Genetic diversity and its impact on disease severity in respiratory syncytial virus subtype-A and -B bronchiolitis before and after pandemic restrictions in Rome.

OBJECTIVES: To scrutinize whether the high circulation of respiratory syncytial virus (RSV) observed in 2021-2022 and 2022-2023 was due to viral diversity, we characterized RSV-A and -B strains causing bronchiolitis in Rome, before and after the COVID-19 pandemic. METHODS: RSV-positive samples, prospectively collected from infants hospitalized for bronchiolitis from 2017-2018 to 2022-2023, were sequenced in the G gene; phylogenetic results and amino acid substitutions were analyzed. Subtype-specific data were compared among seasons. RESULTS: Predominance of RSV-A and -B alternated in the pre-pandemic seasons; RSV-A dominated in 2021-2022 whereas RSV-B was predominant in 2022-2023. RSV-A sequences were ON1 genotype but quite distant from the ancestor; two divergent clades included sequences from pre- and post-pandemic seasons. Nearly all RSV-B were BA10 genotype; a divergent clade included only strains from 2021-2022 to 2022-2023. RSV-A cases had lower need of O2 therapy and of intensive care during 2021-2022 with respect to all other seasons. RSV-B infected infants were more frequently admitted to intensive care units and needed O2 in 2022-2023. CONCLUSIONS: The intense RSV peak in 2021-2022, driven by RSV-A phylogenetically related to pre-pandemic strains is attributable to the immune debt created by pandemic restrictions. The RSV-B genetic divergence observed in post-pandemic strains may have increased the RSV-B specific immune debt, being a possible contributor to bronchiolitis severity in 2022-2023.

Differences in the incidence and clinical outcomes of SARS-CoV-2 infection between Italian and non-Italian nationals using routine data.

OBJECTIVES: This study was to compare the incidence and clinical outcomes of SARS-CoV-2 infection between Italian and non-Italian nationals. STUDY DESIGN: We retrospectively analysed data from the COVID-19 Italian integrated surveillance system (14 September 2020 to 17 October 2021). METHODS: We used multivariable Cox proportional hazards models to estimate the hazard ratio (HR) of infection and, among cases, the HRs of death, hospitalisation and subsequent admission to intensive care unit in non-Italian nationals relative to Italian nationals. Estimates were adjusted for differences in sociodemographic characteristics and in the week and region of diagnosis. RESULTS: Of 4,111,067 notified cases, 336,265 (8.2%) were non-Italian nationals. Compared with Italian nationals, non-Italians showed a lower incidence of SARS-CoV-2 infection (HR = 0.81, 95% confidence interval [CI]: 0.80-0.81). However, once diagnosed, they were more likely to be hospitalised (HR = 1.90, 95% CI: 1.87-1.92) and then admitted to intensive care unit (HR = 1.08, 95% CI: 1.04-1.13), with differences larger in those coming from countries with a lower human development index. Compared with Italian cases, an increased rate of death was observed in non-Italian cases from low-human development index countries (HR = 1.41, 95% CI: 1.23-1.62). The HRs of SARS-CoV-2 infection and severe outcomes slightly increased after the start of the vaccination campaign. CONCLUSIONS: Underdiagnosis and delayed diagnosis in non-Italian nationals could explain their lower incidence compared with Italians and, among cases, their higher probability to present clinical conditions leading to worse outcomes. Facilitating early access to vaccination, diagnosis and treatment would improve the control of SARS-CoV-2 transmission and health outcomes in this vulnerable group.

Temporin G, an amphibian antimicrobial peptide against influenza and parainfluenza respiratory viruses: Insights into biological activity and mechanism of action.

