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Oncolytic viruses (OVs) are anti-cancer therapeutics combining the selective killing of cancer cells with the triggering of an anti-tumoral immune response. The latter effect can be improved by arming OVs with immunomodulatory factors. Due to the heterogeneity of cancer and the tumor microenvironment, it is anticipated that strategies based on the co-expression of multiple therapeutic molecules that interfere with different features of the target malignancy will be more effective than mono-therapies. Here, we show that (i) the simultaneous expression of different proteins in triple-negative breast cancer (TNBC) cells can be achieved through their infection with a combination of OVs based on herpes simplex virus type 1 (oHSV1), each encoding a single transgene. (ii) The level of expressed proteins is dependent on the number of infectious viral particles utilized to challenge tumor cells. (iii) All recombinant viruses exhibited comparable efficacy in the killing of TNBC cells in single and multiple infections and showed similar kinetics of replication. Overall, our results suggest that a strategy based on co-infection with a panel of oHSV1s may represent a promising combinatorial therapeutic approach for TNBC, as well as for other types of solid tumors, that merits further investigation in more advanced in vitro and in vivo models.

Abstract Adult vaccination programs are receiving increasing attention however, little is known regarding the impact of age on the maintenance of the immune response. We investigated this issue in the context of a human papillomavirus (HPV) vaccination program collecting real-world data on the durability of humoral immunity in 315 female subjects stratified according to vaccination age (adolescents and adults) and sampled at early or late time points after the last vaccine dose. HPV-specific IgGs, but not memory B cells, were induced and maintained at higher levels in subjects vaccinated during adolescence. Nonetheless, antibody functions waned over time to a similar degree in adolescents and adults. To shed light on this phenomena, we analyzed quantitative and qualitative properties of lymphocytes. Similar biochemical features were observed between B-cell subsets from individuals belonging to the two age groups. Long term humoral responses toward vaccines administered at an earlier age were comparably maintained between adolescents and adults. The percentages of naïve B and CD4+ T cells were significantly higher in adolescents, and the latter directly correlated with IgG titers against 3 out of 4 HPV types. Our results indicate that age-specific HPV vaccine responsiveness is mostly due to quantitative differences of immune cell precursors rather than qualitative defects in B cells. In addition, our results indicate that adults also have a good humoral immunogenic profile, suggesting that their inclusion in catch-up programmes is desirable.

Here we propose that viruses emerging in the human population undergo an evolution that is conditioned by the rules of chaos. Our data support the notion that the initial growth rate “r” affects the chances of the virus to establish a long-lasting relationship with the new host. Indeed, an emerging virus is able to spread and adapt only when it displays an initial r falling in a range frankly associated with chaotic growth.

Our hypothesis about evolution of the COVID-19 pandemic foresees an inverse relation between infectivity (R0) and lethality (L) of SARS-CoV-2. The above parameters are driven by a continuing mutation process granting the virus a clear survival advantage over virulence. For interpreting this relation we adopted a simple equation, R0 × L ≈ k, by which R0 and L depend upon a constant k, that corresponds to an intrinsic property of the viral species involved. The hypothesis was verified by following changes of the R0 and L terms of the formula in the different variants of SARS-CoV-2 that progressively appeared. A further validation came when the equation was applied to pandemic and epidemic influenza type A viruses, Ebola virus and measles virus. We believe this equation that considers virus biology in Darwinian terms could be extremely useful to better face infectious viral threats and validate virus-host molecular interactions relevant to viral pathogenesis.

Open reading frame 8 (ORF8) shows one of the highest levels of variability among accessory proteins in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits the presentation of viral antigens by the major histocompatibility complex class I (MHC-I), which interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 in evading immunity and plays a role in SARS-CoV-2 replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein, defines the B.1.1.7 lineage of SARS-CoV-2, engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) with the Q27STOP mutations were identified among 49,055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents, which include Africa, Asia, Europe and South America. Based on various quantitative features, such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, nine possible T-ORF8 unique variants were defined. The question as to whether T-ORF8 variants function similarly to the wild type ORF8 is yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.

