Lisa Macera

Torquetenovirus viremia kinetics after autologous stem cell transplantation are predictable and may serve as a surrogate marker of functional immune reconstitution

BACKGROUND It is common experience that retreating patients too early after a course of intensive chemotherapy predisposes to opportunistic infections despite apparently normal lymphocyte levels. OBJECTIVES The extent of replication of persistent viruses that cause no obvious disease (and hence need no treatment) might better define when a patient has recovered from functional immune deficiency. STUDY DESIGN We used real-time polymerase chain reaction to monitor the kinetics of plasma torquetenovirus (TTV) viremia in hematological patients undergoing autologous hematopoietic stem cell transplantation as support to high-dose chemotherapy (HSCT). RESULTS Independently from underlying hematological disease and therapeutic regimen, TTV viremia increased post-HSCT, and this increase paralleled the increase of circulating CD8(+)CD57(+) T lymphocytes, known to represent an indirect marker of functional immune deficiency. Subsequently, within a matter of months, TTV levels returned to baseline values, at a pace that appeared to be constant over time. CONCLUSION Monitoring of TTV viremia represents a unique opportunity to follow functional immune reconstitution in immunosuppressed patients. Also, the size of the TTV viremia increases resulting from immunosuppressive treatments might be of guidance in determining the appropriate time interval before delivery of a next course of therapy.

Genetic variability and phylogeny of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) based on complete genomes

Thirteen genomes of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) were obtained to examine the diversity and evolution of swine TTVs. Despite the low nucleotide identity reported, the genomic organization and transcriptional profiles of TTVs are similar. The nucleotide diversity for TTSuV1 was higher than TTSuV2, and the pattern of mutation among the ORFs was also different. Phylogenetic and genetic analyses support the proposed division of TTV into two species. TTSuV1 showed high levels of variability (>30%), with three different types (the third described for the first time) that may display a geographical structure. In contrast, TTSuV2 showed lower levels of variability (<15%), and no different types could be described. Larger values for the ratios of synonymous (dS) to non-synonymous (dN) base substitutions (dS/dN) were reported for the ORFs pointing to a certain level of selective constraint in TTV genomes.

Torquetenovirus DNA drives proinflammatory cytokines production and secretion by immune cells via toll-like receptor 9

Active infection with torquetenovirus (TTV) has been associated with an increased severity of diseases in which inflammation plays a particularly important pathogenetic role. Here, we report that cloned DNA of a genogroup 4 TTV (ViPiSAL) is an activator of proinflammatory cytokine production by murine spleen cells and that the effect is mediated via toll-like receptor (TLR)9. The same DNA also increased the levels of proinflammatory cytokines induced by two well-characterized TLR9 stimulants. Finally, in silico analyses of the genomes of ViPiSAL and other TTVs revealed marked differences in the representation of CpG motifs known to be most effective at activating immune cells via TLR9. These findings demonstrate for the first time that at least one TTV isolate has the potential to stimulate and co-stimulate inflammatory responses.

Human Gyrovirus DNA in Human Blood, Italy

HGyV in blood suggests the infection might be systemic.

Torque Teno Virus Viremia Correlates With Intensity of Maintenance Immunosuppression in Adult Orthotopic Liver Transplant

