Of the novel circular sequences detected in the survey, 1844 encode genes with similarity to proteins of ssDNA viruses and 55 encode genes with similarity to dsDNA viral proteins (Figure 1A). The large majority of genomes from this study are highly divergent from RefSeq entries (Figure 1—figure supplement 1). We discovered 868 genomes that had similarity to unclassified eukaryotic viruses known as circular replication-associated protein (Rep)-encoding single-stranded DNA (CRESS) viruses. The group is defined by the presence of a characteristic rolling circle endonuclease/superfamily three helicase gene (Rep) (Zhao et al., 2019; Kazlauskas et al., 2019), but has not been assigned to families by the ICTV or RefSeq. We estimate that 199 non-redundant unclassified CRESS virus genomes had been previously deposited in GenBank, and 85 are curated in RefSeq (Figure 1B). Also abundant was the viral family Microviridae, a class of small bacteriophages, with 670 complete genomes. This represents a substantial expansion beyond the 459 non-redundant microvirus genomes previously listed in GenBank (of which 44 were curated in the RefSeq database). Other genomes that were uncovered represent Anelloviridae (n = 170), Inoviridae (n = 70), Genomoviridae (n = 58), Siphoviridae (n = 18), unclassified phage (n = 14), Podoviridae (n = 10), Myoviridae (n = 7) unclassified virus (n = 6), Papillomaviridae (n = 4), Circoviridae (n = 3), unclassified Caudovirales (n = 3), Bacilladnaviridae (n = 2), Smacoviridae (n = 2), and CrAssphage-like (n = 2) (Figure 1B, Supplementary file 2). Viral families were found in association with 23 different animal species (Figure 1C). It was not surprising to find bacterial viruses, as all animals are presumed to have microbial communities and our sampling included tissues where these communities reside.

Figure 1 with 3 supplements with 3 supplements see all Download asset Open asset Novel viruses associated with animal samples. Gross characterization of viruses discovered in this project compared to NCBI RefSeq virus database entries. (A) Pie chart representing the number of viral genomes in broad categories. (B) Bar graph showing the number of new representatives of known viral families or unclassified groups. (C) Heatmap reporting number of genomes found associated with each animal species. Number of samples per species in brackets. Note that genomes in this study were assigned taxonomy based on at least one region with a BLASTX hit with an E value <1 × 10−5, suggesting commonality with a known viral family. Some genomes may ultimately be characterized as being basal to the assigned family.

It is difficult to assign a host to most of the viruses from this study due to their divergence from known viral sequences. However, we searched the CRISPR database at (https://crispr.i2bc.paris-saclay.fr/crispr/BLAST/CRISPRsBlast.php), and three viruses had exact matches to CRISPR spacers in bacterial genomes (Siphoviridae sp. ctcj11:Shewanella sp. W3-18-1, Inoviridae sp. ctce6:Shewanella baltica OS195, Microviridae sp. ctbe523:Paludibacter propionicigenes WB4) and one virus had an exact match to the CRISPR spacer of an archaeon (Caudovirales sp. cthg227:Methanobrevibacter sp. AbM4), implying that these organisms are infected by these viruses. Further, the 142 anelloviruses found in human blood samples (Supplementary file 2) are almost certain to be bona fide human viruses based on their relatedness to known human anelloviruses.

In addition to circular genomes with recognizable similarity to known viruses, 609 circular contigs appeared to represent elements that lacked discernable similarity to known viruses (Figure 1A,C).

The vast majority of the de novo assembled circular genomes were <10 kb in length (Figure 1—figure supplement 2). This is largely due to the fact that large genomes are typically more difficult to de novo assemble from short reads. Despite these technical obstacles, our detection of a new tailed bacteriophage with a 419 kb genome (Myoviridae sp. isolate ctbc_4, GenBank Accession: MH622943), along with 45 other >10 kb circular sequences (Figure 1—figure supplement 2), indicates that the methods used for the current work can detect large viral genomes.

There has been a recent renewal of interest in the hypothesis that viruses may be etiologically associated with degenerative brain diseases, such as Alzheimer's disease (Itzhaki et al., 2016; Eimer et al., 2018). Conflicting literature suggests the possible presence of papillomaviruses in human brain tissue (Coras et al., 2015; Chen et al., 2012). Samples of brain tissue from individuals who died of Alzheimer’s disease (n = 6) and other forms of dementia (n = 6) were subjected to virion enrichment and deep sequencing. Although complete or partial genomes of known papillomaviruses, Merkel cell polyomavirus, and/or anelloviruses were observed in some samples (Supplementary file 3), no novel complete viral genomes were recovered (Supplementary file 2). No viral sequences were detected in a follow-up RNA deep sequencing analysis of the brain samples. It is difficult to know how to interpret these negative data. It is conceivable that the known viral DNA sequences observed in the Optiprep-RCA samples represent virions from blood vessels or environmental sources.

It has recently become apparent that certain nucleic acid extraction reagents are contaminated with viral nucleic acids (Asplund et al., 2019). To ensure we were not merely reporting the sequences of the ‘reagent virome,’ we performed our wet bench and bioinformatic pipeline on three independent replicates of reagent-only samples. We found no evidence of sequences of any viruses reported here or elsewhere. Further, cross-sample comparison of contigs showed that almost no sequences were found in different animal samples, aside from technical replicates. In total, six viral genomes were observed in multiple unrelated samples from at least two sequencing runs (Supplementary file 4). It is unclear whether this small minority of genomes (0.24% of the genomes reported in the current study) represent reagent contamination, lab contamination, or actual presence of the sequences in different types of samples.

Given the stringent requirements for sequences to be considered as belonging to a complete viral genome, as well as the largely unexplored nucleotide space of the virome, it is unsurprising that, in most samples, most reads did not align to the genomes reported in this study or virus genomes from RefSeq (Figure 1—figure supplement 3) (Supplementary file 5).