Abstract Background Few studies focused on longitudinal modifications over time of high-risk HPV (HR-HPV) at anal and oral sites in HIV+ men who have sex with men (MSM). Methods We described patterns and longitudinal changes of HR-HPV detection and the prevalence of HR-HPV covered by the nonavalent HPV vaccine (vax-HPV) at oral and anal sites in 165 HIV+ MSM followed in an Italian hospital. The samples were collected at baseline and after 24 months (follow-up). The presence of HPV was investigated with Inno-LiPA HPV Genotyping Extra II. Results Median age was 44 years (IQR 36–53), median CD4+ cell count at nadir was 312 cells/mm3 (IQR 187–450). A total of 120 subjects (72.7%) were receiving successful antiretroviral therapy (ART). At baseline and follow-up, the frequency of HR-HPV was significantly higher in the anal site (65.4% vs 9.4 and 62.4% vs 6.8%, respectively). Only 2.9% of subjects were persistently HR-HPV negative at both sites. All oral HR-HPV were single at baseline vs 54.6% at baseline at the anal site (p = 0.005), and all oral HR-HPV were single at follow-up vs 54.4% at anal site at follow-up (p = 0.002). The lowest rate of concordance between the oral and anal results was found for HR-HPV detection; almost all HR-HPV positive results at both anal and oral sites had different HR-HPV.The most frequent HR-HPV in anal swabs at baseline and follow-up were HPV-16 and HPV-52.At follow-up at anal site, 37.5% of patients had different HR-HPV genotypes respect to baseline, 28.8% of subjects with 1 HR-HPV at baseline had an increased number of HR-HPV, and patients on ART showed a lower frequency of confirmed anal HR-HPV detection than untreated patients (p = 0.03) over time. Additionally,54.6 and 50.5% of patients had only HR-vax-HPV at anal site at baseline and follow-up, respectively; 15.2% had only HR-vax-HPV at baseline and follow-up. Conclusions We believe that it is important testing multiple sites over time in HIV-positive MSM. ART seems to protect men from anal HR-HPV confirmed detection. Vaccination programmes could reduce the number of HR-HPV genotypes at anal site and the risk of the first HR-HPV acquisition at the oral site.

Abstract Background With few exceptions, current chemotherapy and radiotherapy protocols only obtain a slightly prolonged survival with severe adverse effects in patients with advanced solid tumors. In particular, most solid malignancies not amenable to radical surgery still carry a dismal prognosis, which unfortunately is also the case for relapsing disease after surgery. Even though targeted therapies obtained good results, clinical experience showed that tumors eventually develop resistance. On the other hand, earlier attempts of cancer immunotherapy failed to show consistent efficacy. More recently, a deeper knowledge of immunosuppression in the tumor microenvironment (TME) allowed the development of effective drugs: in particular, monoclonal antibodies targeting the so-called immune checkpoint molecules yielded striking and lasting effects in some tumors. Unfortunately, these monoclonal antibodies are not effective in a majority of patients and are ineffective in several solid malignancies. Furthermore, due to their mechanism of action, checkpoint inhibitors often elicit autoimmune-like disease. Main body The use of viruses as oncolytic agents (OVs) was considered in the past, while only recently OVs revealed a connection with immunotherapy. However, their antitumoral potential has remained largely unexplored, due to safety concerns and some limitations in the techniques to manipulate viruses. OV research was recently revived by a better knowledge of viral/cancer biology and advances in the methodologies to delete virulence/immune-escape related genes from even complex viral genomes or “to arm” OVs with appropriate transgenes. Recently, the first oncolytic virus, the HSV-1 based Talimogene Laherparepvec (T-VEC), was approved for the treatment of non-resectable melanoma in USA and Europe. Conclusion OVs have the potential to become powerful agents of cancer immune and gene therapy. Indeed, in addition to their selective killing activity, they can act as versatile gene expression platforms for the delivery of therapeutic genes. This is particularly true for viruses with a large DNA genome, that can be manipulated to address the multiple immunosuppressive features of the TME. This review will focus on the open issues, on the most promising lines of research in the OV field and, more in general, on how OVs could be improved to achieve real clinical breakthroughs in cancers that are usually difficult to treat by immunotherapy.

