Canonical fibroblast growth factors in viral infection.

Fibroblast growth factors (FGFs) are a family of proteins that play a crucial role in the development and maintenance of various tissues in the body. There are three function-al groups of FGFs: canonical FGFs (cFGFs), intracellularly retained FGFs, and metabolic (also called endocrine) FGFs. cFGFs are secreted and act in an autocrine/paracrine fashion to regulate differentiation during foetal development, as well as tissue repair in adults. Recent studies have also begun to unravel the role of cFGFs during viral infections, suggesting that FGF-2 and other canonical FGFs may have an important virus-specific role, also by the regulation of the immune response. Because dysregulation in the FGF pathways is pivotal in cancer development, FGFs are the target of many anticancer drugs. These drugs may be repurposed to treat viral infection, since dysregulation of FGF signalling has been implicated in the pathogenesis of viral infections, such as hepatitis C. Overall, the role of cFGFs during viral infection is an underrepresented area of current research. This review focuses on overviewing the effects of canonical FGFs during infection by different viruses. Many studies highlight that the effects of FGFs during viral infection may be complex and context-dependent. While there is evidence to suggest that FGFs may have a beneficial impact on the immune response and tissue repair during viral infection, further studies are needed to fully understand the mechanisms underlying these effects and to determine in what cases FGFs could be targeted as a therapeutic approach for viral infection.

SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes.

Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation (Haptoglobin, MCP-1, IL-6, PAI-1), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells.

Specialized metabolites from plants as a source of new multi-target antiviral drugs: a systematic review.

Viral infections have always been the main global health challenge, as several potentially lethal viruses, including the hepatitis virus, herpes virus, and influenza virus, have affected human health for decades. Unfortunately, most licensed antiviral drugs are characterized by many adverse reactions and, in the long-term therapy, also develop viral resistance; for these reasons, researchers have focused their attention on investigating potential antiviral molecules from plants. Natural resources indeed offer a variety of specialized therapeutic metabolites that have been demonstrated to inhibit viral entry into the host cells and replication through the regulation of viral absorption, cell receptor binding, and competition for the activation of intracellular signaling pathways. Many active phytochemicals, including flavonoids, lignans, terpenoids, coumarins, saponins, alkaloids, etc., have been identified as potential candidates for preventing and treating viral infections. Using a systematic approach, this review summarises the knowledge obtained to date on the in vivo antiviral activity of specialized metabolites extracted from plant matrices by focusing on their mechanism of action.

ERN ReCONNET points to consider for treating patients living with autoimmune rheumatic diseases with antiviral therapies and anti-SARS-CoV-2 antibody products.

Recent studies have shown that people who are immunocompromised may inadvertently play a role in spurring the mutations of the virus that create new variants. This is because some immunocompromised individuals remain at risk of getting COVID-19 despite vaccination, experience more severe disease, are susceptible to being chronically infected and remain contagious for longer if they become infected and considering that immunocompromised individuals represent approximately 2% of the overall population, this aspect should be carefully considered. So far, some autoimmune rheumatic disease (ARD) patients with COVID-19 have been treated with antiviral therapies or anti-SARS-CoV-2 antibody products. However, there is no homogeneous approach to these treatment strategies. This issue was addressed within the European Reference Network (ERN) on Rare and Complex Connective Tissue and Musculoskeletal Diseases (ReCONNET) in a discussion among experts and patient's representatives in the context of the rare and complex connective tissue diseases (rCTDs) covered by the Network. ERN ReCONNET is one of the 24 ERNs launched by the European Commission in 2017 with the aim of tackling low prevalence and rare diseases that require highly specialised treatment and promoting concentration of knowledge and resources through virtual networks involving healthcare providers (HCPs) across the European Union (EU). Considering the urgent need to provide guidance not only to the rCTDs community, but also to the whole ARDs community, a multidisciplinary Task Force, including expert clinicians and European Patient Advocacy Group (ePAG) Advocates, was created in the framework of ERN ReCONNET with the aim of developing overarching principles (OP) and points-to-consider (PtC) on a homogenous approach to treat immunocompromised patients with ARDs (with a particular focus on CTDs) affected by COVID-19 using antiviral therapies and anti-SARS-CoV-2 antibody products. The present work reports the final OP and PtC agreed by the Task Force.

Human T-Cell Leukemia Virus Type 1 Oncogenesis between Active Expression and Latency: A Possible Source for the Development of Therapeutic Targets.

