High-Depth 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 abundance of anelloviruses in such samples, which represented >98% 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.

Morphology of blood microbiota in healthy individuals assessed by light and electron microscopy

“…The direct and indirect evidence for the existence of viruses, bacteria and fungi in the blood of healthy individuals, residing as normal microflora has thrown light on their impact on host immune surveillance, and possibly on their role in chronic systemic inflammatory diseases (Potgieter et al, 2015;Cebria-Mendoza et al, 2021). Models describing the mechanisms for bacterial translocation from the gastrointestinal tract to extraintestinal sites, such as the extracellular interstitial space ("tissue space") of the body and bloodstream, predict pathology associated with: (a) disruption of the ecologic gastrointestinal equilibrium allowing intestinal bacterial overgrowth, (b) increased permeability of the intestinal mucosal barrier, and (c) deficiencies in host immune (Schatten et al, 1955;Berg, 1999).…”

“…The term blood microbiome refers to the collection of genomes or genomic fragments from all the microorganisms, that is the DNA and/or RNA, while microbiota refers to viruses (Liang and Bushman, 2021;Cebria-Mendoza et al, 2021), bacteria (Damgaard et al, 2015) and fungi (Panaiotov et al, 2018) found in the blood. Next-generation sequencing technology has been used to identify viruses, bacteria, archaea, and fungi in the blood of healthy individuals (Morgan and Huttenhower, 2012;Paisse et al, 2016;Panaiotov et al, 2021).…”

“…Convincing supportive evidence for the presence of microbiota in the blood of healthy individuals has been a few. (Tedeschi et al, 1969;Domingue and Schlegel, 1977;Kalfin, 1997;Damgaard et al, 2015;Markova et al, 2015;Dimova et al, 2017;Markova, 2017;Panaiotov et al, 2018;Cebria-Mendoza et al, 2021). For several decades, the scientific community debates whether normal microbial flora could be present in the blood of healthy individuals.…”