Remdesivir is the first drug approved for treatment of COVID-19 but current evidence for recommending its use for the treatment of moderate-to-severe disease is still controversial among clinical guidelines. We performed a nationwide, registry-based study including all Italian hospitalized patients with COVID-19 treated with remdesivir to assess the impact of major confounders on crude 15-day and 29-day mortality. Mortality was calculated using the Kaplan–Meier estimator and the Cox proportional-hazards model was applied to analyze the risks by patient’s baseline features. In total, 16,462 patients treated with remdesivir from 29 October 2020 to 17 December 2020 were entered in the study. Crude 15-day and 29-day mortality were 7.1% (95% CI, 6.7–7.5%) and 11.7% (95% CI, 11.2–12.2%), respectively. Being treated within two days of admission reduced the risk of death by about 40% (HR 1.4, 95% CI, 1.2–1.6). Results from the largest cohort of remdesivir-treated patients suggests that mortality in SARS-CoV-2 hospitalized patients is substantially influenced by the days between SARS-CoV-2 diagnosis and drug prescription. Current recommendations and future clinical trials for remdesivir alone or in combination should carefully consider the target population and timing for best efficacy of treatment.

Abstract Background This longitudinal study described Cytomegalovirus (CMV) DNA, Epstein-Barr (EBV) DNA and human herpesvirus 8 (HHV-8) DNA asymptomatic salivary shedding in HIV-positive men who have sex with men (MSM). We aimed to 1-analyze frequency and persistence of herpesvirus shedding, 2-correlate herpesvirus positivity and HIV viroimmunological parameters and 3-assess the association between HIV-RNA suppression and herpesvirus replication. Methods Herpesvirus DNA was tested with an in-house real-time PCR in 2 salivary samples obtained at T0 and T1 (24 months after T0). HIV-RNA was evaluated in the 24 months prior to T0 and in the 24 months prior to T1; MSM were classified as successfully suppressed patients (SSPs), viremic patients (VPs) and partially suppressed patients (PSPs). EBV DNA load was classified as low viral load (EBV-LVL, value ≤10,000 copies/ml) and as high viral load (EBV-HVL,> 10,000 copies/ml). Mann-Whitney U test tested the difference of the median between groups of patients. Chi-squared test and Fisher’s exact test compared categorical variables according to the frequencies. Kruskal-Wallis test compared continuous data distributions between levels of categorical variables. Results Ninety-two patients (median CD4+ count 575 cells/mm3, median nadir 330 CD4+ cells/mm3) were included: 40 SSPs,33 VPs and 19 PSPs. The more frequently single virus detected was EBV, both at T0 and at T1 (in 67.5 and 70% of SSPs, in 84.8 and 81.8% of VPs and in 68.4 and 73.7% of SPSs) and the most frequently multiple positivity detected was EBV + HHV-8. At T1, the percentage of CMV positivity was higher in VPs than in SSPs (36.4% vs 5%, p

Oral mucosa epithelial stem cells from a patient affected by Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome carrying the R279H mutation in the TP63 gene were reprogrammed into human induced pluripotent stem cells (hiPSCs) with episomal vectors. The generated UNIPDi003-A-hPSC line retained the mutation of the parental cells and showed a normal karyotype upon long term culture. Analysis of residual transgenes expression showed that the episomal vectors were eliminated from the cell line. UNIPDi003-A-hiPSCs expressed the undifferentiated state marker alkaline phosphatase along with a panel of pluripotency markers, and formed embryoid bodies capable of expressing markers belonging to all the three germ layers.

Transgene free UNIPDi002-A-human induced pluripotent stem cell (hiPSC) line was generated by Sendai Virus Vectors reprogramming from human oral mucosal epithelial stem cells (hOMESCs) of a patient affected by ectrodactyly-ectodermal dysplasia-clefting (EEC)-syndrome, carrying a mutation in exon 8 of the TP63 gene (R304Q). The UNIPDi002-A-hiPSC line retained the mutation of the parental R304Q-hOMESCs and displayed a normal karyotype. No residual expression of transgenes nor Sendai virus vector sequences were detected in the line at passage 8. UNIPDi002-A-hiPSC expressed a panel of pluripotency-associated markers and could form embryoid bodies expressing markers belonging to the three germ layers ectoderm, endoderm and mesoderm.

Human oral mucosa epithelial stem cells (hOMESCs) were obtained from a fresh oral biopsy collected from a healthy subject at the Fondazione Banca degli Occhi del Veneto (FBOV). An integration-free reprogramming protocol was applied exploiting episomal plasmids transfected into cells using a Nucleofector device. Around day 20 post transfection, several human induced pluripotent stem cell (hiPSC) colonies were manually picked and expanded. One of these (UNIPDi001-A-hiPSCs) expressed undifferentiated state marker alkaline phosphatase along with a panel of pluripotency state markers and was able to differentiate into the derivatives of all the three germ layers.

