Saliva has emerged as a promising diagnostic fluid for viral infections, enabling the direct analysis of viral genetic material and the detection of infection markers such as proteins, metabolites, microRNAs, and immunoglobulins. This comprehensive review aimed to explore the use of saliva as a diagnostic tool for viral infections, emphasizing its advantages and limitations. Saliva stands out due to its simplicity and safety in collection, along with the convenience of self-collection without the need for healthcare supervision, while potentially being comparable to urine and blood in terms of effectiveness. Herein, we highlighted the significant potential of saliva in assessing viral loads and diagnosing viral infections, such as herpesviruses, HPV, PyV, TTV, SARS-CoV-2, and MPXV. The detection of viral shedding in saliva underscores its utility in early diagnosis, the monitoring of infection progression, and evaluating treatment responses. The non-invasive nature of saliva collection makes it an appealing alternative to more invasive methods, promoting better patient compliance and facilitating large-scale screening and surveillance. As such, we further highlight current evidence on the use of saliva as a prognostic tool. Although a significant amount of data is already available, further investigations are warranted to more comprehensively assess the added benefit from the utilization of salivary biomarkers in the clinics. Salivary biomarkers show great promise for the early detection and prevention of viral infection complications, potentially improving disease management and control at the population level. Integrating these non-invasive tools into routine clinical practice could enhance personalized healthcare strategies and patient outcomes. Future studies should focus on establishing standardization protocols, validating the accuracy of salivary diagnostics, and expanding clinical research to enhance the diagnostic and monitoring capabilities of salivary biomarkers.

Torquetenovirus (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. TTV 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. Preliminary 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. Collectively, 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.