Hantavirus Pulmonary Syndrome: CD8+and CD4+Cytotoxic T Lymphocytes to Epitopes on Sin Nombre Virus Nucleocapsid Protein Isolated during Acute Illness

AbstractIn 1993 a number of cases of unexplained adult respiratory syndrome occurred in the southwestern United States. The illness was characterized by a prodrome of fever, myalgia, and other symptoms followed by the rapid onset of a capillary leak syndrome with hemoconcentration, thrombocytopenia, and pulmonary edema. Viral RNA sequences in the lungs identified a new member of the hantavirus genus, Sin Nombre virus (SNV), unique to North America. Pulmonary endothelial cells were heavily infected but were not necrotic. We speculated that this capillary leak syndrome was initiated by immune responses to the SNV-infected pulmonary endothelial cells. We isolated a CD8+cytotoxic T lymphocyte (CTL) clone directly from the blood of a patient with the acute hantavirus pulmonary syndrome (HPS) which recognizes a SNV specific epitope on the virus nucleocapsid protein (aa 234–242) that is restricted by HLA C7 and produces IFNγ but not IL-4. We identified a second CD8+CTL epitope located within another site aa 131–139 on the nucleocapsid protein, which is HLA B35 restricted, and a CD4+CTL epitope located on a third site on nucleocapsid protein aa 372–380 using lymphocytes obtained during HPS from another patient that were stimulatedin vitro.Hantavirus specific CD8+and CD4+CTL may contribute to the immunopathology and capillary leak syndrome observed in the HPS

Crimean Congo hemorrhagic fever virus nucleoprotein and GP38 subunit vaccine combination prevents morbidity in mice.

Immunizing mice with Crimean-Congo hemorrhagic fever virus (CCHFV) nucleoprotein (NP), glycoprotein precursor (GPC), or with the GP38 domain of GPC, can be protective when the proteins are delivered with viral vectors or as a DNA or RNA vaccine. Subunit vaccines are a safe and cost-effective alternative to some vaccine platforms, but Gc and Gn glycoprotein subunit vaccines for CCHFV fail to protect despite eliciting high levels of neutralizing antibodies. Here, we investigated humoral and cellular immune responses and the protective efficacy of recombinant NP, GP38, and GP38 forms (GP85 and GP160) associated with the highly glycosylated mucin-like (MLD) domain, as well as the NP + GP38 combination. Vaccination with GP160, GP85, or GP38 did not confer protection, and vaccination with the MLD-associated GP38 forms blunted the humoral immune responses to GP38, worsened clinical chemistry, and increased viral RNA in the blood compared to the GP38 vaccination. In contrast, NP vaccination conferred 100% protection from lethal outcome and was associated with mild clinical disease, while the NP + GP38 combination conferred even more robust protection by reducing morbidity compared to mice receiving NP alone. Thus, recombinant CCHFV NP alone is a promising vaccine candidate conferring 100% survival against heterologous challenge. Moreover, incorporation of GP38 should be considered as it further enhances subunit vaccine efficacy by reducing morbidity in surviving animals

Sustained replication of synthetic canine distemper virus defective genomes in vitro and in vivo

Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitr

Deep Sequencing of RNA from Blood and Oral Swab Samples Reveals the Presence of Nucleic Acid from a Number of Pathogens in Patients with Acute Ebola Virus Disease and Is Consistent with Bacterial Translocation across the Gut.

