Human enteric viruses–potential indicators for enhanced monitoring of recreational water quality

Recreational waters contaminated with human fecal pollution are a public health concern, and ensuring the safety of recreational waters for public use is a priority of both the Environmental Protection Agency (EPA) and the Centers for Disease Control and Prevention (CDC). Current recreational water standards rely on fecal indicator bacteria (FIB) levels as indicators of human disease risk. However present evidence indicates that levels of FIB do not always correspond to the presence of other potentially harmful organisms, such as viruses. Thus, enteric viruses are currently tested as water quality indicators, but have yet to be successfully implemented in routine monitoring of water quality. This study utilized enteric viruses as possible alternative indicators of water quality to examine 18 different fresh and offshore recreational waters on O‘ahu, Hawai‘i, by using newly established laboratory techniques including highly optimized PCR, real time PCR, and viral infectivity assays. All sample sites were detected positive for human enteric viruses by PCR including enterovirus, norovirus genogroups I and II, and male specific FRNA coliphage. A six time-point seasonal study of enteric virus presence indicated significant variation in virus detection between the rainy and dry seasons. Quantitative PCR detected the presence of norovirus genogroup II at levels at which disease risk may occur, and there was no correlation found between enteric virus presence and FIB counts. Under the present laboratory conditions, no infectious viruses were detected from the samples PCR-positive for enteric viruses. These data emphasize both the need for additional indicators for improved monitoring of water quality, and the feasibility of using enteric viruses as these indicators. Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s12250-015-3644-x and is accessible for authorized users.     

Occurrence of adenovirus and other enteric viruses in limited-contact freshwater recreational areas and bathing waters

Aims: The goal of this study was to characterize enteric virus concentrations and their infectivity in a variety of limited‐contact recreation and bathing waters, including Great Lakes beaches, inland lakes, rivers, and an effluent‐dominated urban waterway. Additionally, we evaluated associations between point sources of human faecal pollution and enterovirus and adenovirus presence and concentrations. Methods and Results: Quantitative polymerase chain reaction (qPCR) and two cell culture lines were used to identify and quantify viruses in water samples. The presence of human adenoviruses F was strongly associated with effluent‐dominated waters (odds ratio 6·1, confidence interval 2·3, 15·7), as was adenovirus concentration; though, neither enterovirus presence nor concentration was associated with an effluent source. Samples with high concentrations of qPCR targets all tested positive by cell culture on both cell lines, although qPCR target concentrations were not correlated with culture values. Conclusions: Adenovirus was strongly associated with point sources of human faecal pollution while enterovirus was not, indicating that adenovirus measured by qPCR is a better target than enterovirus for identifying wastewater discharges in recreational freshwaters. Significance and Impact of the Study: The development of monitoring for enteric human viral pathogens at recreational waters should include adenovirus testing. Further research is needed to interpret the results of qPCR testing in relationship to the presence of infectious viruses using cell culture.     

Prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments

Aims: To study the virological quality of surface water from highly urbanized tropical water catchment areas and to determine predominant enteric viral genotypes in surface water. Methods and Results: A wide range of human pathogenic viruses in urban surface waters was screened by nested PCR assays after concentration by ultrafiltration. Among the 84 water samples collected, at least one virus was detected in 70 (83·3%) of these samples. Noroviruses were determined to be the most prevalent enteric viruses detected in urban surface water samples, followed by astroviruses, enteroviruses, adenoviruses and hepatitis A viruses. The molecular characterization of environmental viral isolates suggested co‐circulation of multiple genotypes of both noroviruses GI and GII, astroviruses and enteroviruses in urban surface waters. Conclusions: Human enteric viruses with great genetic diversity were detected in surface waters, indicating the presence of human origin of faecal contamination in highly urbanized water catchment areas. Significance and Impact of the Study: The present study identifies and characterizes potential viral hazards of source waters for drinking water supply and recreational activities. This will enable scientific decisions to be made regarding the selection and prioritization of human pathogenic viruses to be included in the future risk assessment and treatment evaluation for water and wastewater.     

