Viruses In Water Research Articles

Absence of human astrovirus RNA in sewage and environmental samples.

Over a period of several weeks during the summer of 1996, samples of sewage, sea water, river water, sand and silt were collected from a sewage works at Weston-super-Mare, England and from coastal areas nearby. A sensitive reverse-transcriptase polymerase chain reaction (RT-PCR) was used to search for human astrovirus (HAstV) RNA in concentrates of the samples. No evidence of astrovirus was found in any sample, which suggests that contamination with these viruses is not a problem in this area during the summer holiday season. Furthermore, the single case of astrovirus diarrhoea diagnosed in this laboratory in the summer occurred at the end of the sampling period, and not in the survey area. The primers used sometimes yielded a product two-thirds the expected size but bearing no sequence homology with HAstV. The confirmation that poliovirus adsorbs to sand and silt shows that these materials might be able to concentrate other enteric viruses in water to a level which could be a threat to the health of people coming into contact with it.

Analysis of bacteriophage inactivation and its attenuation by adsorption onto colloidal particles by batch agitation techniques

A batch agitation technique was designed to specify the different parameters that influence the inactivation and adsorption mechanisms of viruses in water. The advantage of this method over the classical procedures is that the kinetic reactions of the different subfractions of the virus population can be described simultaneously. A first set of experiments with phage T7 showed that this phage is rapidly inactivated in a constantly agitated liquid medium. This inactivation rate is highly influenced by temperature, but variation of the pH (from 5 to 9) and increase in salt concentration have no effect on it. The addition of colloidal clay particles (CCPs) of montmorillonite and attapulgite into the liquid medium considerably modifies this behavior, even at very low concentrations (0.025 mg/mL). The experiments show that the viruses react quickly with the particles and that bonding is not permanent. Viruses establish a dynamic equilibrium, which is strongly dependent on physicochemical parameters such as pH, ionic concentrations, and the presence of proteins or protein hydrolysates. A major environmental consequence is that the presence of CCPs seems to effectively protect the coliphage T7 from rapid inactivation.Key words: bacteriophage T7, viruses, inactivation and adsorption kinetics, colloidal particles, protective effect.

RT-PCR amplification detects inactivated viruses in water and wastewater

Nucleic acid (NA) amplification techniques are useful to detect viruses in water and other environmental samples because they are highly sensitive, specific and can detect fastidious enteric viruses that do not grow well or not at all in cell cultures. However, RT-PCR was found to detect inactivated viruses. In terms of risks to public health this constitutes a false positive result, as inactivated viruses are no longer infectious. When poliovirus type 1 and coliphage MS2 were studied for (a) persistence in water and sewage and (b) inactivation in water by free chlorine, chlorine dioxide and UV radiation, RT-PCR assays underestimated virus inactivation. The use of multiple RT-PCR amplification sites, larger RT-PCR genomic targets and immunocapture RT-PCR sometimes reduced, but did not eliminate, the discrepancy between loss of infectivity and loss of RT-PCR titre. Virus presence based on RT-PCR detection must be interpreted with caution when predicting human health risks.

RT-PCR amplification detects inactivated viruses in water and wastewater

Nucleic acid (NA) amplification techniques are useful to detect viruses in water and other environmental samples because they are highly sensitive, specific and can detect fastidious enteric viruses that do not grow well or not at all in cell cultures. However, RT-PCR was found to detect inactivated viruses. In terms of risks to public health this constitutes a false positive result, as inactivated viruses are no longer infectious. When poliovirus type 1 and coliphage MS2 were studied for (a) persistence in water and sewage and (b) inactivation in water by free chlorine, chlorine dioxide and UV radiation, RT-PCR assays underestimated virus inactivation. The use of multiple RT-PCR amplification sites, larger RT-PCR genomic targets and immunocapture RT-PCR sometimes reduced, but did not eliminate, the discrepancy between loss of infectivity and loss of RT-PCR titre. Virus presence based on RT-PCR detection must be interpreted with caution when predicting human health risks.

