SARS-CoV-2 Inhibition by Sulfonated Compounds.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) depends on angiotensin converting enzyme 2 (ACE2) for cellular entry, but it might also rely on attachment receptors such as heparan sulfates. Several groups have recently demonstrated an affinity of the SARS-CoV2 spike protein for heparan sulfates and a reduced binding to cells in the presence of heparin or heparinase treatment. Here, we investigated the inhibitory activity of several sulfated and sulfonated molecules, which prevent interaction with heparan sulfates, against vesicular stomatitis virus (VSV)-pseudotyped-SARS-CoV-2 and the authentic SARS-CoV-2. Sulfonated cyclodextrins and nanoparticles that have recently shown broad-spectrum non-toxic virucidal activity against many heparan sulfates binding viruses showed inhibitory activity in the micromolar and nanomolar ranges, respectively. In stark contrast with the mechanisms that these compounds present for these other viruses, the inhibition against SARS-CoV-2 was found to be simply reversible

Tyrosine decarboxylase activity of Enterococcus mundtii: new insights into phenotypic and genetic aspects

Few information is available about the tyraminogenic potential of the species Enterococcus mundtii. In this study, two plant-derived strains of E. mundtii were selected and investigated to better understand the phenotypic behaviour and the genetic mechanisms involved in tyramine accumulation. Both the strains accumulated tyramine from the beginning of exponential phase of growth, independently on the addition of tyrosine to the medium. The strains accumulated also 2-phenylethylamine, although with lower efficiency and in greater extent when tyrosine was not added. Accordingly, the tyrosine decarboxylase (tyrDC) gene expression level increased during the exponential phase with tyrosine added, while it remained constant and high without precursor. The genetic organization as well as sequence identity levels of tyrDC and tyrosine permease (tyrP) genes indicated a correlation with those of phylogenetically closer enterococcal species, such as E. faecium, E. hirae and E. durans; however, the gene Na+/H+ antiporter (nhaC) that usually follow tyrP is missing. In addition, BLAST analysis revealed the presence of additional genes encoding for decarboxylase and permease in the genome of several E. mundtii strains. It is speculated the occurrence of a duplication event and the acquisition of different specificity for these enzymes that deserves further investigations

Development of Novel Therapeutic Stratégies to Combat Emerging Pathogenic Viruses