The human T-cell leukemia virus type 1 (HTLV-1) is the only known human oncogenic retrovirus. HTLV-1 can cause a type of cancer called adult T-cell leukemia/lymphoma (ATL). The virus is transmitted through the body fluids of infected individuals, primarily breast milk, blood, and semen. At least 5-10 million people in the world are infected with HTLV-1. In addition to ATL, HTLV-1 infection can also cause HTLV-I-associated myelopathy (HAM/TSP). ATL is characterized by a low viral expression and poor prognosis. The oncogenic mechanism triggered by HTLV-1 is extremely complex and the molecular pathways are not fully understood. However, viral regulatory proteins Tax and HTLV-1 bZIP factor (HBZ) have been shown to play key roles in the transformation of HTLV-1-infected T cells. Moreover, several studies have shown that the final fate of HTLV-1-infected transformed Tcell clones is the result of a complex interplay of HTLV-1 oncogenic protein expression with cellular transcription factors that subvert the cell cycle and disrupt regulated cell death, thereby exerting their transforming effects. This review provides updated information on the mechanisms underlying the transforming action of HTLV-1 and highlights potential therapeutic targets to combat ATL.

Incorporation of Copper Nanoparticles on Electrospun Polyurethane Membrane Fibers by a Spray Method.

Electrospinning is an easy and versatile technique to obtain nanofibrous membranes with nanosized fibers, high porosity, and pore interconnectivity. Metal nanoparticles (e.g., Ag, Cu, ZnO) exhibit excellent biocide properties due to their size, shape, release of metal ions, or reactive oxygen species production, and thus are often used as antimicrobial agents. In this study, a combined electrospinning/spray technique was employed to fabricate electrospun polyurethane membranes loaded with copper nanoparticles at different surface densities (10, 20, 25, or 30 µg/cm2). This method allows particle deposition onto the surface of the membranes without the use of chemical agents. SEM images showed that polyurethane fibers own homogeneous thickness (around 650 nm), and that spray-deposited copper nanoparticles are evenly distributed. STEM-EDX demonstrated that copper nanoparticles are deposited onto the surface of the fibers and are not covered by polyurethane. Moreover, a uniaxial rupture test showed that particles are firmly anchored to the electrospun fibers. Antibacterial tests against model microorganisms Escherichia coli indicated that the prepared electrospun membranes possess good bactericidal effect. Finally, the antiviral activity against SARS-CoV-2 was about 90% after 1 h of direct contact. The obtained results suggested that the electrospun membranes possess antimicrobial activities and can be used in medical and industrial applications.

CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats.

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.

Comparative evaluation of molecular methods for the quantitative measure of torquetenovirus viremia, the new surrogate marker of immune competence.

BACKGROUND: Torquetenovirus (TTV) viremia is emerging as a promising tool to assess functional immune competence, to predict posttransplant immune-related complications, and eventually to customize immunosuppression. METHODS: In this study, 327 blood samples were tested using two real-time PCR (rtPCR) assays both targeted to the untranslated region of the TTV genome. The first assay was an in-house rtPCR developed by our group, the second one was the recently marketed TTV R-GENE assay. RESULTS: In the validation study, the TTV R-GENE showed good performances in precision and reproducibility, and sensitivity as low as 12 TTV DNA copies/mL, like previously reported for the in-house rtPCR. The Bland-Altman analysis showed that the mean difference between the two methods was -0.3 log copies/mL. In the comparison study, 69% and 72% of samples were detected positive by rtPCR and TTV R-GENE, respectively (94% concordance, κ = 0.88). Performances did not differ between the two rtPCRs by type of TTV group examined. When a newly-developed in-house digital droplet PCR was applied for TTV quantification and used as an alternative method of comparison on 94 samples, the results strongly correlated with those obtained by the two rtPCR methods (99% concordance). CONCLUSION: In summary, all the molecular methods assayed are highly sensitive and accurate in quantitation of TTV DNA in blood samples.

Positive peritoneal swab in SARS-CoV-2 patients undergoing abdominal emergency surgery: effect or cause?

