Detection of virus in water: sensitivity of the tentative standard method for drinking water.

The sensitivity of several microporous virus-adsorbent media for reliably detecting low levels of poliovirus from 380 and 1,900 liters of drinking water by use of the tentative standard method was investigated. The virus-adsorbent media tested were (i) nitrocellulose membrane filters, (ii) epoxy-fiber glass-asbestos filters, (iii) yarn-wound fiber glass depth filters, and (iv) epoxy-fiber glass filter tubes. Virus was adsorbed to the filter media at pH 3.5 and eluted with glycine buffer, pH 11.5. The results from 44 samples demonstrated that poliovirus was detected with a 95% reliability at mean virus input levels of 3 to 7 plaque-forming units/380 liters when 1,900 liters of water was sampled. At mean virus input levels of less than 1 to 2 plaque-forming units/380 liters, the detection reliability was 66% in 76 samples when 1,900 liters of water was sampled. No significant difference in virus detection sensitivity was observed among the various virus adsorbent media tested. Overall virus recovery efficiency ranged from 28 to 42%, with a grand average of 35%. Members of the coxsackievirus groups A and B, echovirus, and adenovirus were also detected when 380 and 1,900 liters of water were sampled. These experimental observations attest to the sensitivity of the tentative standard method for detecting low levels of virus in large volumes of drinking water.     

Detection of virus in water: sensitivity of the tentative standard method for drinking water.

The sensitivity of several microporous virus-adsorbent media for reliably detecting low levels of poliovirus from 380 and 1,900 liters of drinking water by use of the tentative standard method was investigated. The virus-adsorbent media tested were (i) nitrocellulose membrane filters, (ii) epoxy-fiber glass-asbestos filters, (iii) yarn-wound fiber glass depth filters, and (iv) epoxy-fiber glass filter tubes. Virus was adsorbed to the filter media at pH 3.5 and eluted with glycine buffer, pH 11.5. The results from 44 samples demonstrated that poliovirus was detected with a 95% reliability at mean virus input levels of 3 to 7 plaque-forming units/380 liters when 1,900 liters of water was sampled. At mean virus input levels of less than 1 to 2 plaque-forming units/380 liters, the detection reliability was 66% in 76 samples when 1,900 liters of water was sampled. No significant difference in virus detection sensitivity was observed among the various virus adsorbent media tested. Overall virus recovery efficiency ranged from 28 to 42%, with a grand average of 35%. Members of the coxsackievirus groups A and B, echovirus, and adenovirus were also detected when 380 and 1,900 liters of water were sampled. These experimental observations attest to the sensitivity of the tentative standard method for detecting low levels of virus in large volumes of drinking water.     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters.

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)     

New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water

The methods used to concentrate enteric viruses from water have remained largely unchanged for nearly 30 years, with the most common technique being the use of 1MDS Virozorb filters followed by organic flocculation for secondary concentration. Recently, a few studies have investigated alternatives; however, many of these methods are impractical for use in the field or share some of the limitations of this traditional method. In the present study, the NanoCeram virus sampler, an electropositive pleated microporous filter composed of microglass filaments coated with nanoalumina fibers, was evaluated. Test viruses were first concentrated by passage of 20 liters of seeded water through the filter (average filter retention efficiency was ≥ 99.8%), and then the viruses were recovered using various salt-based or proteinaceous eluting solutions. A 1.0% sodium polyphosphate solution with 0.05 M glycine was determined to be the most effective. The recovered viruses were then further concentrated using Centricon Plus-70 centrifugal ultrafilters to a final volume of 3.3 (±0.3 [standard deviation]) ml; this volume compares quite favorably to that of previously described methods, such as organic flocculation (~15 to 40 ml). The overall virus recovery efficiencies were 66% for poliovirus 1, 83% for echovirus 1, 77% for coxsackievirus B5, 14% for adenovirus 2, and 56% for MS2 coliphage. In addition, this method appears to be compatible with both cell culture and PCR assays. This new approach for the recovery of viruses from water is therefore a viable alternative to currently used methods when small volumes of final concentrate are an advantage.     

