Torquetenovirus detection in exosomes enriched vesicles circulating in human plasma samples

Abstract

BackgroundTorquetenovirus (TTV) belongs to Anelloviridae family, infects nearly all people indefinitely without causing overt disease establishing a fine and successful interaction with the host. Increasing evidence have shown some human viruses exploit extracellular vesicles thereby helping viral persistence in the host. Here, the presence of TTV in extracellular vesicles circulating in human plasma was investigated.MethodsTTV DNA was quantified in plasma-derived exosomes from 122 samples collected from 97 diseased patients and 25 healthy donors. Exosomes enriched vesicles (EEVs) were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81 and annexin II protein and, finally, by electron microscopy (EM). Presence and quantitation of TTV DNA were assessed with an universal single step real-time TaqMan PCR assay.ResultsPreliminary investigation showed that the human plasma extracted extracellular vesicles exhibited a main size of 70 nm, had concentration of 2.5 × 109/ml, and scored positive for tetraspanin CD63, CD81 and annexin II, typical characteristic of the exosomes vesicles. EEVs extracted from pooled plasma with TTV DNA viremia of 9.7 × 104 copies/ml showed to contain 6.3 × 102 TTV copies/ml, corresponding to 0.65% of total viral load. Important, TTV yield changed significantly following freezing/thawing, detergents and DNAse treatment of plasma before EEVs extraction. EEVs purified by sucrose-density gradient centrifugation and analysis of gradient fraction positive for exosomes marker CD63 harbored 102 TTV copies/ml. Moreover, EM evidenced the presence of TTV-like particles in EEVs. Successive investigation of plasma EEVs from 122 subjects (37 HIV-positive, 20 HCV infected, 20 HBV infected, 20 kidney transplant recipients, and 25 healthy) reported TTV DNA detection in 42 (34%) of the viremic samples (37 were from diseased patients and 5 from healthy people) at a mean level of 4.8 × 103 copies/ml. The examination of EEVs selected samples reported the presence of TTV genogroup 1, 3, 4 and 5, with genogroup 3 highly observed.ConclusionsCollectively, although these observations should be confirmed by further studies, circulation of TTV particles in EEVs opens new avenues and mechanistic insights on the molecular strategies adopted by anelloviruses to persist in the host.

Human anelloviruses: diverse, omnipresent and commensal members of the virome

“…Moreover, it was recently found that TTV particles can circulate in the body within exosomes (Martelli et al . 2018 ). Travelling within vesicles can promote spread of the virus by two means: it may enhance infectivity by allowing entry of TTVs in otherwise non-permissive cells, and the virus inside the exosomes may be less exposed to neutralizing antibodies (Martelli et al .…”

“…Travelling within vesicles can promote spread of the virus by two means: it may enhance infectivity by allowing entry of TTVs in otherwise non-permissive cells, and the virus inside the exosomes may be less exposed to neutralizing antibodies (Martelli et al . 2018 ).…”

Cancer Liquid Biopsy Using Integrated Microfluidic Exosome Analysis Platforms

“…Exosomes, small EVs with a diameter of 20–140 nm, [ 5 ] are secreted by most cells and are abundantly present in body fluids such as blood, [ 6 ] where the exosome concentration can exceed 10 9 mL −1 . [ 7 ] Exosomal lipid bilayer membranes permit them to stably carry genetic material and protein from their cells of origin (including early stage cancer cells) [ 8,9 ] and allow exosomes to serve as ideal tools for use in monitoring cancer occurrence and progression. [ 10,11 ]…”

Molecular epidemiology of Anellovirus infection in children's urine: A cross-sectional study

“…ORF1 contains hypervariable regions that are responsible for wide range of genetic variations. [ 4 ] Based on ORF1, the International Committee on Taxonomy of Viruses (ICTVs) defines three genera for human Anelloviruses, including Alpha Torque Teno, Beta Torque Teno, and Gamma Torque Teno viruses. The prototype of this family was isolated by Nishzawa et al .…”

“…Replication of these viruses occurs in various tissues, such as the liver, lymph nodes, spleen, lung, muscles, lymphocytes, and polymorphonuclear cells. [ 7 ] However, the main replication site of the virus has been reported to be in T-cell lymphocytes[ 4 ] and the replication of the virus may be performed by rolling circle mechanisms. [ 8 ]…”