PURPOSE: The presence of the SARS-CoV-2 in the peritoneal fluid is a matter of debate in the COVID-19 literature. The study aimed to report the prevalence of SARS-CoV-2 in the peritoneal fluid of patients with nasopharyngeal swab tested positive for SARS-CoV-2 undergoing emergency surgery and review the literature. METHODS: The present study was conducted between March 2020 and June 2021. Diagnosis of SARS-CoV-2 positivity was confirmed by preoperative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Eighteen patients with positive nasopharyngeal swabs were operated in emergency in two third-level Italian hospitals. In 13 of these patients (72%), a peritoneal swab was analyzed: SARS-CoV-2 RNA was found in the abdominal fluid of two patients (15%). Neither of them had visceral perforation and one patient died. In ten patients with negative peritoneal swabs, visceral perforation and mortality rates were 30% and 20%, respectively. CONCLUSION: SARS-CoV-2 peritoneal positivity is rare. Abdominal surgery can, therefore, be safely performed in patients with COVID-19 using standard precautions. The correlation with a visceral perforation is not evaluable. The clinical outcomes seem uninfluenced by the viral colonization of the peritoneum. Assessment in large series to provide definitive answers about the involvement of the SARS-CoV-2 in the peritoneum will be challenging to coordinate.

Viral infection neutralization tests: A focus on severe acute respiratory syndrome-coronavirus-2 with implications for convalescent plasma therapy.

Viral neutralization tests (VNTs) have long been considered old-fashioned tricks in the armamentarium of fundamental virology, with laboratory implementation for a limited array of viruses only. Nevertheless, they represent the most reliable surrogate of potency for passive immunotherapies, such as monoclonal or polyclonal antibody therapy. The recent interest around therapy with convalescent plasma or monoclonal antibodies for the Covid-19 pandemic has paralleled the revival of VNTs. We review here the available methods by dissecting variations for each fundamental component of the VNT (i.e., virus type and dose, replication-competent cell line, serum, and detection system).

Ictogenesis of viral pneumonia: A comparison between SARS-CoV-2 and H1N1/H3N2.

Several studies reported acute symptomatic seizures as a possible neurological complication of COVID-19 pneumonia. Apart from metabolic imbalances, hypoxia, and fever, other ictogenic mechanisms are likely related to an immune-mediated damage. The same mechanisms are shared by other respiratory viruses. Since neurotropic properties of SARS-CoV-2 have been questioned, we investigated whether SARS-CoV-2 has a similar ictogenic potential to other respiratory non-neurotropic viruses. We conducted a retrospective study identifying 1141 patients with SARS-CoV-2 pneumonia and 146 patients with H1N1/H3N2 pneumonia. We found a similar prevalence of seizures in the two viral pneumonia (1.05% with SARS-CoV-2 vs 2.05% with influenza; p = 0.26). We detailed clinical, electroencephalographic, and neuroradiological features of each patient, together with the hypothesized pathogenesis of seizures. Previous epilepsy or pre-existing predisposing conditions (i.e., Alzheimer's disease, stroke, cerebral neoplasia) were found in one-third of patients that experienced seizures, while two-thirds of patients had seizures without known risk factors other than pneumonia in both groups. The prevalence of pre-existing predisposing conditions and disease severity indexes was similar in SARS-CoV-2 and H1N1/H3N2 pneumonia, thus excluding they could act as potential confounders. Considering all the patients with viral pneumonia together, previous epilepsy (p < 0.001) and the need for ventilatory support (p < 0.001), but not the presence of pre-existing predisposing conditions (p = 0.290), were associated with seizure risk. Our study showed that SARS-CoV-2 and influenza viruses share a similar ictogenic potential. In both these infections, seizures are rare but serious events, and can manifest without pre-existing predisposing conditions, in particular when pneumonia is severe, thus suggesting an interplay between disease severity and host response as a major mechanism of ictogenesis, rather than a virus-specific mechanism.

Predictors of hospital-acquired bacterial and fungal superinfections in COVID-19: a prospective observational study.