Rapid PCR-based monitoring of infectious enteroviruses in drinking water

Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensitivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300–4001 of water were concentrated using charged filters followed by a modified 11, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24–48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48h against 5–16d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

Rapid PCR-based monitoring of infectious enteroviruses in drinking water

Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300–4001 of water were concentrated using charged filters followed by a modified II, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, Conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24–48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus Concentrations as low as 0.001MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48h against 5–l6d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

Removal of virus from water by filtration using microporous membranes made of poly(N-benzyl-4-vinylpyridinium chloride)

Two types of microporous filter materials were developed for removing virus from water by using poly(N-benzyl-4-vinylpyridinium chloride) that captures virus in water. Conventional ultrafiltration using one to three sheets of 145-μm-thick cellulose nitrate membrane with a pore size of 0.45 μm and coated with 1.7 mg/g of poly(N-benzyl-4-vinylpyridinium chloride-co-styrene) showed 99.4–99.998% removal (2.2–4.7 log10-unit reduction in concentration) of bacteriophage T4, whereas the control experiments using noncoated membrane showed 91–96% removal (1.0–1.4 log10-unit reduction in concentration) of the virus. A composite 360-μm-thick microporous membrane with a pore size of 20 ¨︁m was prepared that consisted of connected minute beads of 1.7 ¨︁m in diameter made of crosslinked poly(N-benzyl-4-vinylpyridinium chloride) and reinforced by a nonwoven cloth. Simple filtration using one sheet of the composite membrane at 34.2cm/h showed 99.96–99.9995% removal (3.4–5.3 log10-unit reduction in concentration). The virus was not detected in the filtrate when two sheets of the composite membrane were used. © 1996 John Wiley & Sons, Inc.

Detection of Fastidious Infectious Enteric Viruses in Water

A procedure based on the infection of CaCo-2 cells and molecular hybridization with specific cDNA probes has been developed forthe detection of infectious fastidious enteric viruses in environmental samples. CaCo-2 cells, derived from a human colon adenocarcinoma, showed an increased sensitivity when compared to the usual routine host cell line to laboratory strains of rotavirus 3, reovirus 3, astrovirus 1, poliovirus 1, coxsackievirus A 24, enterovirus 70, and adenovirus 5, 40, and 41. Using this methodology, wild-type rotaviruses, enteric adenoviruses, enteroviruses, and for the first time, astroviruses have been detected in freshwater samples. Direct dot-blot hybridization alone was not sufficient for virus detection from environmental samples. CaCo-2 cells may be used as a universal in vivo amplification system for human enteric viruses, enabling the specific monitoring of infectious viral agents in the environment.

Whole Particle Microelectrophoresis for Small Viruses

The surface-charge characteristics of viruses in water can strongly influence their behavior with respect to physicochemical processes like coagulation and filtration. In the past, characterization of this surface charge by whole-particle microelectrophoresis has been limited to relatively large viruses, primarily due to difficulties in visualizing the individual virus particles. Here, we demonstrate a new approach for performing microelectrophoresis measurements which permits the surface characterization of even very small viruses. Our approach differs from conventional microelectrophoresis measurements in that virus particles are visualized by light scattering, employing a green laser (wavelength = 544 nm) as the light source. We demonstrate this technique by characterizing the electrophoretic mobilities and isoelectric points of two viruses—bacteriophage MS-2 and lambda (λ strain cI47)—which differ in their shape and size.

Validity of serological methods (ELISA) for detecting infectious viruses in water

Detection of cultivable enteric viruses in environmental water samples in tissue culture is time consuming and expensive. Moreover, some important enteric viruses grow very slowly (Hepatitis A virus) or do not grow as yet (Norwalk) in tissue culture. Therefore, sensitive serological and molecular methods have been developed to simplify and speed the detection of viruses in environmental samples. This study was conducted to test the reliability of serological methods to monitor the presence of viable viruses in natural waters. The study was performed with poliovirus purified in CsCl gradients and impure virus. Poliovirus 1 either purified or impure was seeded in raw wastewater, groundwater and phosphate buffered saline (PBS) and incubated for 20 days at 4°C, 20°C and 30°C. Virus survival was monitored by a nylon filter A-ELISA and plaque-assay in BGM cells. In all water samples at 4°C, no die-off was observed neither by A-ELISA nor by plaque-assay. In wastewater and groundwater at 20°C and 30°C, greater die-off was observed with A-ELISA than with plaque-assay. Purified poliovirus was undetectable by the A-ELISA after two days of incubation at 20°C in wastewater and groundwater, whereas under the same conditions, only 2log10 reduction were observed in the titer of poliovirus 1. The data of this study have shown that in all cases, a positive test by A-ELISA was also positive by the plaque-assay. Therefore, a positive result of A-ELISA indicates the presence of viable virus in natural waters.

