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The use of lagooning as a complementary natural method of treating secondary effluents of wastewater treatment plants has been employed as an affordable and easy means of producing reclaimed water. However, using reclaimed water for some purposes, for example, for food irrigation, presents some risks if the effluents contain microbial pathogens. Classical bacterial indicators that are used to assess faecal contamination in water do not always properly indicate the presence of bacterial or viral pathogens. In the current study, the presence of faecal indicator bacteria (FIB), heterotrophic bacterial counts (HBC), pathogens and opportunistic pathogens, such as Legionella spp., Aeromonas spp., Arcobacter spp., free-living amoeba (FLA), several viral indicators (human adenovirus and polyomavirus JC) and viral pathogens (noroviruses and hepatitis E virus) were analysed for 1 year in inlet and outlet water to assess the removal efficiency of a lagooning system. We observed 2.58 (1.17-4.59) and 1.65 (0.15-3.14) log reductions in Escherichia coli (EC) and intestinal enterococci (IE), respectively, between the inlet and outlet samples. Genomic copies of the viruses were log reduced by 1.18 (0.24-2.93), 0.64 (0.12-1.97), 0.45 (0.04-2.54) and 0.72 (0.22-2.50) for human adenovirus (HAdV), JC polyomavirus (JCPyV) and human noroviruses (NoV GI and GII), respectively. No regrowth of opportunistic pathogens was observed within the system. FLA, detected in all samples, did not show a clear trend. The reduction of faecal pathogens was irregular with 6 out of 12 samples and 4 out of 12 samples exceeding the EC and IE values, specified in the Spanish legislation for reclaimed water (RD 1620/2007). This data evidences that there is a need for more studies to evaluate the removal mechanisms of lagooning systems in order to optimize pathogen reduction. Moreover, surveillance of water used to irrigate raw edible vegetables should be conducted to ensure the fulfilment of the microbial requirements for the production of safe reclaimed water.

Disinfection by low-pressure monochromatic ultraviolet (UVC) radiation (253.7 nm) became an important technique to sanitize drinking water and also wastewater in tertiary treatments. In order to prevent the transmission of waterborne viral diseases, the analysis of the disinfection kinetics and the quantification of infectious viral pathogens and indicators are highly relevant and need to be addressed. The families Adenoviridae and Polyomaviridae comprise human and animal pathogenic viruses that have been also proposed as indicators of fecal contamination in water and as Microbial Source Tracking tools. While it has been previously suggested that dsDNA viruses may be highly resistant to UVC radiation compared to other viruses or bacteria, no information is available on the stability of polyomavirus toward UV irradiation. Here, the inactivation of dsDNA (HAdV2 and JCPyV) and ssRNA (MS2 bacteriophage) viruses was analyzed at increasing UVC fluences. A minor decay of 2-logs was achieved for both infectious JC polyomaviruses (JCPyV) and human adenoviruses 2 (HAdV2) exposed to a UVC fluence of 1,400 J/m(2), while a decay of 4-log was observed for MS2 bacteriophages (ssRNA). The present study reveals the high UVC resistance of dsDNA viruses, and the UV fluences needed to efficiently inactivate JCPyV and HAdV2 are predicted. Furthermore, we show that in conjunction with appropriate mathematical models, qPCR data may be used to accurately estimate virus infectivity.

Environmental factors are highly relevant to the global dissemination of viral pathogens. However, the specific contribution of major effectors such as temperature and sunlight on the inactivation of waterborne viruses is not well characterized. In this study, the effect of temperature (7, 20, and 37 °C), UVB and UVA radiation on viral inactivation was evaluated in phosphate buffered saline (PBS), mineral water, wastewater, 1,000-fold diluted wastewater and seawater. The stability of human adenoviruses infectivity, known as human pathogens and indicators of fecal contamination, was monitored during 24 h, both in the dark and exposed to UV radiation by immunofluorescence assays. In the dark, no Human adenovirus (HAdV) inactivation was observed in PBS and mineral water at any of the temperatures studied, whereas at 37 °C in reactors with higher microbial concentration (wastewater, diluted wastewater, and seawater), decays between 2.5 and 5 log were recorded. UVB radiation showed a dramatic effect on HAdV inactivation and 6-log were achieved in all reactors by the end of the experiments. The effect of UVA showed to be dependent on the water matrix analyzed. At 20 °C, HAdV showed a 2-log decay in all reactors radiation while at 37 °C, results in wastewater, diluted wastewater, and seawater reactors were equivalent to those observed in the dark. These results suggest UVB radiation as the major environmental factor challenging viral inactivation, followed by biotic activity indirectly associated to higher temperatures and finally, by UVA radiation.

JC virus is etiologically associated with a fatal demyelinating disease known as PML. JCV produces persistent infections in the kidney and is excreted in the urine of healthy individuals and in the urine of PML patients. The characteristics of the JCV excreted in the environment have been studied by analyzing sewage samples from divergent geographical areas. The intergenic region of JCV strains detected in the sewage of Barcelona (Spain), Umeå (Sweden), Nancy (France), Pretoria (South Africa), Patras (Greece), Cairo (Egypt), Washington, D.C. (USA), and diverse areas of Northern India has been sequenced, and the phylogenetic analysis showed their relationships with JCV strains previously described in urine or clinical samples in these geographic areas. The JCV regulatory region of the JCV DNA detected in sewage presented archetypal or archetypal-like regulatory regions with the exception of one of the twenty clones obtained from a sewage sample of the area of Washington, D.C. that presented a tandem repeated structure. Infectivity studies showed that archetypal JCV present in the urine of a pregnant woman productively infected SVG cells. Also JC viral particles showed considerable stability in sewage at 20 degrees C and in front of treatments with acidic pH and trypsin. The high prevalence of JCV in urine and in sewage and the stability of the viral particles observed suggests that contaminated water, food, and fomites could be the vehicles of JCV transmission through the oral route. Virions partially degraded or noninfectious could be a source of JCV DNA and may represent an additional mechanism of entry of viral genes into cells.

JC virus (JCV) particles have been detected in urban sewage of divergent geographical areas. In this study, the authors evaluate the genetic characteristics and the infective capabilities of JCV strains in relation to the potential oral transmission of JCV in the population. JCV strains excreted in urine and detected in sewage have been described as presenting archetypal structure of the regulatory region of the viral genome. The regulatory region of JCV viral particles detected in two urban sewage samples have been cloned and characterized. From a total of 40 clones tested, 39 presented archetypal-like regulatory regions, whereas 1 of the clones analyzed presented a tandem repeated structure. Archetypal strains present in the urine of a pregnant woman were able to infect SVG cells, producing infectious virions, as demonstrated by confirmative cell culture, electron microscopy, and in situ DNA hybridization. This is the first description of archetypal JCV productive infection of SVG cells. SVG cells were also successfully infected with Mad-4 JCV viral particles subjected to pH 3 for 1 h at 37 degrees C and to 10 microg/ml of trypsin in the same conditions. A decrease in the viral progeny production was observed when Mad-4 was subjected to acidic pH. Mad-4 did not produce any detectable infection in the enteric cell line CaCo-2. The oral route could represent a significant route of transmission of JCV infections because JCV virions have demonstrated relative resistance in the environment and to some of the conditions present in the gastrointestinal tract. The archetypal strains commonly detected in the environment may be implicated in the transmission of JCV among the population. Sporadic infection with strains presenting tandem repeated structures may have implications in pathogenicity.