Infectious Toscana Virus in Seminal Fluid of Young Man Returning from Elba Island, Italy.

We report detecting infectious Toscana virus in the seminal fluid of a 25-year-old man from Italy returning from Elba Island. The presence of infectious virus in human semen adds Toscana virus to the long list of viruses detected in this genital fluid and indicates a potential for sexual transmission.

Antibody response to SARS-CoV-2 in infected patients with different clinical outcome.

Data regarding antibody responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in patients infected with COVID-19 are not yet available. In this study, we aimed to evaluate serum antibody responses in patients regardless of the outcome. We measured the circulating immunoglobulin G (IgG) antibody levels in 60 subjects with a certified history of SARS-CoV-2 infection by using immunoenzymatic, chemiluminescent, and Neutralization assays. Half patients had a severe infection, the other half were pauci-symptomatic. We analyzed their antibody response to see the trend of the humoral response. Our results showed a significant difference in circulating IgG level among the two groups. The neutralizing antibody response against SARS-CoV-2 was significantly higher among those who had severe disease. Furthermore, ten subjects from each group were screened twice, and a declining antibody trend was observed in pauci-symptomatic individuals. These findings provide evidence that humoral immunity against SARS-CoV-2 in pauci-symptomatic people is weak and may not be long-lasting. This may have implications for immunity strategy and prevention, since it is still not clear whether a time-dependent decrease of both circulating and neutralizing antibodies to nonprotective levels could occur in a longer time span and whether potential vaccines are able to induce a herd immunity and a durable response.

Toscana virus infects dendritic and endothelial cells opening the way for the central nervous system.

Toscana virus (TOSV) is a Phlebovirus responsible for human neurological infections in endemic Mediterranean areas. The main viral target is the central nervous system, with viremia as a way of dissemination throughout the host. This study was aimed at understanding the spread of TOSV in the host by identifying the cell population infected by the virus and the vehicle to the organs. In vivo studies provided evidence that endothelial cells are infected by TOSV, indicating their potential role in the diffusion of the virus following viremic spread. These results were further confirmed in vitro. Human peripheral mononuclear blood cells were infected with TOSV; only monocyte-derived dendritic cells were identified as susceptible to TOSV infection. Productive viral replication was then observed in human monocyte-derived dendritic cells (moDCs) and in human endothelial cells by recovery of the virus from a cell supernatant. Interleukin-6 was produced by both cell types upon TOSV infection, mostly by endothelial cells, while moDCs particularly expressed TNF-α, which is known to induce a long-lasting decrease in endothelial cell barrier function. These cells could therefore be implicated in the spread of the virus in the host and in the infection of tissues that are affected by the disease, such as the central nervous system. The identification of in vitro and in vivo TOSV cell targets is an important tool for understanding the pathogenesis of the infection, providing new insight into virus-cell interaction for improved knowledge and control of this viral disease.

Rational Use of Monoclonal Antibodies as Therapeutic Treatment in an Oncologic Patient with Long COVID.

We present the case of a 76-year-old male patient persistently infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the setting of a stage IIIC cutaneous melanoma and non-Hodgkin's lymphoma (NHL). Due to the persistent coronavirus disease 19 (COVID-19), all cancer treatments were discontinued. Because of the worsening of his clinical state and the persistence of SARS-CoV-2 positivity for more than six months, the patient was treated with sotrovimab, which was ineffective due to resistance mutations acquired during that time. In order to resume cancer treatment and make the patient free from SARS-CoV-2, an in vitro screening of Evusheld monoclonal antibodies (tixagevumab-cilgavimab) against the viral strains isolated from the subject was performed. The promising results obtained during in vitro testing led to the authorization of the off-label use of Evusheld, which made the patient negative for SARS-CoV-2, thus, allowing him to resume his cancer treatment. This study highlights the Evusheld monoclonal antibodies' efficacy, not only in prevention but also in successful therapy against prolonged COVID-19. Therefore, testing neutralizing monoclonal antibodies in vitro against SARS-CoV-2 mutants directly isolated from patients could provide useful information for the treatment of people affected by long COVID.