Studies evaluating the applicability of utilising the same concentration techniques for the detection of protozoan parasites and viruses in water

In this study concentration techniques regularly used for viral detection, i.e. flat-bed ultrafiltration and Filterite cartridge filtration, were evaluated for their efficacies in the recovery of protozoan parasites from water. Recovery of cysts was studied using tap water seeded with Giardia muris cysts and compared to methods designed for the detection of protozoan parasites. Recovery of cysts utilizing 1.2µm membrane filters was 11.1% (4.5-23%) compared to 11.6% (2.7-25.5%) with ultrafiltration (pore size 46-50 Å, with a molecular cut off of 50 000 daltons). Comparison of these methods for the isolation of Giardia cysts and Cryptosporidium oocysts from environmental water samples also indicated a similar efficacy. The recovery of cysts from 1001 of seeded samples using a Cuno wynd cartridge filter was 12.2% (1.6-46%) compared to 13.4% (5-24.2%) using a Filterite cartridge filter. Although the results indicated similar recovery efficacies for these two methods, use of Filterite resulted in a more consistent recovery rate. This study also indicated that the use of cartridge filters for the processing of large volume water samples (1001) showed a slightly better recovery efficacy than the flat-bed filtration technique which limits sample volume to about 101. This study shows that concentration techniques utilised for the isolation of enteric viruses can also be applied for the detection of protozoan parasites from water. This procedure allows for co-analysis of both viruses and protozoan parasites and provides a more rapid and cost-effective evaluation of water quality.

Enteric virus removal/inactivation by coal-based media

Four coal-based media, viz. alum-pretreated or ferric hydroxide-impregnated Giridih bituminous coal and lignite (alum-GBC, FeGBC, alum-lignite and FeLignite) were laboratory tested to assess their potential in removing/inactivating enteric viruses in water. Batch-sorption screening tests, employing a poliovirus-spiked (3.10 × 10 7−1.49 × 10 8 PFU 1 −1) canal water, indicated high poliovirus sorption by FeGBC (7.07 × 10 6 PFU g −1) and alum-GBC (5.70 × 10 6 PFU g −1) in a short contact time of 5 min. Based on the results of further batch-sorption tests, using silver-incorporated media (alum/AgGBC, alum-GBCAg and FeGBCAg), as well as aesthetic water quality consideration and previous findings on removal of coliforms and turbidity, alum/AgGBC, alum-GBC and alum-GBCAg were included in downflow column studies (bed depth 10 cm; flow rate 99 ml h −1; bed flowthrough time 4.76 min), employing poliovirus-spiked (1.95 × 10 5−2.68 × 10 7 PFU 1 −1) canal water. All three media showed potential in removing/inactivating enteric viruses. In a separate column study (bed depth 25.4 cm; flow rate 500 ml h −1; flowthrough time 7.7 min), employing a joint challenge of poliovirus and rotavirus (2.47 × 10 7 PFU 1 −1), alum/AgGBC removed 59.3–86.5% of the viruses along with more than 99% reduction in indigenous heterotrophic bacteria. Alum/silver-pretreated bituminous coal medium appears promising for use in household water filters in rural areas of the developing world. However, improved medium preparation to further enhance its efficiency is needed; also, its efficacy in removing/inactivating indigenous enteric bacteria, viruses and protozoa has to be ensured and practicalities or economics of application need to be considered.

