One-year survey of enteroviruses, adenoviruses, and reoviruses isolated from effluent at an activated-sludge purification plant.

Samples of raw sewage, primary effluent, and secondary effluent from a large activated-sludge purification plant near Melbourne (Victoria, Australia) were collected every second week for 1 year. Viruses were detected in all secondary effluent samples and in six of seven samples obtained after final chlorination. Adenoviruses (85% reduction) and reoviruses (28% reduction) were removed less efficiently by this treatment process than were enteroviruses (93% reduction). In addition, 57 of 171 samples of effluent tested were positive for either adenoviruses or reoviruses, or both, when enteroviruses were not isolated. This clearly shows that the use of enteroviruses as sole indicators of viruses in water may miss up to one-third of instances of viral contamination. Enteroviruses and adenoviruses were isolated most frequently in HeLa-R cell cultures, whereas reoviruses were most often isolated in primary monkey kidney cells.     

Enteroviruses in sludge: multiyear experience with four wastewater treatment plants.

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.     

Enteroviruses in sludge: multiyear experience with four wastewater treatment plants

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.     

Detection and molecular identification of human adenoviruses and enteroviruses in wastewater from Morocco

Aims: Reclaimed wastewater is a considerable water resource in Morocco. Its agricultural reuse requires an assessment of viral contamination. The aim of this study was to detect both infectious and noninfectious human adenoviruses (HAdV) and enteroviruses (EV) in raw wastewater and treat effluent from wastewater treatment plants (WWTPs) and domestic sewage in Morocco. Methods and Results: A total of 22 samples were analysed. A polyethylene glycol (PEG) precipitation method was used, followed by integrated cell culture‐PCR (ICC‐PCR) using two cell lines: human rhabdomyosarcoma tumour tissue and laryngeal carcinoma cells (RD and Hep2 cells). Furthermore, viral genome amplification was confirmed by sequencing. HAdV were detected in 10 (45·5%) of the 22 samples involving two species: HAdV‐B and HAdV‐D. EV was detected in 5 (23%) samples belonging to Coxsackievirus B5 and poliovirus vaccine strain (Sabin 2). Conclusions: Human adenoviruses and EV were detected in the analysed samples from two WWTPs and HAdV in domestic sewage. Significance and Impact of the Study: This work is the first study in Morocco using cell culture, PCR and sequencing of enteric viruses from wastewater. The presence of infectious HAdV and EV in treated effluent emphasizes the need of wastewater treatment surveillance.     

Elimination of human enteric viruses during conventional waste water treatment by activated sludge

The present study was undertaken to determine if viruses were selectively eliminated during waste water treatment. Human enteric viruses were detected at all steps of treatment in a conventional activated sludge waste water treatment plant. Liquid overlays and large volume sampling with multiple passages on BGM cells permitted the detection of poliovirus (serotypes 1, 2, and 3), coxsackievirus B (serotypes 1, 2, 3, 4, and 5), and echovirus (serotypes 3, 14, and 22), as well as reoviruses. The mean virus concentration was 95.1 most probable number of infectious units per litre (mpniu/L) in raw sewage, 23.3 in settled water, 1.4 in effluent after activated sludge treatment, and 40.3 mpniu/L in sludge samples. All samples of raw sewage and settled water, 79% of effluent water, and 94% of sludge samples contained viruses. The mean reduction was 75% after settling and 98% after activated sludge treatment. Poliovirus type 3 was rarely isolated after the activated sludge treatment, but was still detected in about one-third of the sludge samples. Reoviruses and coxsackieviruses were detected at similar rates from all samples and appear to be more resistant to the activated sludge treatment than poliovirus type 3. Poliovirus types 1 and 2 were present in almost every sample of raw sewage and settled water and still found in about half of the effluent and sludge samples, indicating a level of resistance similar to that of reoviruses and coxsackieviruses.     