TO THE EDITOR—We read with interest the recent article by Béland et al, detailing torque teno virus (TTV) kinetics in pediatric orthotopic liver transplant (OLT) recipients [1]. Interestingly, the authors show that TTV load increased with the number of immunosuppressive agents received. TTV viremia, which is normally at about 2 log10 in healthy subjects, has been shown to increase (up to 4 log10) in almost every condition of chronic immunosuppression (congenital, acquired, or iatrogenic); our group and others have reported high TTV viremia in congenital mannose-binding lectin deficiencies [2], HIV infection [3], cancer [4], heart and lung transplant [5], and high-dose chemotherapy for lymphomas and myelomas. High TTV viremia correlates with high CD27 dysfunctional Blymphocyte [3] and high CD8CD57 dysfunctional T-lymphocyte [6] counts in peripheral blood. In the setting of solid organ transplant, De Vlaminck et al have recently reported that TTV viremia correlates with intensity of maintenance immunosuppression in heart and lung transplant recipients [5]. In OLT, we and others previously reported that TTV DNA load increased significantly after transplant (P < .001) and that, in accordance with the report by Béland et al, TTV DNA was significantly higher in patients on calcineurin inhibitors plus azathioprine or mycophenolate mofetil (CNI + AZA/MMF) than in patients on CNI alone (P = .04) at 3 months after OLT [7]. Because CNIs have severe toxicities in liver transplant recipients and strong iatrogenic immunosuppression may cause hepatitis B virus (HBV) and hepatitis C virus (HCV) reactivation in OLT recipients, some centers have introduced protocols combining low-dose CNIs and extracorporeal photopheresis (lowCNI + ECP) [8, 9], with positive clinical results (reduced numbers of HCV reactivations [8]).We investigated herewhether such improved hepatitis control throughout the first year after transplant could be due to better immune competence (reduced iatrogenic immunosuppression), using TTV viremia as a surrogate marker. Forty-six adult patients with HBV/ HCV-related cirrhosis undergoing consecutive OLT at the Liver Transplant Centre of the Azienda Ospedaliero– Universitaria Pisana in 2009–2011 were enrolled in the low-CNI + ECP study. Preand posttransplant peripheral blood serum samples were obtained from these patients after they provided written informed consent during ECP visits at 3, 6, and 12 months after transplant. Retrospective preand posttransplant samples from historical controls treated with standard immunosuppression in 1996– 2001, namely, CNI monotherapy (n = 19) or CNI + AZA/MMF (n = 6), were provided by the University of Padua Transplant Centre, as previously published [7]. Cyclosporine A C2 levels were maintained at 800–1200 ng/mL for the first 3 months, then at 600–800 ng/ mL up to 6 months and 400–600 ng/mL up to 12 months after liver transplant. Tacrolimus trough levels were kept at 10–15 ng/mL for the first 6 months, then at 8–12 ng/mL up to 12 months after OLT. Azathioprine (1.5 mg/kg) up until the year 2000 and mycophenolate mofetil (1.5–2 g) thereafter were added when the patient’s serum creatinine level was >200 μmol/L, to minimize the cyclosporine or tacrolimus dosage. In all 3 treatment arms, 70% of all patients received induction immunosuppression

A novel rolling circle amplification assay to detect members of the family Anelloviridae in pigs and humans

The study describes a novel rolling circle amplification (RCA) assay to detect members of the family Anelloviridae from swine and human serum samples. The RCA was carried out using short anellovirus (AV)-specific primers based on a highly conserved region among available AV full-length genomes. Then, RCA products were used as templates to amplify full-length genomes with an AV-specific PCR. Amplification products were separated by agarose gel electrophoresis and full-length genomes were isolated based on the known size. With this novel AV-RCA/PCR approach it was possible to detect Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2) in swine and many species of Torque teno virus (TTV) in human sera, which were previously tested negative by conventional PCRs.

Assessment of the risk of polyomavirus JC reactivation in patients with immune-mediated diseases during long-term treatment with infliximab

Polyomavirus JC (JCV) reactivation causing progressive multifocal leukoencephalopathy is a main concern during biological therapies. Here, JCV reactivation in patients suffering from immune-mediated diseases after a long-term treatment with anti-tumor necrosis factor alpha (TNF-α) inhibitor infliximab was investigated. Peripheral mononuclear blood cells (PBMC), plasma and urine samples were obtained from 61 immune-mediated diseases patients treated or not with infliximab in combination with steroid and other immunomodulators and from 20 healthy donors. JCV DNA was transiently detected in 12 PBMC of 40 patients at different doses of infliximab with a higher prevalence than that of the 21 patients untreated. Conversely, a stable JCV positivity in urine of treated and untreated patients was detected. Sequencing the noncoding control region (NCCR), all samples exhibited the archetype structure with few mutations in transcriptional factor binding regions. The consequence of anti-TNF-α treatment on viral persistence was examined monitoring Torquetenovirus viremia and investigating the TNF-α-induced microRNA regulators of transcriptional factors, with a binding site on NCCR. Although infliximab treatment in this study did not affect directly JCV reactivation, further investigation on host factor(s) regulated by it will be of warranty in the understanding the mechanism(s) that may affect viral persistence.