Treatment of respiratory viral infections remains a global health concern, mainly due to the inefficacy of available drugs. Therefore, the discovery of novel antiviral compounds is needed; in this context, antimicrobial peptides (AMPs) like temporins hold great promise. Here, we discovered that the harmless temporin G (TG) significantly inhibited the early life-cycle phases of influenza virus. The in vitro hemagglutinating test revealed the existence of TG interaction with the viral hemagglutinin (HA) protein. Furthermore, the hemolysis inhibition assay and the molecular docking studies confirmed a TG/HA complex formation at the level of the conserved hydrophobic stem groove of HA. Remarkably, these findings highlight the ability of TG to block the conformational rearrangements of HA2 subunit, which are essential for the viral envelope fusion with intracellular endocytic vesicles, thereby neutralizing the virus entry into the host cell. In comparison, in the case of parainfluenza virus, which penetrates host cells upon a membrane-fusion process, addition of TG to infected cells provoked ~1.2 log reduction of viral titer released in the supernatant. Nevertheless, at the same condition, an immunofluorescent assay showed that the expression of viral hemagglutinin/neuraminidase protein was not significantly reduced. This suggested a peptide-mediated block of some late steps of viral replication and therefore the impairment of the extracellular release of viral particles. Overall, our results are the first demonstration of the ability of an AMP to interfere with the replication of respiratory viruses with a different mechanism of cell entry and will open a new avenue for the development of novel therapeutic approaches against a large variety of respiratory viruses, including the recent SARS-CoV2.

The Amphibian Antimicrobial Peptide Temporin B Inhibits In Vitro Herpes Simplex Virus 1 Infection.

The herpes simplex virus 1 (HSV-1) is widespread in the population, and in most cases its infection is asymptomatic. The currently available anti-HSV-1 drugs are acyclovir and its derivatives, although long-term therapy with these agents can lead to drug resistance. Thus, the discovery of novel antiherpetic compounds deserves additional effort. Naturally occurring antimicrobial peptides (AMPs) represent an interesting class of molecules with potential antiviral properties. To the best of our knowledge, this study is the first demonstration of the in vitro anti-HSV-1 activity of temporin B (TB), a short membrane-active amphibian AMP. In particular, when HSV-1 was preincubated with 20 µg/ml TB, significant antiviral activity was observed (a 5-log reduction of the virus titer). Such an effect was due to the disruption of the viral envelope, as demonstrated by transmission electron microscopy. Moreover, TB partially affected different stages of the HSV-1 life cycle, including the attachment and the entry of the virus into the host cell, as well as the subsequent postinfection phase. Furthermore, its efficacy was confirmed on human epithelial cells, suggesting TB as a novel approach for the prevention and/or treatment of HSV-1 infections.

COS-7-based model: methodological approach to study John Cunningham virus replication cycle.

John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection.Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved.Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.

A candidate anti-HIV reservoir compound, auranofin, exerts a selective 'anti-memory' effect by exploiting the baseline oxidative status of lymphocytes.

Central memory (T(CM)) and transitional memory (T(TM)) CD4(+) T cells are known to be the major cellular reservoirs for HIV, as these cells can harbor a transcriptionally silent form of viral DNA that is not targeted by either the immune system or current antiretroviral drug regimens. In the present study, we explored the molecular bases of the anti-HIV reservoir effects of auranofin (AF), a pro-oxidant gold-based drug and a candidate compound for a cure of AIDS. We here show that T(CM) and T(TM) lymphocytes have lower baseline antioxidant defenses as compared with their naive counterpart. These differences are mirrored by the effects exerted by AF on T-lymphocytes: AF was able to exert a pro-differentiating and pro-apoptotic effect, which was more pronounced in the memory subsets. AF induced an early activation of the p38 mitogen-activated protein kinase (p38 MAPK) followed by mitochondrial depolarization and a final burst in intracellular peroxides. The pro-differentiating effect was characterized by a downregulation of the CD27 marker expression. Interestingly, AF-induced apoptosis was inhibited by pyruvate, a well-known peroxide scavenger, but pyruvate did not inhibit the pro-differentiating effect of AF, indicating that the pro-apoptotic and pro-differentiating effects involve different pathways. In conclusion, our results demonstrate that AF selectively targets the T(CM)/T(TM) lymphocyte subsets, which encompass the HIV reservoir, by affecting redox-sensitive cell death pathways.

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase 1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation,sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Current advances in anti-influenza therapy.