One of the main pathological features of Parkinson’s disease (PD) is a diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 Ub ligase promotes aS degradation by the endosomal–lysosomal route. Interestingly, NEDD4, as well as being a small molecule able to trigger its functions, is protective against human aS toxicity in evolutionary distant models. While pharmacological activation of E3 enzymes is not easy to achieve, their flexibility and the lack of “consensus” motifs for Ub-conjugation allow the development of engineered Ub-ligases, able to target proteins of interest. We developed lentiviral vectors, encoding well-characterized anti-human aS scFvs fused in frame to the NEDD4 catalytic domain (ubiquibodies), in order to target ubiquitinate aS. We demonstrate that, while all generated ubiquibodies bind to and ubiquitinate aS, the one directed against the non-amyloid component (NAC) of aS (Nac32HECT) affects aS’s intracellular levels. Furthermore, Nac32HECT expression partially rescues aS’s overexpression or mutation toxicity in neural stem cells. Overall, our data suggest that ubiquibodies, and Nac32HECT in particular, represent a valid platform for interfering with the effects of aS’s accumulation and aggregation in neurons.

It has been theorized that Calmette-Guérin bacillus may prevent or reduce the severity of COVID-19 through a nonspecific stimulation of the immune system. A preliminary assessment of SARS-CoV-2 infection rates and outcomes among 2803 individuals affected with high risk non-muscle-invasive bladder cancer and treated with intra-bladder instillation of BCG, showed no evidence of a protective effect. However, the interpretation of these data need some caution, due to the low prevalence of infection (

Pneumococcal diseases are associated with a significant clinical and economic burden. The 7-valent pneumococcal conjugate vaccine (PCV-7) has been used for the immunization of newborns against invasive pneumococcal diseases (IPD) in Italy while now, the pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) and the 13-valent pneumococcal conjugate vaccine (PCV-13) are available. The aim of this analysis was to compare the estimated health benefits, cost and cost-effectiveness of immunization strategies vs. non-vaccination in Italy using the concept of overall vaccine effectiveness. A published Markov model was adapted using local data wherever available to compare the impact of neonatal pneumococcal vaccination on epidemiological and economic burden of invasive and non-invasive pneumococcal diseases, within a cohort of newborns from the Italian National Health Service (NHS) perspective. A 18-year and a 5-year time horizon were considered for the base-case and scenario analysis, respectively. PHiD-CV and PCV-13 are associated with the most important reduction of the clinical burden, with a potential marginal advantage of PHiD-CV over PCV-13. Compared with no vaccination, PHiD-CV is found on the higher limit of the usually indicated willingness to pay range (30,000 - 50,000€/quality-adjusted life year [QALY] gained), while the incremental cost-effectiveness ratio (ICER) for PCV-13 is slightly above. Compared with PCV-13, PHiD-CV would provide better health outcomes and reduce costs even at parity price, solely due to its differential effect on the incidence of NTHi acute otitis media (AOM). The analysis on a shorter time horizon confirms the direction of the base-case.

Abstract Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the top 10 causes of death worldwide in 2015. The recent emergence of strains resistant to all current drugs urges the development of compounds with new mechanisms of action. G-quadruplexes are nucleic acids secondary structures that may form in G-rich regions to epigenetically regulate cellular functions. Here we implemented a computational tool to scan the presence of putative G-quadruplex forming sequences in the genome of Mycobacterium tuberculosis and analyse their association to transcription start sites. We found that the most stable G-quadruplexes were in the promoter region of genes belonging to definite functional categories. Actual G-quadruplex folding of four selected sequences was assessed by biophysical and biomolecular techniques: all molecules formed stable G-quadruplexes, which were further stabilized by two G-quadruplex ligands. These compounds inhibited Mycobacterium tuberculosis growth with minimal inhibitory concentrations in the low micromolar range. These data support formation of Mycobacterium tuberculosis G-quadruplexes in vivo and their potential regulation of gene transcription, and prompt the use of G4 ligands to develop original antitubercular agents.

