Finally, the extracted nucleic acids were subjected to random ϕ29-based amplification and library preparation. Pools were filtered to remove bacteria and cellular debris, subjected to high-speed centrifugation to pellet potential viruses and treated with nucleases to digest free nucleic acids, followed by extraction of both viral DNA and RNA independently. Briefly, 60 plasma pools from 8–13 individual plasma samples obtained from healthy donors were analyzed. We used a recently described experimental protocol for viral fraction enrichment. Anellovirus species demarcation was performed by checking nucleotide pairwise identity matrices obtained independently for each genus. The final trees were annotated with EvolView. The reliability of the phylogenetic results was assessed using 1000 bootstrap pseudo-replicates. Analyses were performed using the best-fit nucleotide substitution model, identified as GTR + Г + I using Akaike information criterion. Sequence alignment (based on the amino acid sequences) was performed with MUSCLE as implemented in MEGA version X, and subsequent phylogenetic inference using nucleotide sequences was conducted with the maximum likelihood (ML) method, also implemented in MEGA version X. Regarding HPgV phylogenetic analysis, nucleotide sequences of the complete polyprotein corresponding to isolates available from Genbank by March 2021 were downloaded ( Supplementary Table S3). To study phylogenetic relationships within the family Anelloviridae, nucleotide ORF1 sequences from hominid TTV, TTMV, and TTMDV accepted as reference species by ICTV were downloaded ( Supplementary Table S4). Then, taxonomical information obtained from blanks was bioinformatically subtracted from actual samples. To control for environmental contaminants in materials and reagents, eight blank samples containing 10 mL PBS 1X were processed in parallel with the rest of the samples. Then, 240 µL supernatant was transferred to a new tube and split into two fractions: 200 µL fraction was used for RNA extraction using TRIzol LS reagent (Invitrogen, Carlsbad, USA), followed by purification with the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) and amplification with the QuantiTect Whole Transcriptome kit (Qiagen), and 40 µL fraction was used for DNA extraction with the QIAamp Viral RNA Mini kit and amplification with the TruePrime WGA kit (Sygnis, Heidelberg, Germany). After incubation (1 h, 37 ☌), 20 µL of stop reagent was added, following the manufacturer’s instructions. Then, 5 µL of Turbo DNase, 2 µL of Benzonase (Sigma, Darmstadt, Germany) and 2 µL of micrococcal nuclease (NEB) were added to the sample to remove unprotected nucleic acids. Briefly, plasma pools were processed with 1.0 µM filters to remove cells and other non-viral particles and the filtered fractions were subject to high-speed centrifugation (87,000 g, 2 h, 4 ☌), washed with PBS 1X (87,000 g, 1 h, 4 ☌), and resuspended in 245 µL 1X digestion buffer (Turbo DNA Free kit, Ambion, Carlsbad, CA, USA). The purification protocol has been previously described in detail. To assess viral recovery, each pool was spiked with 10 3 PFU of ϕX174 and 10 4 PFU of vesicular stomatitis virus (VSV). In-depth investigation of the human blood virome should help to elucidate the ecology of these viruses, and to unveil potentially associated diseases.Įach of the 60 pools (SP1-SP60) analyzed in this study was obtained by mixing 1 mL of plasma from a variable number of donors (between 8- and 13-mL total). HPgV was much less frequent, but we, nevertheless, recovered 17 different isolates that we subsequently used for characterizing the diversity of this virus. In total, we assembled 332 complete or near-complete anellovirus genomes, 50 of which could be considered new species. This showed that anelloviruses were clearly the major component of the blood virome and showed remarkable diversity. To shed light on the diversity of the human blood virome, we subjected pooled plasma samples from 587 healthy donors in Spain to a viral enrichment protocol, followed by massive parallel sequencing. However, these studies have revealed the common presence of apparently non-pathogenic viruses in blood, particularly human anelloviruses and, to a lower extent, human pegiviruses (HPgV). In this vein, studies of the human blood virome are often motivated by the search for new viral diseases, especially those associated with blood transfusions. The vast expansion of currently known viral diversity has revealed a large fraction of non-pathogenic viruses, and offers a new perspective in which viruses function as important components of many ecosystems. However, metagenomics has also changed our understanding of viruses in general. Metagenomics is greatly improving our ability to discover new viruses, as well as their possible associations with disease.
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