Human Polymorphonuclear Cells Support Zika Virus to Cross Endothelial Monolayer and Access Bloodstream.

The rapid spread of new outbreaks of human infection caused by Zika virus (ZIKV) has raised many global concerns since 2016. Despite the increasing knowledge of this virus, data on the pathogenesis of ZIKV are still missing. In particular, it is still unknown how the virus crosses the endothelial monolayer and gets access to the bloodstream. In the present work, we used human umbilical vein endothelial cells (HUVECs) as a model to study ZIKV infection in vitro. We demonstrated that HUVECs are an optimal reservoir for viral replication, as they were able to sustain ZIKV infection up to two weeks, without showing a cytopathic effect. In order to evaluate the integrity of endothelial monolayer, immunofluorescence was performed on mock-infected or ZIKV-infected cells ± peripheral blood mononuclear cells (PBMCs) or polymorphonuclear cells (PMN), 48 h p.i., by using an anti-VE-Cadherin antibody, a major adherence protein that maintains the integrity of intercellular junctions. In addition to infection, we noted that the presence of some components of the immune system, such as PMNs, played an important role in altering the endothelial monolayer in cell junctions, suggesting that presence at the site of infection probably promotes the spread of ZIKV in vivo in the bloodstream.

Omicron Infection Evokes Cross-Protection against SARS-CoV-2 Variants in Vaccinees.

Due to the rapid global spread of the Omicron (B.1.1.529) variant, efforts to scale up COVID-19 booster vaccination have been improved, especially in light of the increasing evidence of reduced neutralizing antibody (NT Ab) over time in vaccinated subjects. In this study, neutralizing antibody responses against the Wild-Type, Delta, and Omicron strains were evaluated among vaccinees, both infected with Omicron or uninfected, and non-vaccinated subjects infected with Omicron. The aim of the study was to compare the cross-protective humoral response to the variant strains induced by vaccination and/or Omicron infection. The results showed a significant difference in the neutralizing antibody response between the vaccinees and the Omicron-infected vaccinated subjects against the three tested strains (p < 0.001), confirming the booster effect of the Omicron infection in the vaccinees. By contrast, Omicron infection only did not enhance the antibody response to the other variants, indicating a lack of cross-protection. These results suggest the importance of updating the current formulation of the SARS-CoV-2 vaccine to protect people against the Omicron subvariants. A specific Omicron vaccine, administered as a booster for the previously adopted mRNA vaccines, may protect against a wider range of SARS-CoV-2 variants. However, it is unlikely that the Omicron vaccine alone would be able to protect non-vaccinated subjects against other circulating variants.

Overview of Anti-SARS-CoV-2 Immune Response Six Months after BNT162b2 mRNA Vaccine.

BACKGROUND: We have designed a prospective study aiming to monitor the immune response in 178 health care workers six months after BNT162b2 mRNA vaccination. METHODS: The humoral immune response of all subjects was evaluated by chemiluminescence (CMIA); in 60 serum samples, a live virus-based neutralization assay was also tested. Moreover, 6 months after vaccination, B- and T-cell subsets from 20 subjects were observed by FACS analysis after restimulation with the trimeric SARS-CoV-2 Spike protein as an antigen, thus mimicking reinfection in vitro. RESULTS: A significant decrease of circulating IgG levels and neutralizing antibodies over time were observed. Moreover, six months after vaccination, a variable T-cell immune response after in vitro antigen stimulation of PBMC was observed. On the contrary, the analysis of B-cell response showed a shift from unswitched to switched memory B-cells and an increase of Th17 cells. CONCLUSIONS: Although the variability of the CD4+ and CD8+ immune response and an antibody decline was observed among vaccinated subjects, the increase of switched memory B-cells and Th17 cells, correlating with the presence of neutralizing antibodies, opened the debate on the correct timing of vaccination.

