g Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for highthroughput viral discovery. A metagenomics analysis of purified viral particles in untreated sewage from the United States (San Francisco, CA), Nigeria (Maiduguri), Thailand (Bangkok), and Nepal (Kathmandu) revealed sequences related to 29 eukaryotic viral families infecting vertebrates, invertebrates, and plants (BLASTx E score, <10 ؊4 ), including known pathogens (>90% protein identities) in numerous viral families infecting humans (Adenoviridae, Astroviridae, Caliciviridae, Hepeviridae, Parvoviridae, Picornaviridae, Picobirnaviridae, and Reoviridae), plants (Alphaflexiviridae, Betaflexiviridae, Partitiviridae, Sobemovirus, Secoviridae, Tombusviridae, Tymoviridae, Virgaviridae), and insects (Dicistroviridae, Nodaviridae, and Parvoviridae). The full and partial genomes of a novel kobuvirus, salivirus, and sapovirus are described. A novel astrovirus (casa astrovirus) basal to those infecting mammals and birds, potentially representing a third astrovirus genus, was partially characterized. Potential new genera and families of viruses distantly related to members of the single-stranded RNA picorna-like virus superfamily were genetically characterized and named Picalivirus, Secalivirus, Hepelivirus, Nedicistrovirus, Cadicistrovirus, and Niflavirus. Phylogenetic analysis placed these highly divergent genomes near the root of the picorna-like virus superfamily, with possible vertebrate, plant, or arthropod hosts inferred from nucleotide composition analysis. Circular DNA genomes distantly related to the plantinfecting Geminiviridae family were named Baminivirus, Nimivirus, and Niminivirus. These results highlight the utility of analyzing sewage to monitor shedding of viral pathogens and the high viral diversity found in this common pollutant and provide genetic information to facilitate future studies of these newly characterized viruses.

Nucleic acids from an unidentified virus from ringed seals (Phoca hispida) were amplified using sequenceindependent PCR, subcloned, and then sequenced. The full genome of a novel RNA virus was derived, identifying the first sequence-confirmed picornavirus in a marine mammal. The phylogenetic position of the tentatively named seal picornavirus 1 (SePV-1) as an outlier to the grouping of parechoviruses was found consistently in alignable regions of the genome. A mean protein sequence identity of only 19.3 to 30.0% was found between the 3D polymerase gene sequence of SePV-1 and those of other picornaviruses. The predicted secondary structure of the short 506-base 5-untranslated region showed some attributes of a type IVB internal ribosome entry site, and the polyprotein lacked an apparent L peptide, both properties associated with the A recent analysis of lungs, lymph nodes, and nasal swabs from ringed seals hunted in 2000 to 2002 from Ulukhaktok (formerly known as Holman) on the shore of the Beaufort Sea demonstrated the presence of a virus causing strong cytopathic effects (CPE) in Vero cells. The causative agent of CPE passed through a 0.45-m filter was resistant to detergent inactivation and was therefore thought to be a nonenveloped virus. Here, we analyzed this virus using sequence-independent PCR amplification and sequence similarity searches. We report the full genome sequence of a novel picornavirus with a deep root on the Picornaviridae family phylogenetic tree. Consistent with the nomenclature for other picornaviruses, we suggest the name seal picornavirus 1 (SePV-1) for this new virus and propose that it represent the prototype of a new picornavirus genus. MATERIALS AND METHODS Extraction of viral nucleic acids.A nasal swab from a seal hunted in 2002 was used to inoculate Vero cells, which developed CPE. CPE was transferable to a fresh cell culture. The supernatant from this cell culture was used as the input to nonspecifically amplify viral nucleic acids. For enrichment of viral particles from the supernatant of infected cells, 2 ml of culture supernatant was clarified (5,000 ϫ g for 10 min) and filtered through a 0.45-m-pore-size sterile filter (Millipore HV Durapole) to remove large particles. To concentrate virus particles, 1.5 ml of filtrate was then centrifuged at 35,000 ϫ g for 3 h at 10°C, and the resulting pellet was resuspended in 100 l of water containing 1ϫ Turbo DNase buffer. To remove non-viral-particle-protected DNA from the cultured cells, 20 U of TurboDNase (Ambion) was added, followed by incubation at 37°C for 90 min. Particle-protected nucleic acids were then extracted using a viral RNA extraction kit (Qiagen).Random amplification, subcloning, and sequencing. Viral RNA was mixed with 50 pmol of primer RA01 (GCCGGAGCTCTGCAGATATCNNNNNNN NNN), denatured at 75°C for 5 min, and chilled on ice. A reaction mix of 9 l containing 4 l of 5ϫ first-strand buffer (Invitrogen), 1 l of 100 mM dithiothreitol (DTT), 1 l solution containing each deoxynucleoside triphosphate (dNTP) at 10 mM, 8 units of ...