At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity.Importance At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that the viral universe is far more vast and diverse than previously suspected.

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A novel and simple procedure for concentrating adenoviruses from sea water samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters.

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23 24Viruses are among the most important pathogens present in water contaminated with 25 feces or urine and represent a serious risk to human health. Four procedures for 26 concentrating viruses from sewage have been compared in this work, three of which 27were developed in the present study. Viruses were quantified using PCR techniques. 28According to statistical analysis and the sensitivity to detect human adenoviruses 29 (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII), (i) a 30 new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on 31 the elution of the viruses with glycine-alkaline buffer followed by organic flocculation 32 with skimmed-milk was found to be the most efficient method when compared to (ii) 33 ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) 34 ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate 35 sewage samples, ESMP showed reproducible results with a coefficient of variation 36 (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked 37 samples, the viral recoveries were estimated at 30%-95% for HAdV, 55%-90% for 38JCPyV and 45%-50% for NoV GGII. ESMP was validated in a field study using 39