Objectives: We described Klebsiella pneumoniae producing carbapenemase (CPKP) spread from 01/01/2015 to 13/09/16 in a tertiary level hospital. Methods: The first positive surveillance rectal swab (SRS) or clinical sample (CS) collected in the medical department (MD), surgical department (SD) and intensive care department (ICD) were included in the study. A validated in-house Real-Time PCR method was used to detect carbapenemases; multilocus sequence typing (MLST) was used for further characterization of the strains. Results: 21535 patients were included: 213 CPKP strains from surveillance rectal swab (SRS) and 98 from clinical samples (CS) were collected. The percentage of CPKP detected in SRS with respect to CS increased in the medical MD from 2015 to 2016 (p = 0.01) and in ICD from 2012 to 2015 (p = 0.0001), while it decreased in SD from 2014 to 2016 (p = 0.003); 68.5% of the positive SRS had a previous negative SRS; CPKP was more frequently identified in CS than in SRS in MD. Twelve strains harboured more than one carbapenemase gene. Many other species harbouring a carbapenemase gene were collected. Conclusions: MDs need more inclusive surveillance criteria. The late detection of positive SRS underlined the risk of colonization during hospitalization.

Abstract G-quadruplexes (G4s) are secondary structures of nucleic acids that epigenetically regulate cellular processes. In the human immunodeficiency lentivirus 1 (HIV-1), dynamic G4s are located in the unique viral LTR promoter. Folding of HIV-1 LTR G4s inhibits viral transcription; stabilization by G4 ligands intensifies this effect. Cellular proteins modulate viral transcription by inducing/unfolding LTR G4s. We here expanded our investigation on the presence of LTR G4s to all lentiviruses. G4s in the 5′-LTR U3 region were completely conserved in primate lentiviruses. A G4 was also present in a cattle-infecting lentivirus. All other non-primate lentiviruses displayed hints of less stable G4s. In primate lentiviruses, the possibility to fold into G4s was highly conserved among strains. LTR G4 sequences were very similar among phylogenetically related primate viruses, while they increasingly differed in viruses that diverged early from a common ancestor. A strong correlation between primate lentivirus LTR G4s and Sp1/NFκB binding sites was found. All LTR G4s folded: their complexity was assessed by polymerase stop assay. Our data support a role of the lentiviruses 5′-LTR G4 region as control centre of viral transcription, where folding/unfolding of G4s and multiple recruitment of factors based on both sequence and structure may take place.

Abstract G-quadruplexes (G4s) are nucleic acids secondary structures, epigenetic regulators in cells and viruses. In herpes simplex virus 1 (HSV-1)-infected cells, G4s are massively present during viral replication. We here aimed at investigating the possibility to target the HSV-1 G4s by a core extended naphtalene diimide (c-exNDI) G4 ligand. Biophysical and biomolecular analysis proved that c-exNDI stabilized the HSV-1 G4s in a concentration dependent manner. In MS competition assays, c-exNDI preferentially recognized HSV-1 G4s over cellular telomeric G4s, the most represented G4s within cells; other less abundant cellular G4s were also recognized. Treatment of HSV-1 infected cells with c-exNDI at low nanomolar concentrations induced significant virus inhibition with no cytotoxicity. The mechanism of action was ascribed to G4-mediated inhibition of viral DNA replication, with consequent impairment of viral genes transcription. Our data suggest that the observed potent antiviral activity and low cytotoxicity mainly depend on a combination of c-exNDI affinity for HSV-1 G4s and their massive presence during infection. HSV-1 G4s may thus represent new effective antiviral targets: the fact that no current antiherpetic drug exploits them and their presence at the viral genome, responsible for both active and latent HSV infections, makes them particularly attracting.

One hundred and twenty-two Mycobacterium chimaera strains isolated in Italy from cardiac surgery-related patients, cardiac surgery-unrelated patients and from heater-cooler units, were submitted to whole-genome sequencing and to subsequent SNP analysis. All but one strains isolated from cardiac surgery-related patients belonged to Subgroup 1.1 (19/23) or Subgroup 1.8 (3/23). Only 28 out of 79 strains isolated from heater-cooler units belonged to groupings other than 1.1 and 1.8. The strains isolated from cardiac surgery-unrelated patients were instead distributed across the phylogenetic tree. Our data, the first on isolates from Italy, are in agreement with a recent large genomic study suggesting a common source, represented by strains belonging to Subgroups 1.1 and 1.8, of cardiac surgery-related Mycobacterium chimaera infections. The strains belonging to groupings other than 1.1 and 1.8 isolated from heather-cooler units evidently resulted from contaminations at hospital level and had no share in the Mycobacterium chimaera outbreak. One Mycobacterium chimaera strain investigated in this study proved distant from every previously known Mycobacterium chimaera Groups (1, 2, 3 and 4) and we propose to assign to a novel group, named "Group 5".