Antibody Response against Circulating Omicron Variants 8 Months after the Third Dose of mRNA Vaccine.

The COVID-19 wave is being recently propelled by BA.2 and, particularly, BA.5 lineages, showing clear transmission advantages over the previously circulating strains. In this study, neutralizing antibody responses against SARS-CoV-2 Wild-Type, BA.2 and BA.5 Omicron sublineages were evaluated among vaccinees, uninfected or infected with Omicron BA.1 strain, 8 months after the third dose of SARS-CoV-2 vaccine. The aim of this study was to compare the cross-protective humoral response to the currently circulating variant strains induced by vaccination, followed by Omicron infection in some subjects. Results showed a low antibody titer against all three variants in uninfected vaccinated subjects. On the other hand, vaccinated subjects, infected with BA.1 variant after receiving the third dose (about 40 days later), showed a strong response against both BA.2 and BA.5 strains, albeit with lower titers. This reinforces the concept that vaccination is fundamental to induce an adequate and protective immune response against SARS-CoV-2, but needs to be updated, in order to also widen the range of action towards emerging variants, phylogenetically distant from the Wuhan strain, against which the current formulation is targeted.

Toscana virus induces interferon although its NSs protein reveals antagonistic activity.

Toscana virus (TOSV) is a phlebotomus-transmitted virus that belongs to the family Bunyaviridae and causes widespread infections in humans; about 30 % of these cases result in aseptic meningitis. In the present study, it was shown that TOSV is an inducer of beta interferon (IFN-ß), although its non-structural protein (NSs) could inhibit the induction of IFN-ß if expressed in a heterologous context. A recombinant Rift Valley fever virus expressing the TOSV NSs could suppress IFN-ß expression in infected cells. Moreover, in cells expressing NSs protein from a cDNA plasmid, IFN-ß transcripts were not inducible by poly(I : C). Unlike other members of the family Bunyaviridae, TOSV appears to express an NSs protein that is a weak antagonist of IFN induction. Characterization of the interaction of TOSV with the IFN system will help our understanding of virus-host cell interactions and may explain why the pathogenesis of this disease is mostly mild in humans.

Neutralizing Antibody Response of Vaccinees to SARS-CoV-2 Variants.

Due to their increased transmissibility, three variants of high concern have emerged in the United Kingdom (also known as B.1.1.7 lineage or VOC-202012/01), South Africa (B.1.351 lineage), and Brazil (P1 lineage) with multiple substitutions in the spike protein. Since neutralizing antibodies elicited by vaccination are likely considered as correlates of protection for SARS-CoV-2 infection, it is important to analyze whether vaccinees with mRNA BNT162b2 are equally protected against these emerging SARS-CoV-2 variants. To this aim, we enrolled healthy subjects one month after complete vaccination with Comirnaty and evaluated the neutralizing response against the native Wuhan strain and the emerging B.1.1.7, B.1.351 and P1 lineages, by using the microneutralization assay, currently considered the gold standard test for the evaluation and detection of functional neutralizing antibodies. The most remarkable finding of this study was the significantly lower neutralizing antibody titer against B.1.351 lineage, compared to the wild-type virus. No significant differences were observed with the other two lineages. These findings provide evidence that vaccinated subjects may not be equally protected against all SARS-CoV-2 lineages.

Comparative Performance of a New SARS-CoV-2 Rapid Detection System.