Comparative inactivation of enteroviruses and adenovirus 2 by UV light

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm(2), respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.     

Removal of Enteroviruses from Sewage by Bench-Scale Rotary-Tube Trickling Filters

The efficacy of a rotary-tube type of trickling filter for removing coxsackievirus A9, poliovirus 1, and echovirus 12 suspended in raw settled sewage was investigated. At filtration rates equivalent to about 10 MGD (million gallons per day)/acre (ca. 3,785 m3/day per acre), the filters removed 95% of the poliovirus, 83% of echovirus 12, and 94% of coxsackievirus A9. Coliform, fecal streptococci, biochemical oxygen demand, and chemical oxygen demand removals were remarkably similar, averaging 94, 92, 93, and 95%, respectively. At filtration rates equivalent to about 23 MGD/acre, 59% of the poliovirus, 63% of the echovirus 23, and 81% of the coxsackievirus A9 were removed. Coliform, fecal streptococci, biochemical oxygen demand, and chemical oxygen demand removals at this filtration rate were 68, 75, 72, and 56%, respectively. Viruses were assumed to be adsorbed to the biological slime growing in the filters, but attempts to disassociate the viruses from the slime were unsuccessful, indicating that the slime-virus complex is very stable or that the viruses were somehow inactivated. The data indicate that coliform and fecal streptococci reductions in this type sewage treatment process can be used as an index of virus reduction. Disinfection, however, must be used to ensure a virus-free final effluent.     

Comparative Inactivation of Enteroviruses and Adenovirus 2 by UV Light

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm², respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.     

Comparison of the Elution and Absorption of Picornaviruses by Cotton and Calcium Alginate Wool Swabs

The recovery of picornaviruses by cotton and calcium alginate wool swabs was studied by use of prototype strains of poliovirus 1, echovirus 1, coxsackievirus A9, and rhinovirus 16/60. No significant differences in recovery of viruses by the two types of swabs could be demonstrated. It is suggested that use of cotton swabs in the virus laboratory be continued, since wool swabs may favor the recovery of undesirable bacterial contaminants. Adsorption of viruses by cotton and wool swabs was similar from virus-buffer mixtures at pH 4.0, 7.0, and 8.0 at 24 and 37 C. Elution of virus from cotton and wool swabs was also studied. There was no significant difference in the amount of virus eluted at pH 5.5, 7.1, or 8.4.     

Recovery of viruses from vegetable surfaces

The efficiency of a system developed for the recovery of viruses contaminating large quantities of vegetables was investigated in the laboratory and tested in the field. Viruses seeded onto a number of leafy vegetables in the laboratory were eluted with a phosphate-buffered saline solution (pH 9.0). The eluate was clarified by glass wool filtration, and any viruses present were concentrated by adsorption to a Filterite pleated cartridge filter, eluted with 3% beef extract (pH 9.0), and further concentrated by organic flocculation. At least 24 liters of vegetable eluate could be concentrated to 70 to 80 ml, equivalent to a greater than 99.5% reduction in volume. With this system, poliovirus was recovered with a mean efficiency of 58% for all vegetables tested. Adenovirus was recovered from lettuce with a slightly lower mean efficiency (55%). Poliovirus was recovered from large quantities of cabbage for up to 5 days in the field after spray irrigation of relatively low levels of virus, even when heavy rain fell before sampling.     

Recovery of viruses from vegetable surfaces.

The efficiency of a system developed for the recovery of viruses contaminating large quantities of vegetables was investigated in the laboratory and tested in the field. Viruses seeded onto a number of leafy vegetables in the laboratory were eluted with a phosphate-buffered saline solution (pH 9.0). The eluate was clarified by glass wool filtration, and any viruses present were concentrated by adsorption to a Filterite pleated cartridge filter, eluted with 3% beef extract (pH 9.0), and further concentrated by organic flocculation. At least 24 liters of vegetable eluate could be concentrated to 70 to 80 ml, equivalent to a greater than 99.5% reduction in volume. With this system, poliovirus was recovered with a mean efficiency of 58% for all vegetables tested. Adenovirus was recovered from lettuce with a slightly lower mean efficiency (55%). Poliovirus was recovered from large quantities of cabbage for up to 5 days in the field after spray irrigation of relatively low levels of virus, even when heavy rain fell before sampling.     

