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In Quito, the microbiological contamination of surface water represents a public health problem, mainly due to the lack of sewage treatment from urban wastewater. Contaminated water contributes to the transmission of many enteric pathogens through direct consumption, agricultural and recreational use. Among the different pathogens present in urban discharges, viruses play an important role on disease, being causes of gastroenteritis, hepatitis, meningitis, respiratory infections, among others. This study analyzes the presence of viruses in highly impacted surface waters of urban rivers using next-generation sequencing techniques. Three representative locations of urban rivers, receiving the main discharges from Quito sewerage system, were selected. Water samples of 500 mL were concentrated by skimmed-milk flocculation method and the viral nucleic acid was extracted and processed for high throughput sequencing using Illumina MiSeq. The results yielded very relevant data of circulating viruses in the capital of Ecuador. A total of 29 viral families were obtained, of which 26 species were associated with infections in humans. Among the 26 species identified, several were related to gastroenteritis: Human Mastadenovirus F, Bufavirus, Sapporovirus, Norwalk virus and Mamastrovirus 1. Also detected were: Gammapapillomavirus associated with skin infections, Polyomavirus 1 related to cases of kidney damage, Parechovirus A described as cause of neonatal sepsis with neurological affectations and Hepatovirus A, the etiologic agent of Hepatitis A. Other emergent viruses identified, of which its pathogenicity remains to be fully clarified, were: Bocavirus, Circovirus, Aichi Virus and Cosavirus. The wide diversity of species detected through metagenomics gives us key information about the public health risks present in the urban rivers of Quito. In addition, this study describes for the first time the presence of important infectious agents not previously reported in Ecuador and with very little reports in Latin America.

The application of next-generation sequencing (NGS) techniques for the identification of viruses present in urban sewage has not been fully explored. This is partially due to a lack of reliable and sensitive protocols for studying viral diversity and to the highly complex analysis required for NGS data processing. One important step towards this goal is finding methods that can efficiently concentrate viruses from sewage samples. Here the application of a virus concentration method based on skimmed milk organic flocculation (SMF) using 10 L of sewage collected in different seasons enabled the detection of many viruses. However, some viruses, such as human adenoviruses, could not always be detected using metagenomics, even when quantitative PCR (qPCR) assessments were positive. A targeted metagenomic assay for adenoviruses was conducted and 59.41% of the obtained reads were assigned to murine adenoviruses. However, up to 20 different human adenoviruses (HAdV) were detected by this targeted assay being the most abundant HAdV-41 (29.24%) and HAdV-51 (1.63%). To improve metagenomics' sensitivity, two different protocols for virus concentration were comparatively analysed: an ultracentrifugation protocol and a lower-volume SMF protocol. The sewage virome contained 41 viral families, including pathogenic viral species from families Caliciviridae, Adenoviridae, Astroviridae, Picornaviridae, Polyomaviridae, Papillomaviridae and Hepeviridae. The contribution of urine to sewage metavirome seems to be restricted to a few specific DNA viral families, including the polyomavirus and papillomavirus species. In experimental infections with sewage in a rhesus macaque model, infective human hepatitis E and JC polyomavirus were identified. Urban raw sewage consists of the excreta of thousands of inhabitants; therefore, it is a representative sample for epidemiological surveillance purposes. The knowledge of the metavirome is of significance to public health, highlighting the presence of viral strains that are circulating within a population while acting as a complex matrix for viral discovery. (c) 2017 Elsevier B.V. All rights reserved.

Farmed animals such as sheep, cattle, swine and poultry play an important role in microbial contamination of water, crops and food, and introduce large quantities of pathogens into the environment. The ability to determine the origin of faecal pollution in water resources is essential when establishing a robust and efficient water management system. Animal-specific viruses have previously been suggested as microbial source tracking tools, but specific ovine viral markers have not been reported before now. Previous studies have shown that polyomaviruses are host-specific, highly prevalent and are commonly excreted in urine. Furthermore, they have been reported to infect several vertebrate species but not sheep. That situation encouraged the study of a new putative ovine polyomavirus (OPyV) and its use to determine whether faecal pollution originates from ovine faecal/urine contamination. Putative OPyV DNA was amplified from ovine urine and faecal samples using a broad-spectrum nested PCR (nPCR). Specific nested PCR and quantitative PCR assays were developed and applied to faecal and environmental samples, including sheep slurries, slaughterhouse wastewater effluents, urban sewage and river water samples. Successful amplification by PCR was achieved in sheep urine samples, sheep slaughterhouse wastewater and downstream sewage effluents. The assay was specific and was negative in samples of human, bovine, goat, swine and chicken origin. Ovine faecal pollution was detected in river water samples by applying the designed methods. These results provide a quantitative tool for the analysis of OPyV as a suitable viral indicator of sheep faecal contamination that may be present in the environment.