Human Adenoviruses and Coliphages in Urban Runoff-Impacted Coastal Waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

PCR detection of pathogenic viruses in southern California urban rivers

AIMS: To investigate human viral contamination in urban rivers and its impact on coastal waters of southern California, USA. METHODS AND RESULTS: Three types of human viruses (adeno, entero and hepatitis A) were detected using nested- and RT-PCR from 11 rivers and creeks. Faecal indicator bacteria as well as somatic and F-specific coliphage were also tested. Approximately 50% of the sites were positive for human adenoviruses. However, there was no clear relationship between detection of human viruses and the concentration of indicator bacteria and coliphage. Both faecal indicator bacteria and human viral input at beaches near river mouths were associated with storm events. The first storm of the wet season seemed to have the greatest impact on the quality of coastal water than following storm events. CONCLUSIONS: This study provides the first direct evidence that human viruses are prevalent in southern California urban rivers. Urban run-off impacts coastal water quality most significantly during the storm season. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health during water recreational activities, it is necessary to develop effective strategies to manage urban run-off during storm events.     

Interference between enterovirus and reovirus as a limiting factor in environmental virus detection

AIMS: Faecal material from raw sewage or other sources lacking effective treatment sometimes contaminates water for human consumption. The relevant Italian regulations therefore call for testing drinking and recreational water for the presence of enterovirus. METHODS AND RESULTS: Traditional methods of analysis are based on revealing the typical cytopathic effects of enterovirus on cell cultures. However, the presence in environmental samples of different types of virus may cause interference phenomena that mask such cytopathic effects. The paper reports on an experimental test of this interference hypothesis. Buffalo Green Monkey cell cultures were co-infected via mixed suspensions of the polio type 3 virus and reovirus type 1. Cytopathic effects were then sought and the presence of enterovirus tested for via RT-PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that the normally high sensitivity of tests for the detection of enterovirus in samples is considerably decreased by the simultaneous presence of reovirus.     

Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Donor variability in HIV binding to peripheral blood mononuclear cells

Background

Currently applied indicator organism systems, such as coliforms, are not fully protective of public health from enteric viruses in water sources. Waterborne disease outbreaks have occurred in systems that tested negative for coliforms, and positive coliform results do not necessarily correlate with viral risk. It is widely recognized that bacterial indicators do not co-occur exclusively with infectious viruses, nor do they respond in the same manner to environmental or engineered stressors. Thus, a more appropriate indicator of health risks from infectious enteric viruses is needed.

Presentation of the hypothesis

Torque teno virus is a small, non-enveloped DNA virus that likely exhibits similar transport characteristics to pathogenic enteric viruses. Torque teno virus is unique among enteric viral pathogens in that it appears to be ubiquitous in humans, elicits seemingly innocuous infections, and does not exhibit seasonal fluctuations or epidemic spikes. Torque teno virus is transmitted primarily via the fecal-oral route and can be assayed using rapid molecular techniques. We hypothesize that Torque teno virus is a more appropriate indicator of viral pathogens in drinking waters than currently used indicator systems based solely on bacteria.

Testing the hypothesis

To test the hypothesis, a multi-phased research approach is needed. First, a reliable Torque teno virus assay must be developed. A rapid, sensitive, and specific PCR method using established nested primer sets would be most appropriate for routine monitoring of waters. Because PCR detects both infectious and inactivated virus, an in vitro method to assess infectivity also is needed. The density and occurrence of Torque teno virus in feces, wastewater, and source waters must be established to define spatial and temporal stability of this potential indicator. Finally, Torque teno virus behavior through drinking water treatment plants must be determined with co-assessment of traditional indicators and enteric viral pathogens to assess whether correlations exist.

Implications of the hypothesis

If substantiated, Torque teno virus could provide a completely new, reliable, and efficient indicator system for viral pathogen risk. This indicator would have broad application to drinking water utilities, watershed managers, and protection agencies and would provide a better means to assess viral risk and protect public health.     

Molecular Detection and Characterization of Gastroenteritis Viruses Occurring Naturally in the Stream Waters of Manaus, Central Amazônia, Brazil

To assess the presence of the four main viruses responsible for human acute gastroenteritis in a hydrographic network impacted by a disordered urbanization process, a 1-year study was performed involving water sample collection from streams in the hydrographic basin surrounding the city of Manaus, Amazonas, Brazil. Thirteen surface water sample collection sites, including different areas of human settlement characterized as urban, rural, and primary forest, located in the Tarumã-Açu, São Raimundo, Educandos, and Puraquequara microbasins, were defined with a global positioning system. At least one virus was detected in 59.6% (31/52) of the water samples analyzed, and rotavirus was the most frequent (44.2%), followed by human adenovirus (30.8%), human astrovirus (15.4%), and norovirus (5.8%). The viral contamination observed mainly in the urban streams reflected the presence of a local high-density population and indicated the gastroenteritis burden from pathogenic viruses in the water, principally due to recreational activities such as bathing. The presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.     

