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Viruses are among the most important pathogens present in water contaminated with feces or urine and represent a serious risk to human health. Four procedures for concentrating viruses from sewage have been compared in this work, three of which were developed in the present study. Viruses were quantified using PCR techniques. According to statistical analysis and the sensitivity to detect human adenoviruses (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII): (i) a new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on the elution of the viruses with glycine-alkaline buffer followed by organic flocculation with skimmed-milk was found to be the most efficient method when compared to (ii) ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate sewage samples, ESMP showed reproducible results with a coefficient of variation (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked samples, the viral recoveries were estimated at 30-95% for HAdV, 55-90% for JCPyV and 45-50% for NoV GGII. ESMP was validated in a field study using twelve 24-h composite sewage samples collected in an urban sewage treatment plant in the North of Spain that reported 100% positive samples with mean values of HAdV, JCPyV and NoV GGII similar to those observed in other studies. Although all of the methods compared in this work yield consistently high values of virus detection and recovery in urban sewage, some require expensive laboratory equipment. ESMP is an effective low-cost procedure which allows a large number of samples to be processed simultaneously and is easily standardizable for its performance in a routine laboratory working in water monitoring. Moreover, in the present study, a CV was applied and proposed as a parameter to evaluate and compare the methods for detecting viruses in sewage samples.

Human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) have been proposed as markers of fecal/urine contamination of human origin. An indirect immunofluorescence assay has been developed to quantify infectious human adenoviruses types 2 and 41 and JC polyomaviruses strain Mad-4 in water samples. The immunofluorescence assay was compared with other quantitative techniques used commonly such as plaque assay, tissue culture infectious dose-50 and quantitative PCR (qPCR). The immunofluorescence assays showed to be specific for the detection of infectious viruses, obtaining negative results when UV or heat-inactivated viruses were analyzed. The assays required less time and showed higher sensitivity for the detection of infectious viral particles than other cell culture techniques (1 log(10) more) evaluated. River water samples spiked previously with human adenoviruses and raw sewage samples were also analyzed using the proposed immunofluorescence assay as well as by qPCR. The results show quantitations with 2 log(10) reduction in the numbers of infectious viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is fast, sensitive, specific, and a standardizable technique for the quantitation and detection of infectious viruses in water samples.

Adenoviruses and polyomaviruses are two distinct DNA viral families that are excreted in high concentrations and distributed in human and animal populations. Targeting specific virus included in these families has proved to be a promising and useful tool for tracing specifically sources of environmental contamination. In this study, a quantitative PCR assay that is specific for bovine polyomaviruses was developed and used to determine the excretion level and concentration of bovine polyomaviruses in urine and environmental samples, including urban sewage, slaughterhouse sewage, and river water. A set of primers and a TaqMan probe were designed to target a 77-bp region of the bovine polyomavirus VP1 gene, and the conditions of the reaction were optimized. A detection limit was established at 1-10 genome copies per test tube. The assay was specific and produced negative results when samples containing human or porcine fecal contamination were analyzed. This is, to our knowledge, the first description of bovine polyomaviruses excreted in bovine urine samples (mean values of 10(4) GC/l). Bovine polyomaviruses were also detected and quantified in slaughterhouse wastewater and river waters, which shows the spread of these viruses in many environmental samples containing contamination of bovine origin. The procedure described in this paper provides a quantitative source-tracking tool for the analysis of bovine polyomaviruses as indicators of the presence of bovine contamination in environmental samples.

Human adenovirus and JC polyomavirus have been proposed as viral indicators of human faecal contamination of water. This study compared concentration and nucleic acid extraction methods and defines a protocol for quantifying human adenoviruses (HAdV), JC polyomavirus (JCPyV) and noroviruses (NoV) in source and drinking water. River water samples and spiked tap water samples were used to evaluate virus recovery, applying quantitative PCR (qPCR) to five concentration methods. In the case of 10-L samples, the use of ultrafiltration cartridges produced acceptable recoveries for HAdV and JCPyV, but they were inefficient for noroviruses and could not be applied to high-volume and river water samples with medium turbidity. The glass wool method with pre-acidification gave similar recoveries and made it possible to detect NoV. In the case of 50-L samples, the method that produced the highest recovery efficiency and applicability was glass wool filtration. Comparing different sample volumes of a river used as source water showed that the largest number of viruses were quantified when lower volumes (1L) were tested (1.5 x 10(4) HAdV genome copies (GC)/L and 2.8 x 10(3) JCPyV GC/L). The methods developed are easy to standardize and may be valuable tools for the control of viral contamination in source water and for assessing the efficiency of virus removal in drinking water treatment plants.

