A new perspective on Torquetenovirus DNAemia as a biomarker of immunosuppression and inflammationDavid Navarro1,2,31Microbiology Service, Clinic University Hospital, INCLIVA Health Research Institute, Valencia, Spain.2CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain.3Department of Medicine, School of Medicine, University of Valencia, Valencia, Spain.*Correspondence:David Navarro, Microbiology Service, Hospital Clínico Universitario, Instituto de Investigación INCLIVA, Av. Blasco Ibáñez 17, 46010 Valencia, Spain. Phone: 34(96)1973500; Fax: 34(96)3864173; Email: david.navarro@uv.es.Torquetenovirus (TTV) is a prototypical Anelloviridae species belonging to the genus Alphatorquevirus , which comprise small, non-enveloped particles harboring a single-stranded circular DNA genome of approximately 3,800 nucleotides in length containing at least four overlapping open reading frames (ORFs).1 Most individuals become infected with TTV early in childhood, leading to a lifelong persistent infection at multiple body sites, although no disease has been directly attributed to it;2 in fact, TTV is a highly abundant component of the human virome in healthy individuals and as such viral DNAemia is frequently measurable.3,4 TTV replication in humans is strictly controlled by T-cell–dependent effector mechanisms, which explains the relative stability of viral loads in blood in immunocompetent subjects.5 In contrast, TTV DNA load fluctuates widely in immunosuppressed individuals, such as solid organ transplant (SOT) recipients, showing a clear direct relationship between the magnitude of plasma viral loads and the degree of immunosuppression.6.7 In this sense, peripheral blood TTV DNA load behaves as an “immunometer” in SOT patients, as low viral loads may anticipate allograft rejection, whereas high viral loads may predict the occurrence of infectious events.6,7In non-canonically immunosuppressed populations, such as the elderly, TTV DNA loads are substantially higher compared with younger adults, and their magnitude has been associated with physical frailty and increased mortality risk, thus serving as a marker of immunosenescence.8 Data obtained from in vitro models indicate that TTV may stimulate inflammatory responses; conversely, pro-inflammatory conditions may enhance TTV replication.6 In this context, high plasma TTV DNA loads have been reported in individuals with chronic inflammatory lung disorders—such as asthma, chronic bronchiectasis, or chronic obstructive pulmonary disease—as well as in Crohn’s disease, when compared with healthy individuals.2,8 Moreover, elevated TTV DNAemia has been observed in patients with systemic inflammation, including those with bacterial sepsis or COVID-19.8Alphatorquevirus species exhibit extensive genetic diversity. Based on sequence identity within the ORF1 coding region, at least 20 human-infecting TTV species—which may co-exist within a single individual—have been identified. While certain species appear to be more prevalent in specific clinical contexts (e.g., TTV3, TTV5, and TTV9 in immunocompromised patients, or TTV1 and TTV2 in autoimmune disorders), the clinical relevance, if any, of TTV species diversity remains largely unknown.In this context, a recent study published in the Journal of Medical Virology by Novazzi and colleagues postulates, for the first time, that TTV species diversity may represent a novel marker of immune dysregulation in the elderly.9 The authors collected peripheral blood samples from 300 randomly selected, age-stratified adults as part of a multicenter European project primarily aimed at identifying biomarkers of human aging. Participants were grouped into three categories based on their age, degree of immunocompetence as inferred from the CD4/CD8 ratio and CMV IgG levels, and their overall inflammatory status as determined by several ad-hoc biomarkers such as C-reactive protein (CRP), α-2 macroglobulin, ferritin, ceruloplasmin and the neutrophil-to-lymphocyte ratio, among others.Three cohorts were established: cohort A consisted of seemingly immunocompetent young adults (35–45 years) with no signs of increased inflammation; cohort B comprised older individuals (65–75 years) displaying mildly reduced immunocompetence and low levels of inflammatory markers; and cohort C included aged individuals (65–75 years) with signs of immunosenescence and a relatively high inflammatory status.In addition to quantifying TTV DNA load in plasma samples using a laboratory-developed universal TTV assay, the authors employed a short-read next-generation sequencing approach (Illumina) to assess intra-host TTV species diversity. Interestingly, cohort C exhibited significantly greater richness and diversity compared with cohorts A and B, while these parameters did not differ substantially between cohorts A and B. Importantly, certain TTV species were associated with decreased CD4 and NK cell percentages, a low CD4/CD8 ratio, and a reduction in the number of T-cell receptor excision circles—all hallmarks of immunosenescence. Moreover, some TTV species appeared to be more prevalent in cohort C individuals compared with the other two cohorts, and notably, a direct correlation was found between the number of detected TTV species and age, degree of immune impairment, and TTV load. Additional evidence linking TTV species diversity to immune aging and inflammation was provided by experiments analyzing the association between TTV species diversity and PARP-1 (poly[ADP-ribose] polymerase-1) activity, as measured by flow cytometry (fluorescence-activated cell sorting). In these experiments, the activity of PARP-1—a key cellular stress regulator and strong promoter of inflammation10—was found to be increased in individuals displaying a higher number of TTV species. Finally, although no consistent association was found between species diversity and levels of inflammatory biomarkers, TTV DNA load was directly correlated with CRP and α-2-macroglobulin levels. From a mechanistic standpoint, the authors propose that the relationship between species diversity and age-related immune dysregulation may reflect decreased immune pressure, which would pave the way for the expansion of better-adapted TTV species with enhanced replication capacity or immunoevasion properties. Nevertheless, an enhanced direct pathogenetic effect of certain TTV species over others on immune homeostasis cannot be ruled out.An important drawback of the study is that sequential monitoring of TTV species diversity was not performed; plausibly, TTV species richness or diversity may vary over time even in non-severely immunosuppressed individuals. Despite the limitations of the study—clearly acknowledged by the authors—it paves the way for incorporating TTV species profiling into the toolbox of studies aiming to predict the risk of age-associated diseases pathogenetically linked to immunosenescence and systemic inflammation. Nevertheless, as the authors point out, well-powered prospective longitudinal studies are needed to validate this assumption.