The simultaneous detection of both enteroviruses and adenoviruses in environmental water samples including tap water with an integrated cell culture-multiplex-nested PCR procedure

AIMS: To evaluate an integrated cell culture (ICC)-multiplex-nested PCR using the buffalo green monkey kidney (BGMK) cells for the simultaneous detection of both enteroviruses and adenoviruses in surface water and tap water samples and optimize the procedure for more sensitive detection of virus showing no apparent cytopathic effect (CPE). METHODS AND RESULTS: A total of 69 surface water and 50 tap water samples were analysed by the ICC-multiplex-nested PCR. All the PCRs were performed five times with a cell lysate from each flask after at least 2 weeks incubation. Forty-six surface water samples (66.7%) and 23 tap water samples (46.0%) exhibited CPE by the cell culture method. By using an ICC-multiplex-nested PCR, 53 surface water samples (76.8%) and 29 tap water samples (58.0%) were determined as containing infectious enteric viral particles. CONCLUSIONS: An ICC-PCR method with a long incubation time using BGMK cells enables the simultaneous detection of enteroviruses and adenoviruses from environmental water samples, including tap water, even with low numbers of viruses. SIGNIFICANCE AND IMPACT OF THE STUDY: A method capable of detecting small numbers of viral particles is necessary.     

Quantification and Stability of Human Adenoviruses and Polyomavirus JCPyV in Wastewater Matrices

Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.     

Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices

Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.     

Detection and quantitation of human enteric viruses in waste waters: increased sensitivity using a human immune serum globulin--immunoperoxidase assay on MA-104 cells

This study demonstrates that the most sensitive method for the detection and quantitation of cultivable human enteric viruses in water samples after repassage in the MA-104 cell line is the detection of infected cells by the human immune serum globulin--immunoperoxidase (HISG-IP) method recently described by the authors. This immunoperoxidase method is up to 50 times more sensitive than a liquid overlay assay by cytopathic effect in BGM cells. The viral content of waste waters was evaluated with this new methodology. By this method the average viral content of raw sewage (RS) was 900 mpniu/L (most probable number of infectious units per litre), 1056 mpniu/L in primary effluent (PE), and 106 mpniu/L in secondary effluent (SE). With a cytopathic effect assay on BGM cells, values of 85 (RS), 56 (PE), and 2 (SE) mpniu/L were observed, a striking underestimation of the viral content of secondary effluents.     

ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples

This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of > or =10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after > or =10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required < or =48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.     

Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification.

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.     

Comparison of indirect and sandwich ELISA for the identification of ECHO viruses

An indirect enzyme linked immunosorbent assay (ELISA) was standardized for the identification of ECHO viruses isolated in buffalo green monkey (BGM) kidney cell culture on inoculation of 113 sewage samples. Comparable results were obtained with both indirect and sandwich ELISA for the identification of ECHO viruses in respect of 15 out of 34 sewage samples which showed 75-100% CPE in BGM monolayers.     

Comparison of methods for recovering indigenous viruses from raw wastewater sludge.

Five general methods for recovering indigenous viruses from raw wastewater sludge were compared. Each method included elution, concentration, and disinfection steps. The elution method, found to consistently yield the greatest viral recovery, was a two-phase technique that involved blending sludge with Freon. Other methods, including two being tested as American Society for Testing Materials tentative standard methods, were less effective. Viral recoveries were generally greater (sometimes much greater) if samples were concentrated by high-speed centrifugation rather than by organic flocculation with 3% beef extract. Three cell lines were used to measure viral recoveries by the plaque assay. The efficiency of recovery was greatest on BGM cells, followed by RD and MA-104 cells.     

Functional absence of FADD in PLC/PRF/5 hepatoma cells: possible involvement in the transformation to hepatoma in HBV-infected hepatocytes.