BACKGROUND: Bacterial and fungal superinfections may complicate the course of hospitalized patients with COVID-19. OBJECTIVES: To identify predictors of superinfections in COVID-19. METHODS: Prospective, observational study including patients with COVID-19 consecutively admitted to the University Hospital of Pisa, Italy, between 4 March and 30 April 2020. Clinical data and outcomes were registered. Superinfection was defined as a bacterial or fungal infection that occurred ≥48 h after hospital admission. A multivariate analysis was performed to identify factors independently associated with superinfections. RESULTS: Overall, 315 patients with COVID-19 were hospitalized and 109 episodes of superinfections were documented in 69 (21.9%) patients. The median time from admission to superinfection was 19 days (range 11-29.75). Superinfections were caused by Enterobacterales (44.9%), non-fermenting Gram-negative bacilli (15.6%), Gram-positive bacteria (15.6%) and fungi (5.5%). Polymicrobial infections accounted for 18.3%. Predictors of superinfections were: intestinal colonization by carbapenem-resistant Enterobacterales (OR 16.03, 95% CI 6.5-39.5, P < 0.001); invasive mechanical ventilation (OR 5.6, 95% CI 2.4-13.1, P < 0.001); immunomodulatory agents (tocilizumab/baricitinib) (OR 5.09, 95% CI 2.2-11.8, P < 0.001); C-reactive protein on admission >7 mg/dl (OR 3.59, 95% CI 1.7-7.7, P = 0.001); and previous treatment with piperacillin/tazobactam (OR 2.85, 95% CI 1.1-7.2, P = 0.028). Length of hospital stay was longer in patients who developed superinfections ompared with those who did not (30 versus 11 days, P < 0.001), while mortality rates were similar (18.8% versus 23.2%, P = 0.445). CONCLUSIONS: The risk of bacterial and fungal superinfections in COVID-19 is consistent. Patients who need empiric broad-spectrum antibiotics and immunomodulant drugs should be carefully selected. Infection control rules must be reinforced.

Evolution of viruses and the emergence of SARS-CoV-2 variants.

Life implies adaptation. This is one of the fundamental principles that has permitted most living species to survive through ages in an ever-changing environment. Spontaneously occurring events have shaped also virus populations and their fitness. Thanks to their plasticity, viruses have thrived in extremely dissimilar conditions. Unsurprisingly, SARS-CoV-2, the etiological agent of COVID-19, is no exception. Thanks to an unprecedented rate of molecular tracing and sequence scrutiny, the virus was followed in all its changes and shown to evolve in such a way as to possibly determine subsequent waves of infection after the first global and massive outbreak. This review illustrates the major modifications occurred to the virus since its discovery. We describe the potential advantages that these changes conveyed as regards SARS-CoV-2 transmissibility, resistance to host innate and adaptive barriers and molecular diagnosis.

Ablation of Acid Ceramidase Impairs Autophagy and Mitochondria Activity in Melanoma Cells.

Cutaneous melanoma is often resistant to therapy due to its high plasticity, as well as its ability to metabolise chemotherapeutic drugs. Sphingolipid signalling plays a pivotal role in its progression and metastasis. One of the ways melanoma alters sphingolipid rheostat is via over-expression of lysosomal acid ceramidase (AC), which catalyses the hydrolysis of pro-apoptotic long-chain ceramides into sphingosine and fatty acid. In this report, we examine the role of acid ceramidase in maintaining cellular homeostasis through the regulation of autophagy and mitochondrial activity in melanoma cell lines. We show that under baseline conditions, wild-type melanoma cells had 3-fold higher levels of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3 II), compared to AC-null cells. This difference was further magnified after cell starvation. Moreover, we noticed autophagy impairment in A375 AC-null cells, possibly due to local accumulation of non-metabolized ceramides. Nonetheless, we observed that AC-null cells exhibited a significant increase in mitochondrial membrane potential compared to control cells. Consistent with this observation, we found that, after total starvation, ~30% of AC-null cells undergo apoptosis compared to ~6% of wild-type cells. As expected, AC transfection restored viability in A375 AC-null cells. Together, these findings suggest that AC-null melanoma cells change and adapt their metabolism to survive in the absence of AC, although in a way that does not allow them to cope with the stress of nutrient deprivation.

SARS-CoV-2 Is Present in Peritoneal Fluid in COVID-19 Patients.

BACKGROUND: The excretion pathomechanisms of SARS-CoV-2 are actually unknown. No certain data exist about viral load in the different body compartments and fluids during the different disease phases. MATERIAL AND METHODS: Specific real-time reverse transcriptase-polymerase chain reaction targeting 3 SARS-CoV-e genes were used to detect the presence of the virus. RESULTS: SARS-CoV-2 was detected in peritoneal fluid at a higher concentration than in respiratory tract. CONCLUSION: Detection of SARS-CoV-2 in peritoneal fluid has never been reported. The present article represents the very first positive result describing the presence of the virus in peritoneal fluid during an emergency surgical procedure in a COVID-19 sick patient. This article thus represents a warning for increasing the level of awareness and protection for surgeon especially in emergency surgical setting.