Hepatitis E virus‐based evaluation of a virion concentration method and detection of enteric viruses in environmental samples by multiplex nested RT‐PCR

Aims: The prevalence of enteric viruses in drinking and river water samples collected from Pune, India was assessed. During an outbreak of HEV in a small town near pune, water samples were screened for enteric viruses. Methods and Results: The water samples were subjected to adsorption–elution‐based virus concentration protocol followed by multiplex nested PCR. Among 64 Mutha river samples, 49 (76·56%) were positive for Hepatitis A Virus, 36 (56·25%) were positive for Rotavirus, 33 (51·56%) were positive for Enterovirus and 16 (25%) were positive for Hepatitis E Virus RNA. Only enterovirus RNA was detected in 2/662 (0·3%) drinking water samples, and the samples from the city’s water reservoir tested negative for all four viruses. HEV RNA was detected in three out of four river water samples during HEV outbreak and partial sequences from patients and water sample were identical. Conclusions: The study suggests absence of enteric viruses both in the source and in the purified water samples from Pune city, not allowing evaluation of the purification system and documents high prevalence of enteric viruses in river water, posing threat to the community. Significance and Impact of the Study: The rapid, sensitive and relatively inexpensive protocol developed for virological evaluation of water seems extremely useful and should be adapted for evaluating viral contamination of water for human consumption. This will lead to development of adequate control measures thereby reducing disease burden because of enteric viruses.     

GIV noroviruses and other enteric viruses in bivalves: a preliminary study

We evaluated the presence of the enteric viruses: norovirus, adenovirus, enterovirus, astrovirus, hepatitis A virus, and hepatitis E virus in bivalves using nested PCR methods and cell culture assays. Noroviruses GII.4 and GIV.1, adenoviruses types 1 and 2, hepatitis A, and echovirus type 7 were detected in the shellfish tested, which were often co-infected. This is the first study to detect such a high level of viral contamination in Italian mussels (up to four different viral groups in a single sample), and the first to document the presence of GIV NoV in shellfish.     

Non detection of enterovirus in the bivalve Anadara tuberculosa (Bivalvia:Arcidae) caused by chemical contamination in the Pacific of Costa Rica

Anadara tuberculosa is one of the most abundant mollusks of commercial importance in Costa Rica. Its habitat water is a potential source of fecal and chemical contamination to humans. We wanted to asses enterovirus, mainly poliovirus and hepatitis A virus and chemicals such as sulphates and nitrates in meat and body fluids. Thirteen samples were taken from four sites in Nicoya Gulf, three sites in the Sierpe-Térraba mangrove (Pacific of Costa Rica) and from five fish markets in San José, the capital of Costa Rica. Samples were tested for 1) fecal coliforms (Most Probable Number/100 ml), 2) isolation of enterovirus in cell culture (Hep-2, FrhK-4), 3) cell cytotoxicity in Vero cells and 4) the ability to inactivate 10 ID50% of poliovirus in cell culture. The Most Probable Number/100 ml in surrounding water was higher than the accepted standard for recreational waters, although the number of fecal coliforms in meats and body fluids was lower than in the external water. No cytopathogenic agents were isolated, but we found nitrate and sulphate concentrations that exceeded maxima for human consumption and recreation. The intrinsic cytotoxicity of the samples was at a 1/8 dilution, but some samples were cytotoxic at dilutions of 1/128. Body fluids were more cytotoxic than meats, but a positive correlation between cytotoxicity and chemical contamination was not determined: apparently other pollutants not identified in this study were responsible. Fluid and meat capacity to inactivate 10 ID50% of poliovirus in cell culture was demonstrated. Samples that were toxic for cell cultures also showed a higher percentage of poliovirus inactivation. Monitoring chemical pollution in these waters is highly recommended.