The death receptor Fas transduces apoptotic death signaling upon stimulation by Fas ligand and plays a key role in viral hepatitis. When hepatitis-B virus (HBV) infects hepatocytes, the Fas ligand/Fas system responds as the triggering machinery of hepatitis. However, some HBV-infected cells may circumvent Fas-mediated apoptosis and transform to hepatoma cells, as do PLC/PRF/5 hepatoma cells. Therefore, in the present study, we used PLC/PRF/5 hepatoma cells to investigate this ability to avoid Fas-mediated apoptosis. When the cells were treated with an agonistic Fas antibody, they showed resistance to Fas-mediated apoptosis. In contrast, HepG2 cells of the same hepatoma line succumbed. Caspase 3 and 8, which are essential regulators for Fas-mediated cell death, were expressed in both hepatoma cell lines, but only HepG2 cells showed activation of the caspases. A comparison study of expression of other death-associated factors between PLC/PRF/5 and HepG2 cells revealed no apparent differences. However, Far-Western blotting analysis using the Fas death domain (FDD) showed a significant difference. Molecular weight comparison and immunoblotting analysis revealed that PLC/PRF/5 cells lack the FDD-associated protein FADD. In addition, FDD-injected HepG2 cells showed a resistance to Fas-mediated apoptosis, and PLC/PRF/5 cells acquired Fas-sensitivity by FADD injection. Here, we propose that a functional absence of FADD is one of the pathways for the carcinogenesis of HBV-infected hepatocytes.     

State of hepatitis B viral DNA in a human hepatoma cell line.

PLC/PRF/5, a tissue culture cell line isolated from a human hepatocellular carcinoma and producing hepatitis B surface antigen, was studied for the presence of hepatitis B virus (HBV)-specific DNA and RNA. PLC/PRF/5 cell DNA accelerated the rate of reassociation of HBV [32P]DNA, and quantitative experiments indicated that the cells contained approximately four copies of viral DNA per haploid, mammalian cell DNA equivalent. PLC/PRF/5 DNA accelerated the rate of reassociation of all individual restriction endonucleases HincII and HaeIII fragments of HBV [32P]DNA, indicating that DNA from all regions of the viral genome is present in the cells. This suggests that these cells contain at least most, and possibly all, of the viral genome. Digestion of PLC/PRF/5 cell DNA with restriction endonuclease HindIII (an enzyme found not to cleave the DNA of any HBV isolate so far examined) yielded only three fragments, all larger than virion DNA, which contained HBV DNA base sequences, suggesting that HBV DNA is integrated in high-molecular-weight DNA at three different sites in these cells and that there is no viral DNA in an episomal form. PLC/PRF/5 cell [32P]RNA was found to hybridize with all restriction fragments of HBV DNA adequately tested, indicating that at least most, and possibly all, of the viral DNA in these cells is transcribed.     

Seasonal distribution of enteroviruses and adenoviruses in domestic sewage

A seasonal distribution of enteroviruses and adenoviruses in raw sewage effluents of Athens, Greece, was observed over a 15-month surveillance period. All 36 samples tested were positive for both virus groups. Adenovirus concentration levels ranged from 70 to 3200 cytopathic units per litre of sample, whereas the corresponding values for enteroviruses were 90-900 cytopathic units per litre. Peak values for adenoviruses were recorded during the months of April and June 1983, whereas for enteroviruses the peak was recorded in September 1983. All three types of poliovirus were present. Coxsackievirus types B-1, B-2, B-4, and B-5 and echovirus type 7 were also isolated. Adenovirus types 1, 2, 3, 5, 7, and 15 were detected as well.     

Nine-year study of the occurrence of culturable viruses in source water for two drinking water treatment plants and the influent and effluent of a Wastewater Treatment Plant in Milwaukee, Wisconsin (August 1994 through July 2003)

Reoviruses, enteroviruses, and adenoviruses were quantified by culture for various ambient waters in the Milwaukee area. From August 1994 through July 2003, the influent and effluent of a local wastewater treatment plant (WWTP) were tested monthly by a modified U.S. Environmental Protection Agency Information Collection Rule (ICR) organic flocculation cell culture procedure for the detection of culturable viruses. Modification of the ICR procedure included using Caco-2, RD, and HEp-2 cells in addition to BGM cells. Lake Michigan source water for two local drinking water treatment plants (DWTPs) was also tested monthly for culturable viruses by passing 200 liters of source water through a filter and culturing a concentrate representing 100 liters of source water. Reoviruses, enteroviruses, and adenoviruses were detected frequently (105 of 107 samples) and, at times, in high concentration in WWTP influent but were detected less frequently (32 of 107 samples) in plant effluent and at much lower concentrations. Eighteen of 204 samples (8.8%) of source waters for the two DWTPs were positive for virus and exclusively positive for reoviruses at relatively low titers. Both enteroviruses and reoviruses were detected in WWTP influent, most frequently during the second half of the year.     

