Deep viral blood metagenomics reveals extensive anellovirus diversity in healthy humans

Abstract

Human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. By far, anelloviruses constitute the viral family that is more frequently found in human blood, although amplification biases and contaminations pose a major challenge in this field. To investigate this further, we subjected pooled plasma samples from 120 healthy donors in Spain to high-speed centrifugation, RNA and DNA extraction, random amplification, and massive parallel sequencing. Our results confirm the extensive presence of anelloviruses in such samples, which represented nearly 97% of the total viral sequence reads obtained. We assembled 114 different viral genomes belonging to this family, revealing remarkable diversity. Phylogenetic analysis of ORF1 suggested 28 potentially novel anellovirus species, 24 of which were validated by Sanger sequencing to discard artifacts. These findings underscore the importance of implementing more efficient purification procedures that enrich the viral fraction as an essential step in virome studies and question the suggested pathological role of anelloviruses.

Exploring the Diversity of the Human Blood Virome

“…Sixty-seven of the 69 contigs considered as complete genomes belonged to TTMV genus, and a single contig was assigned to each TTV and TTMDV genera. This is consistent with the presence of shorter GC-rich regions in TTMV 34 , which can increase assembly efficiency, as previously described 18 .…”

“…Finally, although our experimental approach allowed the detection of large viruses 18 , only marginal evidence of the presence of giant blood Marseille-like viruses was obtained (Supplementary Table S1), in agreement with previous studies 31,32 suggesting that this signature could also be a laboratory contaminant.…”

“…The remarkable diversity of this residual fraction strongly suggested that these reads may correspond to taxonomic misidentifications or amplification of small traces of nucleic acids present in the reagents used in our virus-enrichment protocol and that were not efficiently removed computationally. This was supported by the fact that ambiguities in the taxonomical classification of reads were not properly handled by Recentrifuge 18 , limiting our ability to remove potential contaminations corresponding to phylogenetically unclassified reads. A clear example of this is the detection of Circoviridae family, which represented the third most abundant family in our study, and which has been previously associated with contaminating reagents 30 .…”

“…For each of the 60 pools, we generated contigs from all reads regardless of their preliminary taxonomical classification, which is an effective approach for the detection of new anelloviruses 18,33 . Blast analyses allowed the detection of spiked and HPgV viruses, but most contigs corresponded to anelloviruses.…”

“…The methodology established for anellovirus species classification has been modified recently and the number of reference species has been updated accordingly. Consequently, we decided to reevaluate the data of a recent study in which we applied the same viral enrichment experimental and bioinformatics procedures to a smaller number of samples 18 . This reevaluation yielded 26 new species (6, 11, and 9, for TTV, TTMV, and TTMDV, respectively; Table 2 and Supplementary Tables S6-S8), which were subsequently included in the pool of reference species used for characterizing the sequences analyzed in the present study.…”