Levels Of Enteric Viruses Research Articles

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.

Detection of the human specific Bacteroides genetic marker provides evidence of widespread sewage contamination of stormwater in the urban environment

Human sewage contamination of surface waters is a major human health concern. We found urban stormwater systems that collect and convey runoff from impervious surfaces act as a conduit for sewage originating from breeches in sanitary sewer infrastructure. A total of 828 samples at 45 stormwater outfalls were collected over a four-year period and assessed by culture based methods, PCR, and quantitative PCR (qPCR) to test for traditional and alternative indicators of fecal pollution. All outfalls had the HF183 (human) Bacteroides genetic marker detected in at least one sample, suggesting sewage contamination is nearly ubiquitous in the urban environment. However, most outfalls were intermittently positive, ranging from detection in 11%–100% of the samples. Positive results did not correlate with seasonality, rainfall amounts, or days since previous rainfall. Approximately two-thirds of the outfalls had high (>5000 copy number, i.e. CN, per 100 ml) or moderate levels (1000–5000 CN per 100 ml) of the human Bacteroides genetic marker. Escherichia coli ( E. coli) and enterococci levels did not correlate to human Bacteroides. A total of 66% of all outfall samples had standard fecal indicator levels above 10,000 CFU per 100 ml. A tiered assessment using this benchmark to identify high priority sites would have failed to flag 35% of the samples that had evidence of sewage contamination. In addition, high fecal indicators would have flagged 33% of samples as priority that had low or no evidence of sewage. Enteric virus levels in one outfall with high levels of the human Bacteroides genetic marker were similar to untreated wastewater, which illustrates stormwater can serve as a pathway for pathogen contamination. The major source of fecal pollution at four of five river sites that receive stormwater discharge appeared to be from sewage sources rather than non-human sources based on the ratios of human Bacteroides to total Bacteroides spp. This study shows the feasibility and benefits of employing molecular methods to test for alternative indicators of fecal pollution to identify sewage sources and potential health risks and for prioritization of remediation efforts.

Quantification of Enteric Viruses, Pathogen Indicators, and Salmonella Bacteria in Class B Anaerobically Digested Biosolids by Culture and Molecular Methods

The most common class B biosolids in the United States are generated by mesophilic anaerobic digestion (MAD), and MAD biosolids have been used for land application. However, the pathogen levels in MAD biosolids are still unclear, especially with respect to enteric viruses. In this study, we determined the occurrence and the quantitative levels of enteric viruses and indicators in 12 MAD biosolid samples and of Salmonella enterica in 6 MAD biosolid samples. Three dewatered biosolid samples were also included in this study for purposes of comparison. Human adenoviruses (HAdV) had the highest gene levels and were detected more frequently than other enteric viruses. The gene levels of noroviruses (NV) reported were comparable to those of enteroviruses (EV) and human polyomaviruses (HPyV). The occurrence percentages of HAdV, HAdV species F, EV, NV GI, NV GII, and HPyV in MAD samples were 83, 83, 42, 50, 75, and 58%, respectively. No hepatitis A virus was detected. Infectious HAdV was detected more frequently than infectious EV, and all infectious HAdV were detected when samples were propagated in A549 cells. Based on most-probable-number (MPN) analysis, A549 cells were more susceptible to biosolid-associated viruses than BGM cells. All indicator levels in MAD biosolids were approximately 10(4) MPN or PFU per gram (dry), and the dewatered biosolids had significantly higher indicator levels than the MAD biosolids. Only two MAD samples tested positive for Salmonella enterica, where the concentration was below 1.0 MPN/4 g. This study provides a broad comparison of the prevalence of different enteric viruses in MAD biosolids and reports the first detection of noroviruses in class B biosolids. The observed high quantitative and infectivity levels of adenoviruses in MAD biosolids indicate that adenovirus is a good indicator for the evaluation of sludge treatment efficiency.

양전하 및 음전하 필터를 이용한 해수 중 Norovirus Surrogate의 회수

Recently coastal seawater has been contaminated by enteric viruses such as the norovirus via untreated groundwater globally. Accordingly, the consumption of molluscan shellfish from seawater that has been contaminated with fecal material has become an important issues. The levels of enteric viruses in seawater are low and recovery and concentration of the virus from large volumes of water is difficult. We compared the effectiveness of two representative method of concentrating virus using negatively and positively charged filters. The mean retention of seeded FCV by HAMF and NCCF was 48% and 78%, respectively. Overall, the recovery of NCCF was 43.3<TEX>$\pm$</TEX>11% better than that of HAMF. However, the eluate obtained by using beef extract solution in the NCCF procedure caused an inhibitory effect on the RT-PCR; therefore, it was necessary to employ a PCR inhibitor removal procedure. The HAMF eluate contained no PCR inhibitors, but HAMF was not an effective method of concentrating the virus from large volumes of natural seawater due to clogging.