Les virus pathogènes émergents sont responsables de maladies parmi les plus sévères et représentent un problème majeur de santé publique. Les arénavirus et les hantavirus sont des virus enveloppés possédant un génome à ARN monocaténaire négatif et sont considérés comme des agents pathogènes d'importance prioritaire par les Centres pour le contrôle et la prévention des maladies (Centers of Disease Control and Prévention). Il n'y a pas de vaccin homologué contre ces virus et nos options thérapeutiques se limitent actuellement aux soins de soutien et à l'utilisation non indiquée de la ribavirine, entraînant des taux de létalité très élevés. La mise au point de médicaments antiviraux et de vaccins protecteurs efficaces contre ces virus est donc hautement prioritaire. L'attachement et l'entrée du virus représentent des étapes cruciales de l'infection de la cellule hôte et constituent des cibles thérapeutiques prometteuses. Par conséquent, dans cette thèse, nous avons étudié les facteurs cellulaires exploités par les virus pour pénétrer dans les cellules hôtes et nous les avons évalués comme cibles potentielles pour le développement de nouvelles stratégies thérapeutiques contre ces agents pathogènes. Dans le projet 1, nous avons étudié le mécanisme d'entrée de l'arenavirus de l'Ancien Monde Lassa (LASV). Nous avons utilisé le virus de la chorioméningite lymphocytaire recombinante (LCMV) exprimant la glycoprotéine de LASV (rLCMV-LASVGP) comme substitut du niveau de biosécurité (BSL) 2 pour montrer que le LASV pénètre dans les cellules épithéliales humaines via son récepteur dystroglycan (DG) à haute affinité en utilisant une voie associée à la macrôpinocytose. En examinant les inhibiteurs de kinases, nous avons identifié l'inhibiteur de HGFR EMD 1214063 ayant des propriétés prometteuses en tant que médicament antiviral contre le rLCMV-LASVGP. Ensuite, nous avons caractérisé le rôle exact de la kinase TAM Axl dans l'entrée de LASV. Nous avons constaté qu'en absence de dystroglycan fonctionnel, rLCMV- LASVGP peut utiliser Axl comme récepteur d'entrée. Dans les cellules endothéliales qui expriment un faible niveau de DG fonctionnel, les deux récepteurs peuvent favoriser l'entrée du LASV. Ces résultats justifient le ciblage d'Axl dans le traitement antiviral. Dans le projet 2, nous avons identifié le dérivé du clotrimazole TRAM-34, un inhibiteur spécifique du canal KCa3.1, en tant que composé antiviral. Nous avons utilisé des pseudotypes VSV portant la glycoprotéine des aréna, des bunya- et des filovirus et nous avons constaté que TRAM-34 bloquait sélectivement les arénavirus. Nous avons également démontré que l'effet antiviral était indépendant de sa cible pharmacologique KCa3.1. Enfin, nous avons montré que TRAM-34 n'affectait ni la liaison ni l'endocytose du récepteur viral, mais bloquait spécifiquement la fusion des arénavirus. Ces résultats ont révélé un mécanisme d'action antivirale nouveau des clotrimazoles, ouvrant la possibilité de réutiliser ces médicaments pour une intervention thérapeutique. Dans le projet 3, nous avons caractérisé la voie d'entrée utilisée par les hantavirus pour pénétrer dans les cellules des voies respiratoires épithéliales humaines. L'évaluation des inhibiteurs de kinases dans un test de criblage basé sur les pseudotypes du virus Hantaan (HTNV) et du virus Andes (ANDV) a montré un premier lien entre l'entrée des virus hanta et la macropinocytose. Nous avons ensuite testé une sélection d'inhibiteurs «diagnostiques» ciblant les facteurs cellulaires impliqués dans la macropinocytose et nous avons fourni les premières preuves du rôle de cette voie endocytaire dans l'entrée des hantavirus, avec d'importantes différences spécifiques au virus. L'entrée des VSV-HTNVGP et VSV-ANDVGP dépendait de façon critique des échangeurs sodium-proton (NHE) et de l'actine, qui sont les principales caractéristiques de la macropinocytose. Cependant, les pseudotypes HTNV et ANDV ont également montré une dépendance différentielle vis-à-vis des facteurs régulateurs connus de cette voie. Enfin, dans le laboratoire BLS 3, nous avons montré que EIPA, l'inhibiteur dés NHE, bloque l'HTNV authentique au même titre que le virus de substitution, ce qui en fait un composé antiviral potentiel. Pris ensemble, ces résultats permettent de mieux comprendre les mécanismes contrôlant l'entrée dans les cellules hôtes d'importants agents pathogènes émergents, ouvrant la voie à l'identification et au développement de nouveaux traitements antiviraux. --- Emerging human pathogenic viruses are responsible for some of the most severe diseases and represent a major public health concern. Arenaviruses and hantaviruses are enveloped viruses with negative single- stranded RNA genomes and are considered by the Centers of Disease Control and Prévention as category A and C priority pathogens, respectively. There are no licensed vaccines against these viruses and our therapeutic options are currently limited to supportive care and off-label use of ribavirin, resulting in very high case-fatality rates. The development of efficacious antiviral drugs and protective vaccines against these viruses is therefore of high priority. Virus attachment and entry represent crucial steps of host cell infection and represent therefore promising drug targets. Therefore, in this thesis we have studied cellular factors exploited by viruses to enter host cells and evaluated them as target for the development of new stratégies to fight against those pathogens. In project 1, we investigated the mechanism of entry of the Old World arenavirus Lassa virus (LASV). We used recombinant lymphocytic choriomeningitis virus (LCMV) expressing LASV glycoprotein (rLCMV-LASVGP) as a biosafety level (BSL) 2 surrogate to show that LASV enters human epithelial cells via its high affînity receptor dystroglycan (DG) using a macropinocytosis-related pathway. We found that rLCMV-LASVGP hijacks an endocytic route that only partially resembles classical macropinocytosis. By screening of kinase inhibitors, we identified the hepatocyte growth factor receptor (HGFR) inhibitor EMD 1214063 as new antiviral drug candidate against LASV. Next, we characterized the exact rôle of the TAM kinase Axl in LASV entry. We found that in absence of functional dystroglycan, rLCMV-LASVGP is able to use Axl as entry receptor. In endothelial cells that express low level of functional DG, both receptors can promote LASV entiy. These results provide a rationale for targeting Axl in antiviral therapy. In project 2, we identify the clotrimazole-derivate TRAM-34, a specific inhibitor of the KCa3.1 channel, as antiviral compound. We employed a panel of VSV pseudotypes bearing the glycoprotein of arena-, bunya- and filoviruses and found that TRAM-34 blocks selectively cell entry of arenaviruses. We also demonstrate that the antiviral effect is independent of its pharmacological target KCa3.1. Finally, we showed that TRAM-34 neither affected virus-receptor binding nor endocytosis but specifically blocked arenavirus fusion. These results revealed a novel and unexpected mechanism of antiviral action of clotrimazoles, opening the possibility to repurpose these drugs for antiviral therapeutic intervention. In project 3, we wanted to better characterize the largely unknown entry pathway used by hantaviruses to enter human epithelial airway cells. Screening of kinase inhibitors in a semi high-throughput assay based on Hantaan virus (HTNV) and Andes virus (ANDV) pseudotypes suggest macropinocytosis as possible entry pathway. We next tested a well-defïned panel of "diagnostic" inhibitors targeting cellular factors implicated in macropinocytosis and provided first evidence for a rôle of this endocytic route in hantaviruses cell entry with important virus-specific différences. Entry of both, VSV-HTNVGP and VSV- i ANDVGP critically depended on sodium-proton exchangers (NHE) and actin, which are major hallmarks of macropinocytosis. However, HTNV and ANDV pseudotypes further displayed differential dependence on known regulatory factors of this pathway. Finally, in BLS 3 laboratory we showed that the authentic HTNV is blocked by EIPA, the NHE inhibitor, in the same way as the surrogate viruses making this inhibitor a potential antiviral compound. Taken together these results allow a better understanding of mechanism governing entry in host cells of important emerging pathogens pawing the avenue for the identification and development of novel antiviral therapeutics

