzed by performing cell culture assays for enteroviruses and by performing reverse transcription PCR (RT-PCR) analyses for enteroviruses, hepatitis A virus, and rotavirus. Thirteen samples (8.7%) produced cellular cytopathic effects when the Buffalo green monkey cell line was used. When primers specific for enteroviruses were used in RT-PCR, 40 of 133 samples (30.1%) tested positive for the presence of enterovirus RNA. When hepatitis A virus-specific primers were used, 12 of 139 samples (8.6%) were considered positive for the presence of hepatitis A viral RNA. The RT-PCR analysis performed with rotavirus-specific primers identified 18 of 130 samples (13.8%) that were positive for rotavirus RNA sequences. Our sample-processing technique and large-volume PCR protocol (reaction volume, 300 microliter) resulted in sufficient removal or dilution of inhibitors so that more than 95% of the samples could be assayed by PCR. Because of its sensitivity for detecting viral nucleic acid sequences, PCR analysis should produce more positive results than cell culture analysis. Since either cell culture analysis or PCR can reveal only a "snapshot" of the quality of the groundwater being sampled, PCR seems to be a desirable rapid initial screening tool.

Replication of coliphage Q beta as affected by host cell number, nutrition, competition from insusceptible cells and non-FRNA coliphages.

F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, might serve as indicators of human enteric viruses in groundwater, provided these phages do not replicate in groundwater and replicate only to a limited extent in wastewater. Several factors that could influence phage replication in either of these environments were examined. Q beta did not replicate when host cells were fewer than 10(4) cfu ml-1. Replication selected for insusceptible cells when Q beta was incubated with its E. coli host. Loss of Q beta, presumably by inactivation, occurred in autoclaved on-site and urban wastewater, autoclaved groundwater, and in filter-sterilized spent LB broth. Replication did not occur in LB broth diluted with sterile saline to 1% of its original strength, which indicates that replication of FRNA coliphages cannot occur in such nutrient-poor environments as wastewater and groundwater. Competition from non-FRNA coliphages and insusceptible cells tended to reduce Q beta replication, as predicted, but phage yields unexpectedly increased significantly when Enterococcus faecalis was added to cultures.

Effects of temperature and host cell growth phase on replication of F-specific RNA coliphage Q beta.

Human enteric viruses have been found in groundwater in the absence of fecal coliforms. Because detection of human enteric viruses is costly, time-consuming, and lacking in sensitivity, F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, are being examined for suitability as indicators of human enteric viruses in groundwater. Temperatures and host cell growth conditions that constrain F-pilus expression will limit FRNA coliphage replication in groundwater and wastewater, as is desirable in an indicator. Below 25 degrees C F-pilus synthesis ceases; FRNA coliphage Qbeta did not replicate below this temperature in batch cultures. One-step replication studies indicated that the replicative cycle is prolonged and that fewer progeny are released as the temperature decreases. The decreases in phage replication observed in the one-step replication studies were a consequence of fewer cells infected as the temperature was lowered or as host cells entered stationary phase. The numbers of phage particles released from infected cells did not change. The minimum temperature for replication of Qbeta, 25 degrees C, is not maintained in wastewater and does not occur in Wisconsin groundwater. On the basis of temperature and host cell growth phase, we have concluded that extensive replication of FRNA coliphages does not occur in wastewater and groundwater in Wisconsin and areas with similar cool climates.

Coliphages as indicators of human enteric viruses in groundwater

Due to a lack of dependable routine methods for direct analysis of pathogenic microorganisms, tests for bacteria that are supposed to be of intestinal origin are used to indicate the presence and extent of fecal pollution in water. Current indicators are not accurate monitors of fecal pollution and do not adequately reflect the presence of human enteric viruses. Coliphages (viruses that infect the bacterium Escherichia coli which occurs in the feces of all warm‐blooded animals) have been proposed as indices of water quality. Coliphages are readily recovered from sewage from all parts of the world. In most cases, the persistence of coliphages in surface waters, groundwaters, and sewage is greater than that of human enteric viruses and enteric bacteria. Coliphages have a number of unique characteristics which permit selective analytical techniques. On the basis of these techniques, a system for predicting the presence of human enteric viruses in groundwater can be developed.

Viruses in groundwater beneath sewage irrigated cropland

The occurrence of enteric viruses in groundwater and soil was examined beneath three different sites where slow rate sewage irrigation of cropland is practiced. At all sites, the sewage was secondarily treated (aeration) before land application. Enteric viruses were isolated from wells beneath all sites. The lowest frequency of isolation was from wells beneath the one site practicing chlorination before application by spray irrigation. Viruses were detected in wells as deep as 27.5 m.