Effects of freezing and storage temperature on MS2 viability

Monitoring human enteric virus levels in domestic wastewater effluent is crucial to protecting human health. Occasionally, during intensive sampling, wastewater samples must be stored for later viral analysis. Little data exist regarding how enteric viruses survive during storage at different temperatures in secondary treated wastewater. During a field-scale study assessing pathogen removal performance by various onsite treatment technologies, the MS2 bacteriophage, an indicator of enteric viruses, was inoculated into septic tank (STE), sand filter, peat filter and constructed wetland (CW) effluents to determine virus decay at various storage temperatures. Virus stored at temperatures ≥10 °C and at −20 °C decayed nearly twice as fast as those stored at 4 °C or −80 °C. Decreased water quality decreased viral decay rates at 4 °C and −80 °C, with slowest decay occurring in STE and the fastest in sterile PBS and low pH peat effluent. In CW effluent after 8 days, less MS2 was inactivated when stored at 4 °C (20%) compared to −80 °C (58%); however, during extended storage (∼300 days), less MS2 was inactivated at −80 °C (75%) compared) to 4 °C (93%). We recommend that viruses in wastewater be stored in the dark at 4 °C unless storage for >40 days is necessary.

Enteric viruses in drinking water supplies

Two rural spring drinking water supplies were studied for their enteric virus levels. In one, serving about 30 dwellings, the water was chlorinated before distribution; in the other, which served a dairy and six dwellings the water was not treated. Samples of treated (40 l) and untreated (20 l) water were taken under normal and heavy rainfall conditions over a six weeks period and concentrated by adsorption/elution and organic flocculation. Infectious enterovirus in concentrates was detected in liquid culture and enumerated by plaque assay, both in BGM cells, and concentrates were also analysed by RT-PCR. Viruses were found in both raw water supplies. Rural supplies need to be analysed for viruses as well as bacterial and protozoan pathogens if the full microbial hazard is to be determined.

A method to detect low levels of enteric viruses in contaminated oysters.

Direct isolation and identification of pathogenic viruses from oysters implicated in gastroenteritis outbreaks are hampered by inefficient methods for recovering viruses, naturally occurring PCR inhibitors, and low levels of virus contamination. In this study we focused on developing rapid and efficient oyster-processing procedures that can be used for sensitive PCR detection of viruses in raw oysters. Poliovirus type 3 (PV3) Sabin strain was used to evaluate the efficacy of virus recovery and the removal of PCR inhibitors during oyster-processing procedures. These procedures included elution, polyethylene glycol precipitation, solvent extraction, and RNA extraction. Acid adsorption-elution in which glycine buffer (pH 7.5) was used was found to retain fewer inhibitors than direct elution in which glycine buffer (pH 9.5) was used. RNA extraction in which a silica gel membrane was used was more effective than single-step RNA precipitation for removing additional nonspecific PCR inhibitors. The final 10-microl volume of RNA concentrates obtained from 2 g of oyster tissue (concentration factor, 200-fold) was satisfactory for efficient reverse transcription-PCR detection of virus. The overall detection sensitivity of our method was 1 PFU/g of oyster tissue initially seeded with PV3. The method was utilized to investigate a 1998 gastroenteritis outbreak in California in which contaminated oysters were the suspected disease transmission vehicle. A genogroup II Norwalk-like virus was found in two of three recalled oyster samples linked by tags to the harvest dates and areas associated with the majority of cases. The method described here improves the response to outbreaks and can be used for rapid and sensitive detection of viral agents in outbreak-implicated oysters.

A Method To Detect Low Levels of Enteric Viruses in Contaminated Oysters

A Method To Detect Low Levels of Enteric Viruses in Contaminated Oysters

Detection of low levels of enteric viruses in metropolitan and airplane sewage.

