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Abstract 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.

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.

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.

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.

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.

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.

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/cm 2 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.

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.

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 NH 2 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.

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.

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., (© 2021 Wiley Periodicals LLC.)

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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-1β 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.

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.

We report a case of West Nile virus (WNV) infection in a symptomatic woman living in Tuscany in 2007. A retrospective analysis on cerebrospinal fluids drawn from people affected by neurological diseases with unknown etiology allowed the identification of a case of WNV infection before the WNV outbreak in the Northeast Italy in 2008. This emphasizes the importance of maintaining a high level of epidemiological surveillance all over the Italian territory.

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-β), although its non-structural protein (NSs) could inhibit the induction of IFN-β if expressed in a heterologous context. A recombinant Rift Valley fever virus expressing the TOSV NSs could suppress IFN-β expression in infected cells. Moreover, in cells expressing NSs protein from a cDNA plasmid, IFN-β 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.

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.

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., ((c) 2009 Wiley-Liss, Inc.)

Toscana virus (TOSV) is an emerging virus, circulating in the Mediterranean area, that is responsible for aseptic meningitis, meningoencephalitis, and encephalitis. The development of a vaccine that could provide complete protection from TOSV infection is needed. In this study we investigated the capacity of TOSV structural proteins, nucleocapsid protein N and the two Gc and Gn glycoproteins, produced as recombinant proteins, in an animal model. In particular, we investigated their role in inducing specific and protective immune responses against virus infection. Mice were immunized intraperitoneally using TOSV antigens singly or in combination. The results show that only the N-Gc combination was able to protect 100% of animals from a lethal challenge with a neurovirulent strain of TOSV. This potential vaccine induces high serum antibody titres with neutralizing activity and it is safe for animals. Moreover, immunization induces a virus specific cell-mediated immune response, in particular a CD8+ T cell response associated with a marked expression of interferon gamma. These results indicate that the N+Gc viral antigen combination could be useful for future development of a vaccine controlling the spread of this emerging virus that could pose a new threat for humans.

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.

The effects of anticancer chemotherapy on antigen-specific cytotoxic T lymphocytes (CTLs) are mostly unknown. We tested the effects of cytotoxic drugs such as 5-fluorouracil, gemcitabine, and oxaliplatin on the functional activity of antigen-specific CTL cultures derived from the peripheral blood mononuclear cells of human donors. We found that a biweekly drug-exposure of human HLA-A(*)02.01+ CTLs derived from bulk cultures led to completely different effects if occurring early (day second) or late (day thirteenth) after the in vitro stimulations with the cognate peptides. In the first case, there was a significant CTL inhibition, whereas in the second, there was a marked enhancement of the antigen-specific cytolytic activity. Results of immunocytofluorimetric studies and CTL/natural killer inhibition assays suggested that the latter effect could be related to a more selective drug-mediated inhibition of cohabitant T regulatory (reg) cells. These results were translated in an in vivo therapeutic mouse model where humanized HLA-A(*)02.01 transgenic mice inoculated with EL-4/humanized HLA-A(*)02.01 transgenic mice showed a prolonged survival and the greatest rate of cure when receiving a combined treatment with a thymidylate synthase-specific peptide vaccine and a multidrug chemotherapy regimen administered late after immunization. Tumor samples derived from this group of mice showed a reduced expression of the target thymidylate synthase antigen, a marked reduction of T(reg)s, and a noteworthy infiltration of C8+ T cells. These results may have clinical implications for the design of new translational anticancer regimens aimed at combining chemotherapy and immunotherapy.

Virus-specific cytotoxic T lymphocytes (CTLs) are crucial for the control of respiratory syncytial virus (RSV) infection. This study has identified CTL epitopes of the RSV N protein in healthy subjects. We screened the primary structure of the N protein for HLA-A 0201-binding amino acid consensus motifs, identifying three peptides designated as N-RSV1, N-RSV2, and N-RSV3. These peptides were used to generate CTL lines by stimulating human HLA-A 02.01 peripheral blood mononuclear cells (PBMCs) in vitro. These CTL lines were then characterized by performing CTL chromium release assays and IFN-gamma secretion detection by intracellular cytokine staining. N-RSV1 and N-RSV3 peptides elicited the strongest cytolytic activity against RSV-infected cells and they could be useful epitopes for the analysis of CTL responses to RSV and for understanding immune-induced disease pathogenesis.

Background: Human bocavirus (HBoV) is a newly discovered human parvovirus. HBoV was detected in respiratory samples by PCR, but its aetiologic role in the pathogenesis of acute respiratory infectious diseases is still unclear., Results: In this report, we describe an atopic child affected by pneumonia, with a past history of wheezing. A panel of bacteria and respiratory viruses were searched in the nasopharyngeal swab, only human bocavirus was detected by PCR., Conclusions: Detection of HboV, as the only microbial agent, in samples from children with wheezing and acute respiratory diseases supports the assumption that this emerging virus could have an aetiologic role in the pathogenesis of respiratory diseases.