High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage

Abstract

Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for high-throughput 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 plant-infecting 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.

Fig 1

Taxonomic distributions of the eukaryotic virus-related sequences from four sewage samples. Assembled sequences were compared to the sequences in the nonredundant protein database using BLASTx (E score, <10⁻⁴). The number of sequences with identities to eukaryotic viruses is shown. Twenty-nine eukaryotic virus families were detected from human, vertebrate, plant, and insect hosts (as well as unclassified viruses and satellites).

Fig 2

Sequence identity distribution analysis of eukaryotic viral sequences from the sewage metagenomes of four countries. Each dot represents an assembled sequence contig or singlet with the corresponding protein identity (best BLASTx match with an E score of <10⁻⁴) to a human, vertebrate, plant, or insect virus in the GenBank nonredundant database. The original pyrosequences used as anchor points for partial for genomes extensions are indicated by numbers, as follows: 1, KoV-SewKTM; 2, SaliV-SewBKK; 3, SaV-SewSFO; 4, AstV-casa; 5, PicaV; 6, SecaliV; 7, HepeV; 8, NediV; 9, CadiV; 10, NiflaV; 11, BamiV; 12, NimiV; 13, NepaV.

Fig 3

Genome organization and phylogenetic relationships of new kobuvirus and salivirus. (A) Genome organization of the kobuvirus sewage Kathmandu (KoV-SewKTM) and salivirus sewage Bangkok (SaliV-SewBKK). (B) Phylogenetic analysis of the two novel picornaviruses with known members of the Kobuvirus and Salivirus genera in the family Picornaviridae, using translated protein sequence alignments of P1 capsid and 3D polymerase regions and the maximum likelihood method. Aligned regions are shown by black bars.

Fig 4

Genome organization and phylogenetic relationship of the casa astrovirus (AstV-casa). (A) Genome organization of the casa astrovirus compared with human and turkey astroviruses; (B) phylogenetic analysis of the translated partial RdRP and the full capsid protein sequences of AstV-casa and representatives of the Mamastrovirus and Avastrovirus of the Astroviridae family using the maximum likelihood method.

Fig 5

Genome organization, phylogenetic analyses, and conserved motifs of the picalivirus (PicaV) with members of the Picornavirales and Caliciviridae. (A) Genome organization and conserved helicase and RdRP motifs of the picalivirus; (B) phylogenetic analyses of picalivirus using the maximum likelihood method; (C) conserved helicase and RdRP motifs of PicaV compared with those of members of the Picornavirales and Caliciviridae.

Fig 6

(A) Genome organization of secalivirus (SecaliV) compared with Norwalk virus (Caliciviridae). The partial genome of secalivirus consists of a partial capsid ORF, followed by two ORFs of unknown function. (B) Sequence identity comparisons of secalivirus and calicivirus diversity through sliding-window analyses of pairwise translated protein p distances of the capsid. (C) Phylogenetic analysis of the partial capsid region of secalivirus and representatives of the Caliciviridae and Picornavirales using the maximum likelihood method.

Fig 7

(A) Genome organization of hepelivirus (HepeV) compared with that of human hepatitis E virus. The partial genome of hepelivirus consists of a partial ORF1b encoding RdRP, followed by ORF2 encoding a capsid protein. (B) Sequence identity comparisons of hepelivirus and hepevirus diversity through sliding-window analyses of pairwise translated protein p distances of the RdRP and capsid. (C) Phylogenetic analysis of the partial RdRP region of hepelivirus and representatives of the Hepeviridae and other ssRNA viruses using the maximum likelihood method.

Fig 8

Phylogenetic relationships of nedicistrovirus, cadiscistrovirus, niflavirus, and other ssRNA viruses, based on the amino acid alignment of the RdRP region using the maximum likelihood method.

Fig 9

(A) Genome organization of the novel geminivirus-related viruses (baminivirus, niminivirus, and nepavirus) and two geminiviruses (reference species of begomoviruses and mastreviruses). Baminivirus and niminivirus both contained the nonanucleotide TAATATTAC within a stem-loop. (B) Phylogenetic analysis of the translated Rep protein sequences among niminivirus, baminivirus, and nepavirus members and representatives of Geminiviridae and other ssDNA viruses with circular ssDNA genomes using the maximum likelihood method. (C) Sequence identity comparisons of baminivirus and geminivirus diversity through sliding-window analysis of pairwise translated protein p distances in the Rep gene alignment. Niminivirus showed a similar result in sliding-window analysis (data not shown).

Fig 10

NCA of the genomes of novel viruses. Projections of the first two (most significant) canonical factors that differentiate host origins of the control sequences with known cellular hosts using mononucleotide and dinucleotide frequencies are shown. Points represent values for individual sequences, with 95% confidence ellipses positioned around the centroid of each group. Positions of the RNA virus sequences obtained in the current study are labeled A to L, and their provisional assignment by NCA is indicated in the color-coded key.