To detect less prevalent viruses, such as wild-type polioviruses in sewage from a highly immunized community, a method was developed to efficiently recover viruses and remove PCR inhibitors. The method consisted of initial separation of solids from liquid, followed by solvent extractions, polyethylene glycol precipitations, Sephadex G-200 chromatography, and guanidinium isothiocyanate (GIT) extraction. To elute viruses from the separated solids, 0.5 M threonine (pH 7.5) was as efficient as 3% beef extract but conferred no PCR inhibition. In samples that were concentrated approximately 1,000-fold, 21% of the initially seeded viruses were recovered. When poliovirus type 3 (PV3) Sabin strain at low levels and PV1 LSc strain at high levels were seeded in raw sewage, PV3 was specifically detected in the final sample concentrates at sensitivities of 14 PFU by direct PCR and 0.7 PFU by GIT extraction-PCR. While applying the method to international airplane sewage, which contains high levels of solids as well as commercial sanitizers, 44% (7 of 16) of the samples were found to harbor enteroviruses by both cell culture infectivity and pan-enterovirus PCR analyses. Nucleotide sequencing of the PCR products revealed that multiple enterovirus genotypes were amplified from each final sewage concentrate, whereas the fewer virus genotypes detected by cell culture infectivity were probably the better growing strains. By this method, we demonstrated that air travel may contribute to the intercontinental dissemination of enteric pathogens.

Detection of low levels of enteric viruses in metropolitan and airplane sewage.

Detection of low levels of enteric viruses in metropolitan and airplane sewage.

Development of a PCR-based method for the detection of enteroviruses and hepatitis a virus in molluscan shellfish and its application to polluted field samples1

The use of the polymerase chain reaction (PCR) for detection of low levels of enteric viruses in bivalve shellfish is hindered by the presence of potent amplification inhibitors. A procedure previously developed for removing the majority of these amplification inhibitors is applied to the detection of enteroviruses and hepatitis A virus in naturally polluted field samples. Quantification of PCR inhibition showed that PCR sample tolerance ranged from 2 to 4.7g shellfish for highly polluted samples. These results indicate the need for adequate controls for PCR inhibition, particularly for negative samples. Reverse transcription (RT)-PCR results were compared with conventional enterovirus isolation for a range of naturally contaminated shellfish. All enterovirus isolation positive samples were also positive by enterovirus RT-PCR. At one field site shellfish were positive by enterovirus RT-PCR but negative for virus isolation. All shellfish tested were negative for hepatitis A by RT-PCR. The procedure for removal of PCR amplification inhibitors should be equally applicable to the detection of Norwalk and related Small Round Structured Viruses (SRSVs) in shellfish.

Development of a PCR-based method for the detection of enteroviruses and hepatitis A virus in molluscan shellfish and its application to polluted field samples

The use of the polymerise chain reaction (PCR) for detection of low levels of enteric viruses in bivalve shellfish is hindered by the presence of potent amplification inhibitors. A procedure previously developed for removing the majority of these amplification inhibitors is applied to the detection of enteroviruses and hepatitis A virus in naturally polluted field samples. Quantification of PCR inhibition showed that PCR sample tolerance ranged from 2 to 4.7g shellfish for highly polluted samples. These results indicate the need for adequate controls for PCR inhibition, particularly for negative samples. Reverse transcription (RT)-PCR results were compared with conventional enterovirus isolation for a range of naturally contaminated shellfish. All enterovirus isolation positive samples were also positive by enterovirus RT-PCR. At one field site shellfish were positive by enterovirus RT-PCR but negative for virus isolation. All shellfish tested were negative for hepatitis A by RT-PCR. The procedure for removal of PCR amplification inhibitors should be equally applicable to the detection of Norwalk and related Small Round Structured Viruses (SRSVS) in shellfish.

Enteric virus levels in river water

During a survey conducted from January 1988 to December 1989, enteric virus levels in river water ranged from 13 to 192 plaque forming units per liter (pfu/l) on a monthly average. Enteric virus levels were high in winter (Jan. to Mar.) and summer (Jun. to Aug.). Isolation of polioviruses coincided with the administration of polio vaccine. Coxsackie group B viruses, particularly type 4, were isolated over a long period in 1988 and 1989. Echoviruses and adenoviruses were isolated throughout the year, though in small numbers. Reoviruses were isolated most frequently throughout the year. Among the five cell lines used in virus isolation. HEp-2 cells were the most sensitive to polioviruses, coxsackie group B viruses and adenoviruses. RD-18S and Vero cells were useful for isolation of echoviruses and reoviruses, respectively.