Validity of serological methods (ELISA) for detecting infectious viruses in water

Detection of cultivable enteric viruses in environmental water samples in tissue culture is time consuming and expensive. Moreover, some important enteric viruses grow very slowly (Hepatitis A virus) or do not grow as yet (Norwalk) in tissue culture. Therefore, sensitive serological and molecular methods have been developed to simplify and speed the detection of viruses in environmental samples. This study was conducted to test the reliability of serological methods to monitor the presence of viable viruses in natural waters. The study was performed with poliovirus purified in CsC1 gradients and impure virus. Poliovirus i either purified or impure was seeded in raw wastewater, groundwater and phosphate buffered saline (PBS) and incubated for 20 days at 4°C, 20°C and 30°C. Virus survival was monitored by a nylon filter A-ELISA and plaque-assay in BGM cells. In all water samples at 4°C, no die-off was observed neither by A-ELISA nor by plaque-assay. In wastewater and groundwater at 20°C and 30°C, greater die-off was observed with A-ELISA than with plaque-assay. Purified poliovirus was undetectable by the A-ELISA after two days of incubation at 20°C in wastewater and groundwater, whereas under the same conditions, only 2log10 reduction were observed in the titer of poliovirus 1. The data of this study have shown that in all cases, a positive test by A-ELISA was also positive by the plaque-assay. Therefore, a positive result of A-ELISA indicates the presence of viable virus in natural waters.

Studies evaluating the applicability of utilising the same concentration techniques for the detection of protozoan parasites and viruses in water

In this study concentration techniques regularly used for viral detection, i.e. flat-bed ultrafiltration and Filterite cartridge filtration, were evaluated for their efficacies in the recovery of protozoan parasites from water. Recovery of cysts was studied using tap water seeded with Giardia muris cysts and compared to methods designed for the detection of protozoan parasites. Recovery of cysts utilizing 1.2µm membrane filters was 11.1% (4.5–23%) compared to 11.6% (2.7–25.5%) with ultrafiltration (pore size 46–50 A, with a molecular cut off of 50 000 daltons). Comparison of these methods for the isolation of Giardia cysts and Cryptosporidium oocysts from environmental water samples also indicated a similar efficacy. The recovery of cysts from 1001 of seeded samples using a Cuno wynd cartridge filter was 12.2% (1.6–46%) compared to 13.4% (5–24.2%) using a Filterite cartridge filter. Although the results indicated similar recovery efficacies for these two methods, use of Filterite resulted in a more consistent recovery rate. This study also indicated that the use of cartridge filters for the processing of large volume water samples (1001) showed a slightly better recovery efficacy than the flat-bed filtration technique which limits sample volume to about 101.This study shows that concentration techniques utilised for the isolation of enteric viruses can also be applied for the detection of protozoan parasites from water. This procedure allows for co-analysis of both viruses and protozoan parasites and provides a more rapid and cost-effective evaluation of water quality.

Prevalence and fate of hepatitis a virus in water

Abstract Hepatitis A virus (HAV) is a major waterborne disease agent with worldwide distribution. The main transmission route of HAV is direct person‐to‐person contact. However, hepatitis A (HA) outbreaks associated with the consumption and use of fecally contaminated water were reported from many countries. Studies on the environmental behavior of HAV were feasible only after developing techniques for its cultivation and enumeration in tissue culture. This study reviews data on the extent of HAV prevalence and persistence in the environment and water. HA is highly prevalent in low socioeconomic populations as determined by seroepidemiologic studies. HAV is excreted for long periods by infected individuals, but it is also shed by healthy persons. HAV has been detected in concentrated wastewater and natural waters. However, in most cases the natural waters were monitored for the presence of HAV after the occurrence of HA outbreak. HAV persists for months at temperatures below 10°C and for at least 1 month at ambient temperature (20 to 25°C). Physical, biological, and chemical factors that influence the survival of enteric viruses — such as temperature, pH, salt concentration, microbial activity, and humidity — have similar effects on HAV. Drinking water treatment processes such as coagulation, high rate filtration, and disinfection seem to be effective in removing HAV from water.

Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine

The efficacy of copper and silver ions, in combination with low levels of free chlorine (FC), was evaluated for the disinfection of hepatitis A virus (HAV), human rotavirus (HRV), human adenovirus, and poliovirus (PV) in water. HAV and HRV showed little inactivation in all conditions. PV showed more than a 4 log10 titer reduction in the presence of copper and silver combined with 0.5 mg of FC per liter or in the presence of 1 mg of FC per liter alone. Human adenovirus persisted longer than PV with the same treatments, although it persisted significantly less than HRV or HAV. The addition of 700 micrograms of copper and 70 micrograms of silver per liter did not enhance the inactivation rates after the exposure to 0.5 or 0.2 mg of FC per liter, although on some occasions it produced a level of inactivation similar to that induced by a higher dose of FC alone. Virus aggregates were observed in the presence of copper and silver ions, although not in the presence of FC alone. Our data indicate that the use of copper and silver ions in water systems may not provide a reliable alternative to high levels of FC for the disinfection of viral pathogens. Gene probe-based procedures were not adequate to monitor the presence of infectious HAV after disinfection. PV does not appear to be an adequate model viral strain to be used in disinfection studies. Bacteroides fragilis bacteriophages were consistently more resistant to disinfection than PV, suggesting that they would be more suitable indicators, although they survived significantly less than HAV or HRV.

Detection and occurrence of waterborne bacterial and viral pathogens

Detection and occurrence of waterborne bacterial and viral pathogens

Disinfection of Virus in Water by Active Oxygen Species

Identification of the species of active oxygen generated in water by chlorination or ozonation was carried out by the spin trapping method using DMPO as the spin trap agent with ESR spectrometry. On chlorinating with NaOCl solution (15 mg/litre), typical ESR signals for DMPO-OH adduct were observed. Increasing the concentration of NaOCl, however, caused complex spectra to appear. The addition of thiourea or D-mannitol, as a radical scavenger, did not significantly affect on the intensity of the signals. On ozonation, two radicals, DMPO-OH and an unknown radical, were always observed. These results indicate the generation of several radicals in the solution treated with NaOCl and ozone. An interaction of the polio virus with ♦OH was also studied. The preliminary test showed that the ♦OH generated by Fenton's reaction slightly disinfected the virus. Precise tests changing the concentration of active oxygen and the reaction condition are currently in progress.

Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water.

The potential for human immunodeficiency virus (HIV) to enter domestic sewers via contaminated body fluids such as blood has spurred interest in the survival of this virus in water and wastewater. This study focused on establishing the inactivation of HIV and productively infected lymphocytes in dechlorinated tap water. In addition, HIV survival was compared with that of poliovirus. Results indicated that either free HIV or cell-associated HIV was rapidly inactivated, with a 90% loss of infectivity within 1 to 2 h at 25 degrees C and a 99.9% loss by 8 h. In comparison, poliovirus showed no loss of infectivity over 24 h. The presence of human serum in tap water slowed the rate of HIV inactivation through 8 h but did not stabilize the virus through 24 h. In addition, blood from stage IV AIDS patients was introduced into tap water, and the recovery of HIV was monitored by using both an infectivity assay and polymerase chain reaction amplification of viral sequences. Virally infected cells were no longer detectable after 5 min in dechlorinated tap water, while little diminution in amplifiable sequences was observed over 2 h. Thus, detection of viral sequences by polymerase chain reaction technology should not be equated with risk of exposure to infectious HIV.

A Nylon Filter A-ELISA for Detecting Viruses in Water

This study was designed to develop a modification of A-ELISA performed in microtitre plates. Nylon filters have been utilized successfully as a solid phase for the performance of A-ELISA. The use of nylon filters resulted in lower background than nitro-cellulose and paper filters, indicating their suitability as a solid phase for developing A-ELISA. With enteric viruses, human rotaviruses and MS-2 coliphage, negative results were obtained, suggesting high specificity of the developed technique for poliovirus 1. The sensitivity of the developed A-ELISA has been shown to be at least one order of magnitude greater than ordinary ELISA. A positive result with the nylon A-ELISA can be obtained with samples containing 100-1000 pfu/ml of poliovirus. Up to date methods used for detecting viruses in water are elaborate, time consuming and costly. Applying the nylon A-ELISA may overcome some of these disadvantages.