In this study, samples from the 2013-2016 West African Ebola virus outbreak from patients in Guinea with Ebola virus disease (EVD) were analyzed to discover and classify what other pathogens were present. Throat swabs were taken from deceased EVD patients, and peripheral blood samples were analyzed that had been taken from patients when they presented at the treatment center with acute illness. High-throughput RNA sequencing (RNA-seq) and bioinformatics were used to identify the potential microorganisms. This approach confirmed Ebola virus (EBOV) in all samples from patients diagnosed as acute positive for the virus by quantitative reverse transcription-PCR in deployed field laboratories. Nucleic acid mapping to Plasmodium was also used on the patient samples, confirming results obtained with an antigen-based rapid diagnostic test (RDT) conducted in the field laboratories. The data suggested that a high Plasmodium load, as determined by sequence read depth, was associated with mortality and influenced the host response, whereas a lower parasite load did not appear to affect outcome. The identifications of selected bacteria from throat swabs via RNA-seq were confirmed by culture. The data indicated that the potential pathogens identified in the blood samples were associated with translocation from the gut, suggesting the presence of bacteremia, which transcriptome data suggested may induce or aggravate the acute-phase response observed during EVD. Transcripts mapping to different viruses were also identified, including those indicative of lytic infections. The development of high-resolution analysis of samples from patients with EVD will help inform care pathways and the most appropriate general antimicrobial therapy to be used in a resource-poor setting. IMPORTANCE Our results highlight the identification of an array of pathogens in the blood of patients with Ebola virus disease (EVD). This has not been done before, and the data have important implications for the treatment of patients with EVD, particularly considering antibiotic stewardship. We show that EVD patients who were also infected with Plasmodium, particularly at higher loads, had more adverse outcomes than patients with lower levels of Plasmodium. However, the presence of Plasmodium did not influence the innate immune response, and it is likely that the presence of EBOV dominated this response. Several viruses other than EBOV were identified, and bacteria associated with sepsis were also identified. These findings were indicative of bacterial translocation across the gut during the acute phase of EVD

Host-Species Transferrin Receptor 1 Orthologs Are Cellular Receptors for Nonpathogenic New World Clade B Arenaviruses

The ability of a New World (NW) clade B arenavirus to enter cells using human transferrin receptor 1 (TfR1) strictly correlates with its ability to cause hemorrhagic fever. Amapari (AMAV) and Tacaribe (TCRV), two nonpathogenic NW clade B arenaviruses that do not use human TfR1, are closely related to the NW arenaviruses that cause hemorrhagic fevers. Here we show that pseudotyped viruses bearing the surface glycoprotein (GP) of AMAV or TCRV can infect cells using the TfR1 orthologs of several mammalian species, including those of their respective natural hosts, the small rodent Neacomys spinosus and the fruit bat Artibeus jamaicensis. Mutation of one residue in human TfR1 makes it a functional receptor for TCRV, and mutation of four residues makes it a functional receptor for AMAV. Our data support an in vivo role for TfR1 in the replication of most, if not all, NW clade B arenaviruses, and suggest that with modest changes in their GPs the nonpathogenic arenaviruses could use human TfR1 and emerge as human pathogens

Recombinant Lloviu virus as a tool to study viral replication and host responses

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness

Παιδικός Μουσικός Αυτοσχεδιασμός στη προσχολική ηλικία: τραγούδι, ρυθμός και οι επιρροές του περιβάλλοντος

Η εργασία αναφέρεται στον τομέα της μουσικής εκπαίδευσης και ερευνά τον μουσικό αυτοσχεδιασμό σε παιδιά προσχολικής ηλικίας 3-5 ετών. Στο 1ο κεφάλαιο αναλύουμε τη μουσική ανάπτυξη των παιδιών , τα στάδια εξέλιξης της φωνής, το παιδικό και ομαδικό τραγούδι και την αντίληψη της κίνησης και του ρυθμού σε αυτές τις ηλικίες. Έπειτα, στο 2ο κεφάλαιο, αναφέρουμε την έννοια του αυτοσχεδιασμού, τα επίπεδά του, καθώς και τη δημιουργικότητα που συνδέεται άμεσα μαζί του. Μαζί με τα παραπάνω, εξετάζουμε τον ρόλο του δασκάλου στην μουσική εκπαίδευση και της οικογένειας στην καθημερινότητα των παιδιών καθώς και τα ψηφιακά παιχνίδια που ενισχύουν το εκπαιδευτικό και οικογενειακό περιβάλλον. Στο 3ο κεφάλαιο πραγματοποιείται η έρευνα του μουσικού αυτοσχεδιασμού μέσα από ρυθμικές και τραγουδιστικές δραστηριότητες σε παιδιά νηπιακού σταθμού, βλέποντας την εξέλιξη τους σε διάστημα 10 ημερών, συνέντευξη με τη δασκάλα τους για το μουσικό της υπόβαθρο και το πώς λειτουργεί στο μάθημα, καθώς και ένα ερωτηματολόγιο που προωθήθηκε στους γονείς που αφορά τη μουσική τους καθημερινότητα μαζί με το παιδί.The work refers to the field of music education and investigates musical improvisation in preschool children 3-5 years old. In the first chapter we analyze the musical development of children, the stages of voice development, children's and group singing and the perception of movement and rhythm at these ages. Then, in the 2nd chapter, we mention the concept of improvisation, its levels, as well as the creativity that is directly related to it. Along with the above, we examine the role of the teacher in music education and the family in the daily lives of children as well as digital games that enhance the educational and family environment. Chapter 3 conducts a research on children’s musical improvisation through rhythmic and singing activities in Kindergarten, observing their development over a ten day period, interviewing their teacher about her musical background and how this affects the lessons, as well as a questionnaire which was completed by the parents in regards to their musical daily life together with the child