Detection of enteroviruses and mammalian reoviruses in Korean environmental waters

Using the standard total culturable virus assay-most probable number (TCVA-MPN) method, we evaluated a total of 348 samples, including surface water, finished water, and tap water samples, collected from randomly selected water treatment plants in Korea from August 2001 through July 2005 according to the Information Collection Rule. All the TCVA-positive samples were also subjected to integrated cell culture-PCR (ICC-PCR) methods for the detection of enteroviruses, hepatitis A virus, adenoviruses, and reoviruses. The most probable number of infectious units per 100 liters for the environmental water samples ranged from 0.5 to 47.3. Nine of the 13 TCVA-positive samples (69.2%) were found by ICC-PCR to be positive for human enteroviruses, which were confirmed to be coxsackievirus type B3, coxsackievirus type B4, coxsackievirus type B6, echovirus type 30, and vaccine strain poliovirus type 3 by direct sequencing. Eleven of the 13 TCVA-positive samples (84.6%) were found by ICC-PCR assay to be positive for reoviruses. The serotype of all the reoviruses was the same as reovirus type 1 by direct sequencing. Both enteroviruses and reoviruses were concurrently detected in seven TCVA-positive samples (53.8%).     

Effects of Source Water Quality on Chlorine Inactivation of Adenovirus,Coxsackievirus, Echovirus,and Murine Norovirus

More information is needed on the disinfection efficacy of chlorine for viruses in source water. In this study, chlorine disinfection efficacy was investigated for USEPA Contaminant Candidate List viruses coxsackievirus B5 (CVB5), echovirus 1 (E1), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated groundwater source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate, using 0.2 and 1 mg/liter free chlorine at 5 and 15°C. The efficiency factor Hom (EFH) model was used to calculate disinfectant concentration × contact time (CT) values (mg·min/liter) required to achieve 2-, 3-, and 4-log₁₀ reductions in viral titers. In all water types, chlorine disinfection was most effective for MNV, with 3-log₁₀ CT values at 5°C ranging from ≤0.020 to 0.034. Chlorine disinfection was least effective for CVB5 in all water types, with 3-log₁₀ CT values at 5°C ranging from 2.3 to 7.9. Overall, disinfection proceeded faster at 15°C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but no single source water had consistently different inactivation rates than another. CT values for CVB5 in one type of source water exceeded the recommended CT values set forth by USEPA''s Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources. The results of this study demonstrate that water quality plays a substantial role in the inactivation of viruses and should be considered when developing chlorination plans.Disinfection processes are critical for the reduction of infectious virus concentrations in source water, because viruses are less efficiently removed by primary treatment of drinking water (e.g., coagulation and filtration) than are other pathogen types of concern (e.g., bacteria and protozoa). Over the years, many disinfection studies have focused on the inactivation of viruses in purified and buffered, demand-free, reagent-grade water (RGW). However, relatively few investigators have examined the impact of water quality during the disinfection process, even though water quality has been found to be a significant factor for inactivation of viruses.Several researchers found that the inactivation rate of poliovirus by free chlorine increased as the ionic concentration of water increased. In one study, poliovirus 1 was inactivated three times faster in boric acid buffer than in purified water (3). In addition, several investigators found that when the ionic content of buffered water was raised by the addition of NaCl or KCl, poliovirus 1 was inactivated two to four times faster than in the buffered water alone (2, 16, 17). In another study, poliovirus 1 was inactivated 10 times more rapidly in drinking water than in purified water (4).Studies conducted with natural waters have demonstrated both increased and decreased disinfection efficacy of chlorine in these waters compared to purified or buffered waters. In a study comparing chlorine disinfection in purified water and Potomac estuarine water, coxsackievirus A9 was inactivated more rapidly in the source water. The remaining study viruses (coxsackievirus B1, echovirus 7, adenovirus 3, poliovirus 1, and reovirus 3) were all inactivated more slowly in the source water (13). Bacteriophage MS2 was inactivated more slowly by free chlorine in two types of surface water than in buffered, demand-free water. However, there was no difference between the inactivation rates of this virus in the buffered water and groundwater (10). In another study, both feline calicivirus and adenovirus 40 were inactivated more slowly in treated groundwater than in buffered, demand-free water (21).The United States Environmental Protection Agency''s (USEPA) Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources (Guidance Manual) recommends disinfectant concentration × contact time (CT) values of 4, 6, and 8 to achieve 2-, 3-, and 4-log₁₀ inactivation, respectively, with chlorine at 5°C and pH 6 to 9 (23). These CT values, which incorporate a safety factor of 3, were obtained from inactivation experiments conducted with monodispersed hepatitis A virus (HAV) in buffered, demand-free water. As water quality can significantly affect the disinfection efficacy of chlorine, it is unclear whether these CT value recommendations are sufficient for inactivation of viruses in source water. More information is needed to systematically examine the role of water quality in chlorine disinfection of viruses.The objective of the present study was to examine the disinfection efficacy of free chlorine on selected viruses from USEPA''s Contaminant Candidate List (CCL) (22) in one untreated and two partially treated source waters from distinct geographical regions. By comparing the efficacy of chlorine disinfection in the source water types to disinfection in buffered, chlorine-demand-free RGW (7), the impact of water quality could be examined. The four representative CCL viruses selected for this study included human adenovirus 2 (HAdV2), echovirus 1 (E1), coxsackievirus B5 (CVB5), and murine norovirus (MNV), a surrogate for human norovirus (22). The viruses were selected because they were previously found to be the least effectively inactivated viruses of their type in RGW (6). Disinfection experiments were carried out in duplicate in pH 7 and 8 source water at 5 and 15°C using 0.2 and 1 mg/liter free chlorine. Inactivation curves were plotted using Microsoft Excel, and CT values were calculated using the efficiency factor Hom (EFH) model (9).     