Chronic HCV infection is characterized by several immunological alterations, such as the accumulation of suppressor cells and of hyperactivated T lymphocytes. However, it is unclear whether direct-acting antiviral (DAA)-mediated HCV clearance restores immune dysfunctions. We performed a phenotypic characterization by flow cytometry of different immune cell subsets, including monocytic myeloid-derived suppressor cells (M-MDSCs) and T lymphocytes in 168 patients with persistent HCV infection not treated, under DAA therapies and sustained virological responders. Chronic HCV infection prompted the accumulation of M-MDSCs independently of patient and clinical characteristics, and altered their metabolic properties. HCV RNA was undetectable in the majority of patients just after few weeks of DAA therapy, whereas M-MDSC levels normalized only 6 months after therapy. In addition, HCV infection deeply perturbed the T cell compartment since a re-distribution of memory CD4+ and CD8+ T cells was observed at the expenses of naïve cells, and memory T lymphocytes displayed increased activation. Notably, these features were only partially restored by DAA therapies in the CD4, but not in the CD8, compartment as high immune activation levels persisted in the terminally differentiated memory CD8+ T cells even more than 1 year after sustained virological response. Together, these results suggest that successful DAA therapies do not lead to full immunological reconstitution as fast as viral clearance.

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.

The estimated smooth curve of the percentage of subjects positive to SARS-CoV-2 started decreasing in Italy at the beginning of January 2021, due to the government containment measures undertaken from Christmas until 7 January. Approximately two weeks after releasing the measures, the curve stopped to decrease and remained approximately constant for four weeks to increase again in the middle of February. This epidemic phase had a public health care impact since, from the beginning of the fourth week of February, the curve of the intensive care unit’s occupancy started to grow. This wave of infection was characterized by the presence of new virus variants, with a higher than 80% dominance of the so-called “English” variant, since 15 April. School activities in Italy started at different times from 7 January until 8 February, depending on every region’s decision. Our present data on the incidence of SARS-CoV-2 in different age groups in Italy are in agreement with literature reports showing that subjects older than 10 years are involved in virus transmission. More importantly, we provide evidence to support the hypothesis that also individuals of age 0–9 years can significantly contribute to the spread of SARS-CoV-2.

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited HCMV AD169 strain in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.

Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on a single isolate of bat coronaviruses (bat CoVs) which does not adequately represent genetically related coronaviruses (CoVs) [...]

(1) Background: A better understanding of COVID-19 dynamics in terms of interactions among individuals would be of paramount importance to increase the effectiveness of containment measures. Despite this, the research lacks spatiotemporal statistical and mathematical analysis based on large datasets. We describe a novel methodology to extract useful spatiotemporal information from COVID-19 pandemic data. (2) Methods: We perform specific analyses based on mathematical and statistical tools, like mathematical morphology, hierarchical clustering, parametric data modeling and non-parametric statistics. These analyses are here applied to the large dataset consisting of about 19,000 COVID-19 patients in the Veneto region (Italy) during the entire Italian national lockdown. (3) Results: We estimate the COVID-19 cumulative incidence spatial distribution, significantly reducing image noise. We identify four clusters of connected provinces based on the temporal evolution of the incidence. Surprisingly, while one cluster consists of three neighboring provinces, another one contains two provinces more than 210 km apart by highway. The survival function of the local spatial incidence values is modeled here by a tapered Pareto model, also used in other applied fields like seismology and economy in connection to networks. Model’s parameters could be relevant to describe quantitatively the epidemic. (4) Conclusion: The proposed methodology can be applied to a general situation, potentially helping to adopt strategic decisions such as the restriction of mobility and gatherings.