Every year, influenza epidemics cause numerous deaths and millions of hospitalizations, but the most frightening effects are seen when new strains of the virus emerge from different species (e.g. the swine-origin influenza A/H1N1 virus), causing world-wide outbreaks of infection. Several antiviral compounds have been developed against influenza virus to interfere with specific events in the replication cycle. Among them, the inhibitors of viral uncoating (amantadine), nucleoside inhibitors (ribavirin), viral transcription and neuraminidase inhibitors (zanamivir and oseltamivir) are reported as examples of traditional virus-based antiviral strategies. However, for most of them the efficacy is often limited by toxicity and the almost inevitable selection of drug-resistant viral mutants. Thus, the discovery of novel anti-influenza drugs that target general cell signaling pathways essential for viral replication, irrespective to the specific origin of the virus, would decrease the emergence of drug resistance and increase the effectiveness towards different strains of influenza virus. In this context, virus-activated intracellular cascades, finely regulated by small changes in the intracellular redox state, can contribute to inhibit influenza virus replication and pathogenesis of virus-induced disease. This novel therapeutic approach involves advanced cell-based antiviral strategies. In this review current advances in the anti-influenza therapy for both traditional virus-based antiviral strategies as well as for alternative cell-based antiviral strategies are described focusing on the last 10 years. Anti-influenza compounds are classified on the basis of their chemical structure with a special attention to describe their synthetic pathways and the corresponding structure activity relationships.

In vitro detection of herpes simplex virus -1 and -2 infection with immunospecific GD3+-CL6-enhanced magnetic resonance imaging.

Herpes simplex virus infections are prevalent viral infections in humans. HSVs are also the most common cause of sporadic viral encephalitis (HSE). Magnetic resonance is the imaging method of choice for HSE because it provides the most sensitive method for detecting early lesions. The objective of this study is to set-up and in vitro test an experimental contrast agent specific for antigens present on HSV-infected cells, bound with a paramagnetic agent detectable by MR imaging. A selected anti-HSV HrFab was labelled with Alexa Fluor 488, 125I and Gd3+Cl6. In order to assess anti-HSV affinity and specificity, ELISA assays were performed. Vero cells infected with HSV strains were visualized by MRI using anti-HSV HrFab/Gd3+Cl6 complex. Results of the ELISA tests demonstrated that the anti-HSV HrFab labelled with Gd3+Cl6 showed similar affinity for the antigens while the 125I immunoconjugate showed reduced affinity. MRI confirmed high affinity and specificity of antibody for the detection of HSV infections.

Antiviral and immunomodulatory properties of new pro-glutathione (GSH) molecules.

Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro-GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.

Glucan-associated protein modulations and ultrastructural changes of the cell wall in Candida albicans treated with micafungin, a water-soluble, lipopeptide antimycotic.

The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet beta-glucan synthesis inhibitory concentration (0.01 microg/ml) of FK463 (micafungin). Namely, a decrease in enzymes of glucose metabolism (mostly enolase and a novel 40 kDaltons component, here identified as the enzyme fructose-1,6-biphosphate aldolase) was observed, and this was coupled with an increase in two beta1-3 exo-glucanase isoforms (34 and 44 kDa, respectively). No GAP changes were detected in the same strain of the fungus made resistant to the drug, attesting to the specificity of the observed cell wall protein modulation. In addition, GAP changes were accompanied by marked ultrastructural alterations upon treatment with the sub-MIC dose of the drug, the majority of which was an aberrant cell surface morphology and a derangement of the normal layering of the cell wall. Our data demonstrate that sub-MIC doses of micafungin do critically affect not only the beta-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

Synthesis, biological evaluation, and pharmacophore generation of uracil, 4(3H)-pyrimidinone, and uridine derivatives as potent and selective inhibitors of parainfluenza 1 (Sendai) virus.

Several new 6-oxiranyl-, 6-oxiranylmethyluracils, and pyrimidinone derivatives, synthesized by lithiation-alkylation sequence of 1,3,6-trimethyluracil, 1,3-dimethyl-6-chloromethyluracil, and 2-alkoxy-6-methyl-4(3H)-pyrimidinones, showed a potent and selective antiviral activity against Sendai virus (SV) replication. To gain insight into the structural features required for SV inhibition activity, the new compounds were submitted to a pharmacophore generation procedure using the program Catalyst. The resulting pharmacophore model showed high correlation and predictive power. It also rationalized the relationships between structural properties and biological data of these inhibitors of SV replication.