Comfort termico e qualita dell'aria interna negli edifici scolastici: analisi sperimentale dei modelli di previsione della sensazione termica e del legame tra controllo percepito e soddisfazione degli occupanti

Fornire alti livelli di Indoor Environmental Quality (IEQ) nelle aule scolastiche e conciliarli con l’esigenza di risparmio energetico, è essenziale per garantire il comfort di studenti ed insegnanti e le prestazioni di apprendimento. Il presente lavoro di tesi, frutto della collaborazione con l’Istituto di Ricerca “Eurac Research” di Bolzano, analizza un data set di 1171 risposte soggettive e di parametri ambientali interni raccolti in ventisei aule scolastiche ed universitarie della Provincia di Pisa con il fine di esplorare l’accuratezza dei principali modelli di comfort (modello PMV-PPD e modello adattivo BS EN 16798) nello stimare la sensazione, la preferenza e l’accettabilità termica in ognuno degli educational stages e con il fine di esplorare l’influenza del controllo percepito, in qualità di fattore di adattamento psicologico, sul comfort termico e sulla percezione della qualità dell’aria interna e del rischio di infezione da COVID-19. Infine, prendendo a riferimento un caso studio, il lavoro stima il possibile aumento di soddisfazione degli intervistati ed i risparmi energetici legati all’implementazione di soluzioni che migliorino la percezione del controllo da parte degli occupanti. Questo studio vuole fornire un contributo fortemente innovativo al panorama scientifico esistente, essendo uno dei pochi a coinvolgere contemporaneamente tutti gli educational stages di una stessa area geografica ed essendo il primo a porre l’attenzione sul problema del controllo percepito nell’ambito dell’edilizia scolastica. High levels of Indoor Environmental Quality (IEQ) in classrooms and their conciliation with the need for energy saving are essential to ensure students’ and teachers’ comfort and learning performance. This work, which was made thanks to the collaboration with "Eurac” Research Institute of Bolzano, analyzes a data set of 1171 subjective responses and internal environmental parameters collected in twenty-six school and university classrooms in Pisa Province, in order to explore the accuracy of the existing comfort models (PMV-PPD model and BS EN 16798 adaptive model) in estimating sensation, preference and thermal acceptability in each of the educational stages and to explore the influence of perceived control, as a psychological adaptation, on thermal comfort and on the perception of indoor air quality and the risk of COVID-19 infection. Lastly, analyzing a case study, the work estimates the possible increase in respondents' satisfaction and energy savings related to the implementation of solutions which are able to improve the perceived control. This study provides a highly innovative contribution to the existing scientific landscape, being one of the few to involve every educational stage in the same geographical area and being the first to focus on the problem of perceived control in schools