The extraordinary global demand for reagents and diagnostic instruments needed for timely detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly affected their availability. In order to meet diagnostic needs, it has been necessary to develop new diagnostic procedures. To date, molecular diagnostic tools have represented the gold standard for diagnosis of SARS-CoV-2 infection, and thus an alternative and real-time PCR system was required. To this aim, a molecular rapid test which works with direct real-time RT-PCR may be a relevant aid. In the present work, the accuracy, sensitivity, and specificity of the bKIT Virus Finder COVID-19 rapid molecular test by Hyris Ltd. was evaluated. Moreover, the influence of a different swab storage medium composition was examined relative to that of a routinely used comparator assay. The Hyris Ltd. assay showed an overall agreement of 100% with the comparator based on a panel consisting of 74 retrospective positive nasopharyngeal swabs (NPSs), collected either in universal transport medium (UTM) or using ESwab. No false-positive result was achieved on samples that previously tested negative. Cross-reactivity screening on microorganisms that commonly colonize the human upper respiratory tract was not detected, excluding the risk of false-positive results. Simultaneously, drugs frequently administered to cure respiratory diseases did not interfere with the analytical performance of the assay. Our results showed that the Hyris Ltd. bKIT Virus Finder COVID-19 is a reliable assay for rapid qualitative detection of SARS-CoV-2, providing the advantage of less complex and unambiguous interpretation of results. Indeed, skilled technicians are not required, and thus the Hyris system is suitable as a rapid and easy system for SARS-CoV-2 diagnosis. IMPORTANCE In order to overcome the increased demand for diagnostic tools for the timely detection of SARS-CoV-2 infection, we tested the bKIT Virus Finder COVID-19 molecular rapid test by Hyris Ltd. The new system was confirmed as a reliable assay for rapid SARS-CoV-2 detection, since sensitivity and specificity parameters were fully satisfied. Moreover, the bKIT Virus Finder COVID-19 provides the advantage of easy results interpretation, since skilled technicians are not required, and thus the Hyris system is a valuable SARS-CoV-2 rapid diagnosis system.

Efficient Inactivation of SARS-CoV-2 and Other RNA or DNA Viruses with Blue LED Light.

Blue LED light has proven to have a powerful bacteria-killing ability; however, little is known about its mechanism of virucidal activity. Therefore, we analyzed the effect of blue light on different respiratory viruses, such as adenovirus, respiratory syncytial virus and SARS-CoV-2. The exposure of samples to a blue LED light with a wavelength of 420 nm (i.e., in the visible range) at 20 mW/cm2 of irradiance for 15 min appeared optimal and resulted in the complete inactivation of the viral load. These results were similar for all the three viruses, demonstrating that both enveloped and naked viruses could be efficiently inactivated with blue LED light, regardless of the presence of envelope and of the viral genome nature (DNA or RNA). Moreover, we provided some explanations to the mechanisms by which the blue LED light could exert its antiviral activity. The development of such safe and low-cost light-based devices appears to be of fundamental utility for limiting viral spread and for sanitizing small environments, objects and surfaces, especially in the pandemic era.

Identification of a Neutralizing Epitope on TOSV Gn Glycoprotein.

Emerging and re-emerging viral infections have been an important public health problem in recent years. We focused our attention on Toscana virus (TOSV), an emergent neurotropic negative-strand RNA virus of the Phenuiviridae family. The mechanisms of protection against phlebovirus natural infection are not known; however, it is supposed that a virus-neutralizing antibody response against viral glycoproteins would be useful to block the first stages of infection. By using an improved memory B cell immortalization method, we obtained a panel of human mAbs which reacted with TOSV antigens. We identified three epitopes of TOSV Gn glycoproteins by neutralizing mAbs using synthetic peptide arrays on membrane support (SPOT synthesis). These epitopes, separated in primary structure, might be exposed near one another as a conformational epitope in their native structure. In vivo studies were conducted to evaluate the humoral response elicited in mice immunized with the identified peptides. The results underlined the hypothesis that the first two peptides located in the NH2 terminus could form a conformational epitope, while the third, located near the transmembrane sequence in the carboxyl terminus, was necessary to strengthen neutralizing activity. Our results emphasize the importance of identifying neutralizing epitopes shared among the various phleboviruses, which could be exploited for the development of a potential epitope-based diagnostic assay or a polyvalent protective vaccine against different phleboviruses.