Nine-Year Study of the Occurrence of Culturable Viruses in Source Water for Two Drinking Water Treatment Plants and the Influent and Effluent of a Wastewater Treatment Plant in Milwaukee, Wisconsin (August 1994 through July 2003)

Reoviruses, enteroviruses, and adenoviruses were quantified by culture for various ambient waters in the Milwaukee area. From August 1994 through July 2003, the influent and effluent of a local wastewater treatment plant (WWTP) were tested monthly by a modified U.S. Environmental Protection Agency Information Collection Rule (ICR) organic flocculation cell culture procedure for the detection of culturable viruses. Modification of the ICR procedure included using Caco-2, RD, and HEp-2 cells in addition to BGM cells. Lake Michigan source water for two local drinking water treatment plants (DWTPs) was also tested monthly for culturable viruses by passing 200 liters of source water through a filter and culturing a concentrate representing 100 liters of source water. Reoviruses, enteroviruses, and adenoviruses were detected frequently (105 of 107 samples) and, at times, in high concentration in WWTP influent but were detected less frequently (32 of 107 samples) in plant effluent and at much lower concentrations. Eighteen of 204 samples (8.8%) of source waters for the two DWTPs were positive for virus and exclusively positive for reoviruses at relatively low titers. Both enteroviruses and reoviruses were detected in WWTP influent, most frequently during the second half of the year.     

Trypsin-treated Ma-104: a sensitive cell line for isolating enteric viruses from environmental samples.

During a 1-year survey of enteroviruses in wastewater samples from the Lorraine area, three widely used continuous monkey kidney cell lines were tested: BGM, Vero, and trypsin-treated Ma-104. Decontaminated samples from secondary wastewater treatment plants (influent or effluent) were directly inoculated onto cells, and viruses were revealed after two passages with a liquid medium technique. Out of the total percentage of positive isolates with the three systems (32.7) 24.7% were found with Ma-104, 14.1% with BGM, and only 1.7% with Vero cells. Poliovirus was recovered more frequently with Ma-104 (12.3%) than with BGM (1.7%). Reovirus (3.5%) and echovirus (1.7%) were only found with Ma-104 cells; however, BGM cells allowed the isolation of a few group B coxsackieviruses (5.9%). It must be pointed out that 7.0% of samples with an unconfirmed cytopathic effect were found with BGM against 3.4% found with Ma-104, but they did not have significant differences. Because of its large spectrum of sensitivity, easy maintenance, and resistance to toxic effects, trypsin-treated Ma-104 may be recommended in conjunction with other cell lines for the detection of viruses from environmental samples, especially with the use of a liquid method.     

Trypsin-treated Ma-104: a sensitive cell line for isolating enteric viruses from environmental samples

During a 1-year survey of enteroviruses in wastewater samples from the Lorraine area, three widely used continuous monkey kidney cell lines were tested: BGM, Vero, and trypsin-treated Ma-104. Decontaminated samples from secondary wastewater treatment plants (influent or effluent) were directly inoculated onto cells, and viruses were revealed after two passages with a liquid medium technique. Out of the total percentage of positive isolates with the three systems (32.7) 24.7% were found with Ma-104, 14.1% with BGM, and only 1.7% with Vero cells. Poliovirus was recovered more frequently with Ma-104 (12.3%) than with BGM (1.7%). Reovirus (3.5%) and echovirus (1.7%) were only found with Ma-104 cells; however, BGM cells allowed the isolation of a few group B coxsackieviruses (5.9%). It must be pointed out that 7.0% of samples with an unconfirmed cytopathic effect were found with BGM against 3.4% found with Ma-104, but they did not have significant differences. Because of its large spectrum of sensitivity, easy maintenance, and resistance to toxic effects, trypsin-treated Ma-104 may be recommended in conjunction with other cell lines for the detection of viruses from environmental samples, especially with the use of a liquid method.