In this study, the investigation of TTV status in the plasma of 56 subjects infected with SARS-CoV-2 developing different grades of COVID-19 severity had a prevalence of 59% of TTV DNA positivity, showing expression of at least one TTV miRNA in 94% of cases. The miRNA tth8 was detected in 91% of subjects, followed by miRNAs t3b (64%) and miRNA t1a (30%). These data confirmed that TTV miRNA expression can be present not only in TTV DNA-positive subjects but also in those where TTV DNA was not revealed [
11,
13]. These results are consistent with the presence of TTV miRNA expression independent of TTV replication. Such a model has been previously proposed for other persistent viruses [
23,
24]. Notably, although TTV DNA status was not associated with COVID-19 severity, miRNA tth8 was significantly associated.
COVID-19 has been associated not only with acute respiratory distress and pneumonia but also with chronic renal failure and myocardial inflammation [
14,
15,
24,
25]. The most common symptoms have been attributed to the elevation of a broad spectrum of inflammatory mediators associated with poor disease outcomes. In this context, the direct or indirect viral interaction with transcription factors regulating the inflammatory response in COVID-19 is not completely understood. As demonstrated for other viruses, studies on SARS-CoV-2 have explored the modification of the miRNA processing pathway as a potential key player in the pathogenesis of COVID-19 [
26,
27]. Thus, it cannot be ruled out that viral miRNAs encoded by persistent viruses to regulate their replicative cycle and escape immune response are also modulated, introducing additional cofactors in COVID-19 evolution. TTV miRNAs have been poorly investigated; although miRNA tth8 was initially associated with the regulation of the interferon pathway, such an association was not confirmed [
10,
28]. In this context, persistent viruses constituting the human virome (such as herpesvirus and Polyomavirus) were shown to express viral miRNAs that control host physiology by targeting multiple processes, including the immune response [
29]. Thus, viral modification of the miRNA processing pathway has been described as a potential key player in the regulation of the inflammatory response [
26]. Several studies have suggested that there is an association between TTV miRNA expression and inflammatory status. For instance, it was reported that TTV miRNAs exhibit differential expression in healthy or diseased patients, with the highest values in transplant recipients [
11,
13]. The miRNA t1a from TTV genogroup 1 was more prevalent in the plasma of diseased patients than of healthy donors (56% vs. 32%, respectively), whereas miRNAs of TTV genogroups 3 and 5 were not significantly different (87% and 51%, vs. 100% and 44%, for diseased and healthy subjects, respectively). Importantly, the overall prevalence of TTV genogroup 1 miRNAs was significantly higher in transplant recipients than in HIV- and HBV-infected patients [95% vs. 55% (11)]. Additionally, TTV miRNA t3b correlates with serum IL-6 levels, a marker of systemic inflammation, in elderly patients [
12]. Of note, in the present study, TTV miRNA t3b status was associated with COVID-19 severity, although a small sample size prevented reaching statistical significance. Notably, the association of SARS-CoV-2 and TTV with the expression of human miR-181a (a regulator of cell proliferation, apoptosis, mitochondrial function, and immune response) has been investigated in patients with acute lymphoblastic leukemia [
30,
31]. In that study, the expression of miR-181a was significantly increased in patients with COVID-19, whereas it was decreased in TTV-positive patients [
31]. Moreover, the human virome in nasopharyngeal swab samples of SARS-CoV-2 patients has been recently investigated, showing TTV positivity in two subjects [
32]. Thus, although it remains to be demonstrated, miRNA expression of a particular TTV genogroup present in the infected host might differentially modulate immune functionality, playing a role as an additional cofactor to the severity of the COVID-19 outcome.
Recently, oligoadenylate synthase-like (OASL) 1 protein, which is involved in the host's innate response against virus infection [
33], has been identified as a risk factor for COVID-19 susceptibility and severity [
34]. To date, TTV association with the inflammatory response has been reported for TTV ORF2 protein, which potentially interferes with the activity of NF-kB, a well-characterized transcription factor known to play a role in immune regulation [
35]. Additionally, TTV DNA was associated with dose-dependent expression and production of proinflammatory cytokines by robust activation of TLR-9 in ex vivo grown mouse spleen cells [
20].
Our study has several limitations, such as the small number of subjects enrolled. However, the relationship of TTV miRNA with the severity of the COVID-19 outcome should guide future studies investigating the role of the host virome at early and late time points after SARS-CoV-2 infection. This effort could be clinically relevant in the potential use of TTV miRNA expression to monitor host virome genetically acquired factor determining susceptibility to development of severe COVID-19 outcomes.