Residual SARS-CoV-2 RNA in nasal swabs of convalescent COVID-19 patients: Is prolonged quarantine always justified?

Real-time reverse transcription PCR is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Defining whether a patient could be contagious or not contagious in the presence of residual SARS-CoV-2 RNA is of extreme importance in the context of public health. In this prospective multicenter study, virus isolation was prospectively attempted in 387 nasal swabs from clinically recovered patients showing low viral load (quantification cycle, Cq, value greater than 30). The median Cq value was 36.8 (range 30.0-39.4). Overall, a cytopathic effect was detected in nine samples, corresponding to a culture positivity rate of 2.3% (9/387). The results of this study help to dissect true virus replication and residual viral RNA detection in recovered patients.

Age related changes in T cell mediated immune response and effector memory to Respiratory Syncytial Virus (RSV) in healthy subjects.

Respiratory syncytial virus (RSV) is the major pathogen causing respiratory disease in young infants and it is an important cause of serious illness in the elderly since the infection provides limited immune protection against reinfection. In order to explain this phenomenon, we investigated whether healthy adults of different age (20-40; 41-60 and > 60 years), have differences in central and effector memory, RSV-specific CD8+ T cell memory immune response and regulatory T cell expression status. In the peripheral blood of these donors, we were unable to detect any age related difference in term of central (CD45RA-CCR7+) and effector (CD45RA-CCR7-) memory T cell frequency. On the contrary, we found a significant increase in immunosuppressive regulatory (CD4+25+FoxP3+) T cells (Treg) in the elderly. An immunocytofluorimetric RSV pentamer analysis performed on these donors' peripheral blood mononuclear cells (PBMCs), in vitro sensitized against RSV antigen, revealed a marked decline in long-lasting RSV specific CD8+ memory T cell precursors expressing interleukin 7 receptor α (IL-7Rα), in the elderly. This effect was paralleled by a progressive switch from a Th1 (IFN-γ and TNF-α) to a Th2 (IL-10) functional phenotype. On the contrary, an increase in Treg was observed with aging. The finding of Treg over-expression status, a prominent Th2 response and an inefficient RSV-specific effector memory CD8+ T cell expansion in older donors could explain the poor protection against RSV reinfection and the increased risk to develop an RSV-related severe illness in this population. Our finding also lays the basis for new therapeutic perspectives that could limit or prevent severe RSV infection in elderly.

Seroprevalence to Measles Virus after Vaccination or Natural Infection in an Adult Population, in Italy.

An increase in measles cases worldwide, with outbreaks, has been registered in the last few years, despite the availability of a safe and highly efficacious vaccine. In addition to an inadequate vaccination coverage, even in high-income European countries studies proved that some vaccinated people were also found seronegative years after vaccination, thus increasing the number of people susceptible to measles infection. In this study, we evaluated the immunization status and the seroprevalence of measles antibodies among 1092 healthy adults, either vaccinated or naturally infected, in order to investigate the persistence of anti-measles IgG. Among subjects who received two doses of measles vaccine, the neutralizing antibody titer tended to decline over time. In addition, data collected from a neutralization assay performed on 110 healthy vaccinated subjects suggested an inverse correlation between neutralizing antibody titers and the time elapsed between the two vaccinations, with a significant decline in the neutralizing titer when the interval between the two doses was ≥11 years. On the basis of these results, monitoring the serological status of the population 10-12 years after vaccination could be important both to limit the number of people who are potentially susceptible to measles, despite the high efficacy of MMR vaccine, and to recommend a booster vaccine for the seronegatives.

Epidemiological and clinical study of viral respiratory tract infections in children from Italy.