Intracellular organelles enwrapped in membranes along with a complex network of vesicles trafficking in, out and inside the cellular environment are one of the main features of eukaryotic cells. Given their central role in cell life, compartmentalization and mechanisms allowing their maintenance despite continuous crosstalk among different organelles have been deeply investigated over the past years. Here, we review the multiple functions exerted by the endosomal sorting complex required for transport (ESCRT) machinery in driving membrane remodeling and fission, as well as in repairing physiological and pathological membrane damages. In this way, ESCRT machinery enables different fundamental cellular processes, such as cell cytokinesis, biogenesis of organelles and vesicles, maintenance of nuclear–cytoplasmic compartmentalization, endolysosomal activity. Furthermore, we discuss some examples of how viruses, as obligate intracellular parasites, have evolved to hijack the ESCRT machinery or part of it to execute/optimize their replication cycle/infection. A special emphasis is given to the herpes simplex virus type 1 (HSV-1) interaction with the ESCRT proteins, considering the peculiarities of this interplay and the need for HSV-1 to cross both the nuclear-cytoplasmic and the cytoplasmic-extracellular environment compartmentalization to egress from infected cells.

Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity.Emerging Microbes & Infections (2017) 6, e99; doi:10.1038/emi.2017.87; published online 8 November 2017

Ectrodactyly‐ectodermal dysplasia‐clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild‐type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K‐p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K‐p63 OMESCs were able to generate well‐organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K‐p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. Significance This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K‐p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly‐ectodermal dysplasia‐clefting syndrome.

SARS-CoV-2 is highly contagious, rapidly turned into a pandemic, and is causing a relevant number of critical to severe life-threatening COVID-19 patients. However, robust statistical studies of a large cohort of patients, potentially useful to implement a vaccination campaign, are rare. We analyzed public data of about 19,000 patients for the period 28 February to 15 May 2020 by several mathematical methods. Precisely, we describe the COVID-19 evolution of a number of variables that include age, gender, patient’s care location, and comorbidities. It prompts consideration of special preventive and therapeutic measures for subjects more prone to developing life-threatening conditions while affording quantitative parameters for predicting the effects of an outburst of the pandemic on public health structures and facilities adopted in response. We propose a mathematical way to use these results as a powerful tool to face the pandemic and implement a mass vaccination campaign. This is done by means of priority criteria based on the influence of the considered variables on the probability of both death and infection.

After a linear growth during September, the diffusion in Italy of SARS-CoV-2, responsible for COVID-19, has been growing exponentially since the end of that month with a doubling time approximately equal to one week [...]

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

Introduction: Crimean–Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF orthonairovirus (CCHFV), a class 4 pathogen. Hyalomma ticks are the viral reservoir, and they represent the main vector. CCHFV can be transmitted to its hosts during tick blood feeding. We have previously shown that CCHFV can persistently infect Hyalomma-derived tick cell lines without any cytopathic effect. However, the mechanism allowing for the establishment of a persistent viral infection in ticks is still unknown. It has been recently reported that Hazara orthonairovirus (HAZV) can be used as a BSL-2 model virus instead of CCHFV to study viral/vector interaction. The aim of our study is to elucidate the mechanism that allows establishment of persistent CCHFV infection in ticks using HAZV as a model. Methods: We used classical and molecular methods applied to virology to characterize the establishment of persistent HAZV infection in two Hyalomma anatolicum-derived cell lines—HAE/CTVM8 and HAE/CTVM9. Results: As for CCHFV, we showed that HAZV persistently infects tick cells without any sign of cytopathic effect and that infected cells can be cultured for more than one year. The persistent infection is characterized by a low viral titer compared to the initial time points. Interestingly, short viral-derived DNA forms (vDNAs) start to be detected in parallel with the beginning of viral replication and are maintained in persistently-infected cells. Experiments with the antiretroviral drug AZT suggest that vDNAs are produced by retrotranscriptase activity. Furthermore, we collected evidence that vDNAs are not integrated and seem to be involved in the downregulation of viral replication by promoting cell survival. Conclusion: vDNA synthesis might represent a strategy to control the replication of RNA viruses in ticks, as recently demonstrated in insects, allowing for persistent infection of virus vectors.