The Adenoviridae family comprises a wide diversity of viruses that may be excreted for long periods in feces or urine. Previous studies have suggested that the detection of human and animal adenoviruses as well as human and animal polyomaviruses by PCR could be used as an index of fecal contamination of human and animal origin. In this study, quantitative PCR assays targeting specifically porcine adenoviruses have been developed and applied to fecal and environmental samples, including pig slurries, urban sewage, slaughterhouse sewage and river water samples. To develop real-time quantitative PCR for the detection and quantitation of porcine adenoviruses, primers and a TaqMan probe targeting a 68-bp region of the porcine adenovirus hexon gene were designed to amplify specifically porcine adenovirus, and the conditions of the reaction were optimized. The assay detected 1-10 genome copies per test tube and was specific in showing no positive results when samples containing human or bovine adenoviruses were analyzed. Fecal samples contained mean concentrations of porcine adenoviruses of 10(5) GC/g while slaughterhouse wastewater samples showed mean values of 10(3) GC/ml. The assay detected porcine fecal pollution in samples that were highly diluted and had been collected at a considerable distance from the input source, such as river water. In general, the data presented here provide a quantitative tool for the analysis of porcine adenoviruses as indicators of the presence of porcine contamination in the environment, and support the detection of porcine adenoviruses by real-time quantitative PCR as a promising and valuable tool for source-tracking studies.

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Viral outbreaks attributed to the consumption of contaminated shellfish have been clearly demonstrated. Thirty-five samples of mussels collected from areas with two different levels of faecal pollution were analysed for somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis HSP40 and RYC2056 following standardised protocols, and for enterovirus, human adenovirus and hepatitis A virus by nucleic acid amplification (Nested-PCR and RT-PCR). Four methods for viral recovery from shellfish have been compared. The first method is based on borate buffer at pH 9.5 as eluent, the second is based on glycine buffer at pH 10 as eluent, a third method is based on glycine buffer at pH 7.5 and changes in conductivity and the fourth method on nutritive broth with Tween 80 as eluent. The results obtained were analysed statistically and the method based in glycine buffer at pH 10 seems to be the most efficient and useful for the recovery of phages and human viruses. The results also show a different pattern in the proportions between the viral parameters when the source of the faecal pollution is close to or distant from the shellfish growing area.

A molecular test based on DNA amplification by PCR was developed for the detection of bacteriophages of Bacteroides fragilis strain HSP40 in the environment. These specific phages are associated with faecal contamination of human origin. A homologous DNA region of 1.5 kb, identified previously by hybridisation, was used to design primers for the detection of B. fragilis HSP40 phages. A nested-PCR procedure for the DNA amplification of those phages was developed. The sensitivity of the nested-PCR was between 10−1 and 10−2 PFU for purified HSP40 phage solutions, sewage and seawater samples, and between 1 and 10 PFU for river water samples. Specific amplification of HSP40 phages was observed when viral suspensions of 103 PFU/ml or lower were used. Common levels of B. fragilis phages found in sewage are 101–102 PFU/ml. A total of 24 water samples (sewage, river water and seawater) were tested both by PCR and by plaque assay, to evaluate the efficiency of the molecular method in field samples. The data obtained by PCR in environmental samples showed good concordance with the PFU counts and a higher sensitivity.

Nine bacteriophage isolates of Bacteroides fragilis, obtained from urban sewage and pig faeces samples using four different host strains (HSP40, RYC4023, RYC2056 and RYC3318), were compared on the basis of morphology, host range, DNA restriction patterns, DNA hybridisation and protein composition. All the phages are siphovirus and, as judged from cleavage by restriction endonucleases, their genome is composed of double-stranded DNA of similar size ( approximately 51-kb). Host range analysis differentiated two types of phages: (1) phages that clearly infect B. fragilis strains HSP40 (B40-2, B23-1, B23-2, B23-3 and B23-4, of which B40-8 is the phage type); and (2) the group of bacteriophages that were not infectious for HSP40 (B56-1, B56-2 and B18-1). Similarity in DNA restriction patterns and protein characteristics was found in the HSP40 infectious phages. Anti-B40-8 serum recognised only the proteins of the phages of this type. Although all phages showed similar major protein sizes, minor specific bands were detected. Bacteriophages B56-1, B56-2 and B18-1 showed heterogeneity in their DNA restriction profiles although some degree of DNA-DNA homology between all genomes was observed. Southern blot analysis with phage B40-8 DNA based probes identified a 1.5-kb DNA region homologous for all HSP40 phage isolates, but absent in the genome of the other phage isolates that did not infect this bacterial strain. The homologous region was used as a specific probe to specifically detect B. fragilis HSP40 phages.