Influence of pH, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment.

This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

Recovery of Small Quantities of Viruses from Clean Waters on Cellulose Nitrate Membrane Filters

A method is described for quantitatively recovering small amounts of viruses from large volumes of buffered, distilled water. Development of the method was motivated by the anticipated need for testing large volumes of renovated sewage for viruses. The method consists of adsorbing viruses onto cellulose nitrate membrane filters (0.45 mum pore size) from water containing sufficient Na(2)HPO(4) to produce a molarity of 0.05 and sufficient citric acid to produce a pH of 7, and eluting the adsorbed viruses in 3% beef extract under extended sonic treatment. Complete recovery of poliovirus 1, echovirus 7, and coxsackievirus B3 resulted when less than 100 plaque-forming units were added to 1-liter quantities of water. Recoveries of reovirus 1 were almost as good. Preliminary studies indicate that good recoveries can be made from 25-gal quantities of water. The method described is efficient in waters of high quality and may be useful for recovering viruses in renovated, and perhaps in tap waters, but not in waters containing certain organic matter unless that matter is first removed.     

Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria,protozoa, and viruses from water

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10(5) to 10(6) CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 x 10(5) and 2.4 x 10(3) per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 x 10(5) CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10(5) PFU/liter), and phage PP7 (10(5) PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.     

Viral pollution of the rivers in Toyama City

Viral pollution of the river water in Toyama City was surveyed during the two-year period from July 1979 to July 1981, and the ecology of viruses in the river water is discussed. Virus isolation from the river water samples, or from the water squeezed from cotton pads that were immersed in the stream for 3 days, was carried out by the "filter adsorption/elution" method. River waters were found to be contaminated with various species of enteric viruses, that is, poliovirus, echovirus, coxsackievirus, adenovirus, and reovirus. Poliovirus was isolated during the period immediately after the oral administration of polio vaccine, and coxsackie B virus was frequently isolated all year around. The enterovirus concentration in the river water was significantly high with a maximum of five plaque-forming units of coxsackie B2 virus per 250 ml. The species and type distribution of enteroviruses isolated from the river water coincided well with that of viruses isolated from inhabitants of Toyama Prefecture, with the exception of reovirus which was the largest population of virus species in the river water.