Lack of Marseillevirus DNA in immunocompetent and immunocompromised Italian patients.

Marseilleviridae is a family of viruses which have only been propagated in acanthamoeba. Marseillevirus sequences have been recently detected in different human matrices by viral metagenomics. Single-center studies worldwide have estimated a low prevalence of marseillevirus both in symptomatic patients and in healthy donors but, to date, no informations are available on the prevalence of this giant virus in Italy. By a polymerase chain reaction targeting the ORF152 viral sequence, we tested sera from 197 immunosuppressed patients and 285 healthy donors, and 63 and 30 respiratory and cerebrospinal fluid samples, respectively, of patients with various clinical conditions and referring the Virology Division for diagnostic purposes. We observed no evidence of Marseillevirus DNA in all 575 samples tested. Marseillevirus probably does not cause infection in human.

Efficacy, Safety, and Predictors of Direct-acting antivirals in Hepatitis C Virus Patients with Heterogeneous Liver Diseases.

Safety, efficacy, and predictor factors of sustained-virological-response after 24 weeks of new direct-acting antivirals were evaluated in hepatitis C virus patients with different stages of hepatic disease. 260 patients, median age 60 years, of whom 48.1% cirrhotics, 17.7% liver transplant recipients, and 45.7% naïve were treated with Sofosbuvir+Ribavirine, Sofosbuvir+Simeprevir±Ribavirine, Sofosbuvir+Daclatasvir± Ribavirine, Sofosbuvir+Ledispavir±Ribavirine, Ombitasvir/Paritaprevir/Ritonavir+Ribavirine and Ombitasvir/Paritaprevir/Ritonavir+Dasabuvir±Ribavirine. Therapy outcomes, hematochemical parameters, viral replication, genotype, and resistance-associated-mutations were analyzed retrospectively. Sustained virological response was 90.4% in the whole population, 83.2% in cirrhotics, 85% in patients with previous virological failure, 93.6% in patients >60 years, and 95.6% in liver transplant recipients. SVR24 for each drug regimen was 75% Sofosbuvir+Ribavirine, 80.4% Sofosbuvir+Simeprevir±Ribavirine, 94.3% Sofosbuvir+Daclatasvir±Ribavirine, 98.7% Sofosbuvir+Ledispavir±Ribavirine, 100% Ombitasvir/ Paritaprevir/Ritonavir+Ribavirine and Ombitasvir/Paritaprevir/Ritonavir+Dasabuvir±Ribavirine. The highest sustained virological response rates were obtained with genotype-1b (95.9%). Twenty-five patients, mostly cirrhotics or suffering from severe liver complications, manifested relapse (84%), breakthrough (12%), or non-response (4%). Mild side effects were observed in 41.1% of patients. Model-for-End-Liver- Disease score <10 and alanine aminotransferase ≤20 U/L at week 8 of therapy proved positive predictors of sustained virological response. Direct-acting antiviral therapy is efficacious and safe even in patients with advanced liver disease and/ or previous virological failure; Model-for-End-Liver-Disease <10 and alanine aminotransferase reduction during therapy were found to be reliable predicting markers of sustained-virological-response.

Low prevalence of Gemycircularvirus DNA in immunocompetent and immunocompromised subjects.

Gemycircularviruses (GemyCV) are a vast array of viruses belonging to the Genomoviridae family. Prevalence and pathogenesis in humans are still poorly understood. Different GemyCV species were investigated in 661 Italian subjects by species-specific PCRs. Only the GemyCV-C1c species was detected, with low prevalence and the highest rate in HIV immunosuppressed patients.

Sphingolipid/Ceramide Pathways and Autophagy in the Onset and Progression of Melanoma: Novel Therapeutic Targets and Opportunities.

Melanoma is a malignant tumor deriving from neoplastic transformation of melanocytes. The incidence of melanoma has increased dramatically over the last 50 years. It accounts for most cases of skin cancer deaths. Early diagnosis leads to remission in 90% of cases of melanoma; conversely, for melanoma at more advanced stages, prognosis becomes more unfavorable also because dvanced melanoma is often resistant to pharmacological and radiological therapies due to genetic plasticity, presence of cancer stem cells that regenerate the tumor, and efficient elimination of drugs. This review illustrates the role of autophagy in tumor progression and resistance to therapy, focusing on molecular targets for future drugs.

Torquetenovirus detection in exosomes enriched vesicles circulating in human plasma samples.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.