Identification of Novel Antiviral Compounds Targeting Entry of Hantaviruses

Hemorrhagic fever viruses, among them orthohantaviruses, arenaviruses and filoviruses, are responsible for some of the most severe human diseases and represent a serious challenge for public health. The current limited therapeutic options and available vaccines make the development of novel efficacious antiviral agents an urgent need. Inhibiting viral attachment and entry is a promising strategy for the development of new treatments and to prevent all subsequent steps in virus infection. Here, we developed a fluorescence-based screening assay for the identification of new antivirals against hemorrhagic fever virus entry. We screened a phytochemical library containing 320 natural compounds using a validated VSV pseudotype platform bearing the glycoprotein of the virus of interest and encoding enhanced green fluorescent protein (EGFP). EGFP expression allows the quantitative detection of infection and the identification of compounds affecting viral entry. We identified several hits against four pseudoviruses for the orthohantaviruses Hantaan (HTNV) and Andes (ANDV), the filovirus Ebola (EBOV) and the arenavirus Lassa (LASV). Two selected inhibitors, emetine dihydrochloride and tetrandrine, were validated with infectious pathogenic HTNV in a BSL-3 laboratory. This study provides potential therapeutics against emerging virus infection, and highlights the importance of drug repurposing

Culture-Competent SARS-CoV-2 in Nasopharynx of Symptomatic Neonates, Children, and Adolescents

Children do not seem to drive transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We isolated culture-competent virus in vitro from 12 (52%) of 23 SARS-CoV-2-infected children; the youngest was 7 days old. Our findings show that symptomatic neonates, children, and teenagers shed infectious SARS-CoV-2, suggesting that transmission from them is plausible

New insights in thermal resistance of staphylococcal strains belonging to the species Staphylococcus epidermidis, Staphylococcus lugdunensis and Staphylococcus aureus

In this study the thermal resistance of seven strains of staphylococci was studied. Three of them (two Staphylococcus epidermidis and one Staphylococcus lugdunensis) were isolated from pasteurized tofu, while one Staphylococcus aureus from fermented sausages. Also the corresponding type strains were tested. Their heat resistance was assessed at 80 C also in relation to different growth conditions (high NaCl concentrations and low pH). Moreover, the effect of two terpenes (carvacrol and citral) on their survival during thermal treatment was assessed. All the strains were able to survive a 20 min thermal treatment and showed a similar behavior, i.e. an inactivation of about 6 log units in the first 2e4 min of treatment, followed by constant survivor counts (2e4 log CFU/ml). The pre-culture of staphylococci under stressing conditions (high NaCl concentration and low pH) increased the susceptibility to thermal treatment in three strains isolated from food matrices. The addition of carvacrol and citral during thermal treatment influenced the number of survived cells at the end of the treatment. This new insight in the thermal resistance of staphylococci needs to be better investigated, in order to study proper strategies to control their occurrence and behavior in real food systems

Sequential infections with rhinovirus and influenza modulate the replicative capacity of SARS-CoV-2 in the upper respiratory tract

Although frequently reported since the beginning of the pandemic, questions remain regarding the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interaction with circulating respiratory viruses in coinfected patients. We here investigated dual infections involving early-pandemic SARS-CoV-2 and the Alpha variant and three of the most prevalent respiratory viruses, rhinovirus (RV) and Influenza A and B viruses (IAV and IBV), in reconstituted respiratory airway epithelial cells cultured at air–liquid interface. We found that SARS-CoV-2 replication was impaired by primary, but not secondary, rhino- and influenza virus infection. In contrast, SARS-CoV-2 had no effect on the replication of these seasonal respiratory viruses. Inhibition of SARS-CoV-2 correlated better with immune response triggered by RV, IAV and IBV than the virus entry. Using neutralizing antibody against type I and III interferons, SARS-CoV-2 blockade in dual infections could be partly prevented. Altogether, these data suggested that SARS-CoV-2 interaction with seasonal respiratory viruses would be modulated by interferon induction and could impact SARS-CoV-2 epidemiology when circulation of other respiratory viruses is restored