Impact of recently discovered viruses on epidemiology of acute respiratory tract infections (ARTI) is still unclear. We studied the impact of recently discovered human metapneumovirus (hMPV), human bocavirus (HBoV), and new coronaviruses (HCoV-NL63 and HKU1) on the global epidemiology of ARTI. From October 2006 to April 2007, 237 pediatric patients affected by ARTI were enrolled in our study. Specimens were tested for respiratory viruses by polymerase chain reaction. One hundred twenty-four out of 237 samples (52.3%) were positive for one or more viruses. Picornaviruses were the most prevalent viruses (n = 61, 43.6%), followed by respiratory syncytial virus (n = 34, 24.3%) and Adenovirus (n = 25, 17.9%); hMPV (n = 9, 6.4%) was the fourth most common virus detected. HBoV and HCoV showed a low prevalence (respectively 2.9% and 2.1%). RSV was the prevalent agent of LRTI (38%). Viruses were identified in more than 50% of the studied ARTI, providing useful information on clinical features and epidemiology of specific agents affecting children in cold months. Although routine surveillance of respiratory viruses does not seem cost-effective, continuous monitoring of ARTI etiology could be a useful tool for planning resources for the development of new vaccines and antiviral agents.

Beyond the Biological Effect of a Chemically Characterized Poplar Propolis: Antibacterial and Antiviral Activity and Comparison with Flurbiprofen in Cytokines Release by LPS-Stimulated Human Mononuclear Cells.

Bee propolis, especially Euro-Asian poplar propolis, is among the most well-known natural products traditionally used to treat pharyngitis and minor wounds. The aim of this research was to investigate the pharmacological properties responsible for poplar propolis effectiveness using, for the first time, different in vitro approaches applied to a chemically characterized sample. The anti-inflammatory activity was compared with flurbiprofen by determining pro-inflammatory cytokines released by lipopolysaccharide-stimulated human peripheral blood mononuclear cells (PBMC). The antibacterial activity against Gram+ and Gram- bacteria was assessed, as well as antiviral effects on H1N1 influenza a virus. Poplar propolis (5 and 25 µg/mL) exerted a concentration-dependent anti-inflammatory activity. In this range of concentrations, propolis effect was not inferior to flurbiprofen on cytokines released by lipopolysaccharide (LPS)-stimulated human PBMC. Poplar propolis was found to upregulate IL-6 and IL-1ß in non-stimulated PBMC. S. aureus, S. pyogenes, and S. pneumoniae were the most susceptible bacterial strains with inhibitory concentrations ranging from 156 to 625 µg/mL. A direct anti-influenza activity was not clearly seen. Effective anti-inflammatory concentrations of propolis were significantly lower than the antibacterial and antiviral ones and results suggested that the anti-inflammatory activity was the most important feature of poplar propolis linked to its rationale use in medicine.

Anti-angiogenetic effects of immune-reconstituted influenza virosomes assembled with parathyroid hormone-related protein derived peptide vaccine.

BACKGROUND: C-IRIV/PTR-4 is a novel anticancer vaccine construct composed of immune-reconstituted influenza virosomes (IRIV) assembled with the PTH-rP derived peptide (PTR)-4, a synthetic CTL epitope with HLA-A(*)02.01 amino acid binding motifs. This peptide is able to generate a human PTH-rP specific CTL response with anti-tumor activity in vitro and in mice. MATERIALS AND METHODS: We have investigated the immunological and preventive anti-tumor activity of C-IRIV/PTR-4 compared with the soluble PTR-4 peptide, in HHD mice inoculated with autologous PTH-rP+ tumor cells. RESULTS: Peptide vaccination with either a soluble and an IRIV formulation showed similar immunological activity and the ability to purge the tumor tissue of tumor cell clones able to produce the target antigen (PTR-rP). The most efficient protection from tumor growth was however observed in animals vaccinated with C-IRIV/PTR-4 in which an additional IRIV related anti-angiogenetic effect was detected in the tumor tissue. CONCLUSIONS: These results confirm the immunological activity of PTR-4 vaccination and suggest a more efficacious therapeutic potential of C-IRIV/PTR-4 against bone metastases and malignancies like breast, prostate and lung which very often over-express PTH-rP.