Nerve growth factor inhibits apoptosis in memory B lymphocytes via inactivation of p38 MAPK, prevention of Bcl-2 phosphorylation, and cytochrome c release.

Survival of memory B lymphocytes is tightly linked to the integrity of the Bcl-2 protein and is regulated by a nerve growth factor (NGF) autocrine circuit. In factor-starved memory B cells, the addition of exogenous NGF promptly induced p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK), dephosphorylation. Conversely, withdrawal of endogenous NGF was followed by p38 MAPK activation and translocation onto mitochondria, whereby it combined with and phosphorylated Bcl-2, as assessed by co-immunoprecipitation and kinase assays in vivo and in vitro. Mitochondria isolated from human memory B cells, then exposed to recombinant p38 MAPK, released cytochrome c, as did mitochondria from Bcl-2-negative MDCK cells loaded with recombinant Bcl-2. Apoptosis induced by NGF neutralization could be blocked by the specific p38 MAPK inhibitor SB203580 or by Bcl-2 mutations in Ser-87 or Thr-56. These data demonstrate that the molecular mechanisms underlying the survival factor function of NGF critically rely upon the continuous inactivation of p38 MAPK, a Bcl-2-modifying enzyme.

Imbalance in corneal redox state during herpes simplex virus 1-induced keratitis in rabbits. Effectiveness of exogenous glutathione supply.

A significant decrease in the antioxidant glutathione (GSH) was found in the corneal tissue of rabbits with Herpes Simplex 1 (HSV-1)-induced keratitis. Such a decrease was due to a loss of the reduced species, since no increase in its oxidized form was observed. Topical administration of purified GSH was able to reduce the virus titre in corneal tissue and, at the same time, was effective in reducing the severity and progression of keratitis and conjunctivitis. This effect was paralleled by a partial recovery in the corneal GSH content. In vitro experiments performed on HSV-1 infected corneal-derived rabbit cells showed that exogenous GSH reduced virus titre in the supernatant of infected cells. These results are in agreement with our previous findings that an oxidative environment, due to GSH depletion, is necessary for virus replication and suggest that topical GSH treatment could be considered as complementary therapy in HSV-1-induced keratitis.

Delta(12)-prostaglandin J(2) is a potent inhibitor of influenza A virus replication.

9-Deoxy-Delta(9),Delta(12)-13,14-dihydro-prostaglandin D(2) (Delta(12)-PGJ(2)), a natural cyclopentenone metabolite of prostaglandin D(2), is shown to possess therapeutic efficacy against influenza A virus A/PR8/34 (H1N1) infection in vitro and in vivo. The results indicate that the antiviral activity is associated with induction of cytoprotective heat shock proteins and suggest novel strategies for treatment of influenza virus infection.

Increased replication of Sendai virus in morphine-treated epithelial cells: evidence for the involvement of the intracellular levels of glutathione.

This paper shows that morphine increases Sendai virus replication in cultured epithelial cells. The effect was maximal when it was added before viral infection. Morphine also reduced the intracellular level of glutathione, namely, the oxidative and most abundant cell thiol. Altered intracellular redox status has recently been proposed as a factor influencing viral infection. Support for this view was provided by our data showing that inhibition of de novo glutathione synthesis, using L-buthionine sulfoximine, increased virus replication. These findings provide the first evidence that morphine increases the susceptibility to virus infection by altering the intracellular levels of glutathione.

Synthesis, cytotoxic effect and antiviral activity of 1-(beta-D-arabinofuranosyl)-5-bromo-N4-substituted cytosine and 1-(beta-D-arabinofuranosyl)-5-bromo-4-methoxypyrimidin-2(1H)-one derivatives.

A convenient and mild synthesis of 5-bromo-N4-substituted-1-(beta-D-arabinofuranosyl)cytosine and 5-bromo-O4-methyl-1-(beta-D-arabinofuranosyl)pyrimidin-2(1H)-one derivatives by selective oxyfunctionalization of the corresponding 4-thionucleosides with 3,3-dimethyldioxirane is reported. The cytotoxicity and the antiviral activity against parainfluenza 1 (Sendai virus) of all new synthesized products are also reported.

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice.

We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.