Microbial Purification of Shellfish: A Review of Depuration and Relaying

A review of the literature on shellfish depuration and relaying revealed wide diversity in microbial uptake and elimination among shellfish species and for different microorganisms. Information on relaying of five commercial shellfish species and on controlled purification (depuration) of 11 species indicates that such processes are effective in reducing the levels of bioconcentrated bacteria and viruses from shellfish. The degree of bacterial and viral bioconcentration varies with shellfish species; however, the primary sites of bioconcentration are the hepatopancreas and digestive diverticula. Low levels of enteric viruses and coliphage may be sequestered in shellfish hemolymph and tissues, thus protecting them from elimination through depurative processes. Vibrio spp. appear to proliferate when closely associated with intestinal cells of shellfish. Shellfish relaying techniques offer effective microbial depletion provided water quality is acceptable and shellfish remain physiologically active. The current body of literature on controlled purification demonstrates a broad spectrum of conditions under which shellfish are depurated. Optimal times, temperatures and salinities for effective depuration vary among shellfish species. Proper design and operation of depuration plants is crucial to insure process integrity. Recirculating and flow-through purification systems are effective in reducing the levels of pathogenic and indicator microorganisms from shellfish, but the extent to which they reduce viruses from shellfish is uncertain. Studies are needed to validate the effectiveness of depuration processes in eliminating pathogenic viruses and to address the adequacy of indicator bacteria as measures of enteric virus contamination.

Enteric virus levels in wastewater effluents and surface waters in the severn trent water authority 1979–1981

Viral surveillance of wastewater effluents and surface waters was carried out between January 1979 and July 1981. Enteric viruses were detected in 45% of 381 samples of effluent and in 48% of 533 samples of surface waters. Viral levels in effluents varied considerably with the highest recovered being 31,000 plaque forming units per litre (pfu l −1). A substantial number of effluent samples gave levels lower than the practical detection limit of 100 pfu l −1. Levels were markedly lower in surface waters, the highest being 647 pfu l −1. River water which had received long-term bankside storage only yielded virus infrequently. The narrow range of serotypes identified probably reflects the limitations of the BGM cell system used in the assay procedure. Serotypes belonged to the polio and coxsackie B groups with only a low incidence of echovirus.

Evaluation of the overland runoff mode of land wastewater treatment for virus removal.

The removal of enteric and tracer viruses by the overland runoff mode of domestic wastewater treatment was evaluated. Raw and primary and secondary treated wastewaters were sprayed onto grass-covered, 36-m soil plots of fine, sandy loam overlying an impermeable clay subsoil. Tracer bacteriophage f2 was seeded into the applied wastewaters, which were subsequently sampled at several points along the length of the plots. Assay of effluent samples revealed modest tracer virus removals of 30 to 60%. Data from timed experiments indicated that advancement of tracer virus to the bottom of the slopes proceeded at the same rate as wastewater, reaching the plot effluents within 50 to 90 min after application. Indigenous enteric virus levels were reduced by approximately 68 to 85% during migration down the treatment slopes. Soil sampling revealed that, although some f2 virus was found associated with the wastewater-saturated topsoil, little penetration of virus into the soil profile occurred. Laboratory soil adsorption studies revealed that poliovirus I was adsorbed much more readily than f2 virus. Comparison of virus removal characteristics during overland runoff with chemical removal characteristics of wastewater did not reveal any obvious correlations that could be used to predict virus removal.

Field survey of enteric viruses in solid waste landfill leachates.

Because municipal solid waste may contain fecal material from a variety of sources, there is concern that the leachate discharged from some solid waste landfills may contain enteric pathogens, including enteric viruses. In this study, 22 leachate samples from 21 different landfills in the United States and Canada were examined for enteric viruses. The sites represented a broad range of conditions for solid waste landfills and the leachate samples ranged from 10.3 to 18 liters in volume. Enteric viruses were found in only one of the 22 leachate samples examined. Two viruses, identified as poliovirus types 1 and 3, were found in an 11.8 liter sample obtained from a site where solid waste landfill practice was deficient. The low levels of enteric viruses detected in field samples of raw leachate and the opportunities for further reductions in the virus concentration of leachates by such processes as thermal inactivation, removal by soil and dilution in ground and surface waters, suggest that leachates from properly operated solid waste landfills do not constitute an environmental or public health hazard due to enteric viruses.