Dynamics of Torque Teno virus plasma DNAemia in allogeneic stem cell transplant recipients

BACKGROUND Torque Teno virus (TTV) plasma DNA load directly correlate with the level of immunosuppresion in different clinical settings. It is uncertain whether this may be the case in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). OBJECTIVES We characterized the dynamics of TTV DNAemia in patients undergoing T-cell replete allo-SCT. STUDY DESIGN Retrospective single-center observational study including 72 allo-HSCT patients. Plasma TTV DNA loads were quantified before initiating the conditioning regimen and at different time-points after transplant by real-time PCR. White blood cells (WBC) and absolute lymphocyte counts (ALC) were measured by flow cytometry. RESULTS A dramatic drop in plasma TTV DNA load was observed shortly after conditioning. The TTV DNA load increased steadily after engraftment reaching its peak at day +90 after transplant. The increase in TTV DNA load paralleled that of ALC, and was of greater magnitude in patients who developed severe (grades II-IV) acute graft vs. host disease. CONCLUSION Repopulation of lymphocytes early after allo-HSCT correlates with an increase of plasma TTV DNA load. Prospective studies are nevertheless needed to determine whether the kinetics of TTV DNAemia may allow inference of the degree of overall immunocompetence in these patients.

Investigation on torquetenovirus (TTV) microRNA transcriptome in vivo.

Torquetenovirus (TTV) is a widespread anellovirus that establishes persistent infections in human showing an increased viremia in immunosuppressed patients. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Here, the presence of TTV DNA and miRNAs expression was investigated in plasma samples of 77 diseased (20 infected with human immunodeficiency virus (HIV), 18 infected with hepatitis B (HBV) virus, 18 infected with hepatitis C (HCV) virus, 21 solid organ transplanted) patients, and 25 healthy controls. TTV prevalence was significantly different in healthy controls (60%, 15/25) versus diseased patients (80%, 62/77), showing the highest TTV loads in transplant recipients. Genetic TTV analysis showed the highest prevalence of group 1, followed by groups 3, 4 and 5, and a lack of isolates of group 2. The expression of at least one TTV miRNAs of group 1, 3 and 5 was found in exosomes of plasma of the great majority of individuals (96%, 98/102 subjects) showing the higher prevalence of miRNAs of TTV group 3 (90%, 92/102), followed by miRNAs of group 1 (66%, 67/102), and miRNA of group 5 (49%, 50/102). TTV miRNAs expression and TTV viremia were not always directly correlated, and significant differences appeared in production of some TTV miRNAs between healthy controls and diseased patients. The reported TTV miRNAs status in exosomes encourages further investigation to understand their potential role in the expansion of anelloviruses upon immunosuppression.

Cyclovirus Vietnam DNA in immunodeficient patients

BACKGROUND Cyclovirus Vietnam (CyCV-VN) is a CyCV detected in 2013 from cerebrospinal fluid (CSF) samples of patients with neurological disorders. Information on prevalence, pathogenesis and disease association of CyCV-VN is still very patchy. OBJECTIVES AND STUDY DESIGN In this study, we have used a PCR assay targeting the Rep gene to investigate the prevalence of CyCV-VN infection in blood and CSF samples of 346 Italian subjects. RESULTS Overall, 7% of blood samples were positive for CyCV-VN while the virus was not detected in any of the CSF samples. The prevalence of CyCV-VN was relatively high in HIV positive patients (21%), modest in patients with HBV or HCV infection (6%), and low in transplant recipient patients (2%). Positive patients showed low levels of CyCV-VN viremia. The virus was not detected in serum samples from healthy individuals. Longitudinal analysis of serum samples obtained from selected patients showed a stable or transient presence of circulating CyCV-VN. CONCLUSIONS The present study is the first to demonstrate CyCV-VN DNA circulation in Italy and to cast light on some biological aspects of this novel virus of men.