Drug-loaded, PEGylated, organic-modified silica (ORMOSIL) nanoparticles prepared by microemulsion condensation of vinyltriethoxysilane (VTES) were investigated as potential nanovectors for cancer therapy. To target cancer stem cells, anti-CD44v6 antibody and hyaluronic acid (HA) were conjugated to amine-functionalized PEGylated ORMOSIL nanoparticles through thiol-maleimide and amide coupling chemistries, respectively. Specific binding and uptake of conjugated nanoparticles were studied on cells overexpressing the CD44v6 receptor. Cytotoxicity was subsequently evaluated in the same cells after the uptake of the nanoparticles. Internalization of nanocarriers loaded with the anticancer drug 3N-cyclopropylmethyl-7-phenyl-pyrrolo- quinolinone (MG2477) into cells resulted in a substantial increase of the cytotoxicity with respect to the free formulation. Targeting with anti-CD44v6 antibodies or HA yielded nanoparticles with similar effectiveness, in their optimized formulation.

G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in organisms. G-quadruplexes are present in eukaryotes, prokaryotes, and viruses. In the latter, mounting evidence indicates their key biological activity. Since data on viruses are scattered, we here present a comprehensive analysis of potential quadruplex-forming sequences (PQS) in the genome of all known viruses that can infect humans. We show that occurrence and location of PQSs are features characteristic of each virus class and family. Our statistical analysis proves that their presence within the viral genome is orderly arranged, as indicated by the possibility to correctly assign up to two-thirds of viruses to their exact class based on the PQS classification. For each virus we provide: i) the list of all PQS present in the genome (positive and negative strands), ii) their position in the viral genome, iii) the degree of conservation among strains of each PQS in its genome context, iv) the statistical significance of PQS abundance. This information is accessible from a database to allow the easy navigation of the results: http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=g4virus. The availability of these data will greatly expedite research on G-quadruplex in viruses, with the possibility to accelerate finding therapeutic opportunities to numerous and some fearsome human diseases.

Herpes Simplex Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection and allow recognition of an original pathophysiologic mechanism underlying gastrointestinal diseases as well as identification of novel therapeutic targets.

The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host–pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance.

Objectives: Effective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV–HCV co-infected patients. Methods: This study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV–HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing. Results: DCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA

Objectives: Very few data exist on viral meningitis and encephalitis in elderly patients (>65 years old). Methods: This study investigated the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, cytomegalovirus (CMV), Epstein–Barr virus (EBV), enterovirus (EV), human adenovirus (HAdV), human parechoviruses (HPeVs), and tick-borne encephalitis virus (TBEV) through real-time PCR (RT-PCR) in patients >65 years old who had cerebrospinal fluid (CSF) tested for a suspected central nervous system infection. Results: A total of 2868 RT-PCRs were performed on 502 CSF samples. Overall, 65 positive RT-PCRs were found: 23 for HSV (35.4% of positives), 15 for EV (23.1% of positives), 14 for EBV (21.5% of positives), 12 for VZV (18.5% of positives), and one for CMV (1.5% of positives). A positive RT-PCR in CSF was detected in 24 (17.4%) patients aged ≥80 years and in 35 (9.6%) patients aged 65–79 years (p = 0.02). VZV was more frequently detected in the oldest subjects (5.9% vs. 1.6%, p = 0.03). Conclusions: HSV was the most common viral aetiology identified in the study, with VZV infection being recognized more frequently in those patients aged ≥80 years.

Human papillomavirus (HPV) persistent infections are associated with cervical cancer and other HPV-related diseases and tumors. Thus, the characterization of long lasting immunity to currently available HPV vaccines is important. A total of 149 female subjects vaccinated with Cervarix or Gardasil participated to the study and they were stratified according to age (10−12-year-old and 16−20-year-old). Humoral immune responses (IgG and neutralizing antibody titers, antibody avidity) and circulating memory B cells were analyzed after an average of 4−6 years from the third immunization. The humoral responses against HPV-16 and HPV-18 (and HPV-6 and HPV-11 for Gardasil) were high in both age groups and vaccines up to six years from the third dose. However, Cervarix induced significantly higher and more persistent antibody responses, while the two vaccines were rather equivalent in inducing memory B cells against HPV-16 and HPV-18. Moreover, the percentage of subjects with vaccine-specific memory B cells was even superior among Gardasil vaccinees and, conversely, Cervarix vaccinated individuals with circulating antibodies, but undetectable memory B cells were found. Finally, a higher proportion of Cervarix-vaccinated subjects displayed cross-neutralizing responses against non-vaccine types HPV-31 and HPV-45. Gardasil and Cervarix may, thus, differently affect long-lasting humoral immunity from both the quantitative and qualitative point of view.

Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells.

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013–2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.