Η διασύνδεση της Περιβαλλοντικής Υγείας με την αειφορία και τη σύγχρονη βιοοικονομία. Ποιος ο ρόλος της βιοοικονομίας στη μείωση των περιβαλλοντικών επιπτώσεων στην ανθρώπινη υγεία και την προστασία του κλίματος

Στο δεύτερο μισό του 20ου αιώνα η δημόσια υγεία υπέστη σημαντική επιβάρυνση, η οποία σχετίζεται με τη ρύπανση και μόλυνση του περιβάλλοντος, την κλιματική αλλαγή και την ανεξέλεγκτη σπατάλη μη ανανεώσιμων φυσικών πόρων. Τα παραπάνω σε συνδυασμό με άλλα προβλήματα, όπως ενδεικτικά νέες ασθένειες οι οποίες ταλαιπωρούν τον πλανήτη, συνέβαλαν στην αύξηση συγκεκριμένων προβλημάτων υγείας, τόσο στις χώρες του δυτικού κόσμου, όσο και στις αναπτυσσόμενες χώρες. Από τη δεκαετία του ‘90 παρατηρείται παγκοσμίως μία στροφή στη ρύθμιση των περιβαλλοντικών επιπτώσεων οι οποίες προκαλούνται από τις επιχειρηματικές και άλλες ανθρώπινες δραστηριότητες, διαμορφώνεται η απαίτηση της κοινής γνώμης για μείωση των εκπομπών των αερίων του θερμοκηπίου και αποκατάσταση του κλίματος και πολλοί αναπτυξιακοί τομείς, κυρίως οι κατασκευές, οι μεταφορές και η ενέργεια, προσανατολίζονται πλέον στην εκμετάλλευση των ανανεώσιμων πηγών. Τα παραπάνω έχουν καθοριστεί μέσα από διεθνείς συμβάσεις με πρωτοβουλία του Οργανισμού Ηνωμένων Εθνών (ΟΗΕ), αλλά και της Ευρωπαϊκής Ένωσης. Η βιώσιμη ανάπτυξη η οποία αφορά την επίτευξη μιας αρμονικής ισορροπίας ανάμεσα στην οικονομία, την κοινωνία και το περιβάλλον, αποτελεί το σημαντικό στοίχημα του πλανήτη για τον 21ο αιώνα και ήδη παρατηρούνται σημαντικά βήματα αλλαγής προς επίτευξη της βιωσιμότητας. Εκτός από τους παραπάνω τρεις πυλώνες της βιώσιμης ανάπτυξης, ο ΟΗΕ έχει καθορίσει ρητά και 17 συγκεκριμένους στόχους οι οποίοι αφορούν τη γενικότερη αειφορία των ανθρώπων, της οικονομίας αλλά και της γης. Σε αυτό το πλαίσιο η Ευρωπαϊκή Ένωση έχει διαμορφώσει μία σειρά συγκεκριμένων πολιτικών προστασίας του περιβάλλοντος, που ρυθμίζουν τις δυνητικά επιβαρυντικές για αυτό ανθρωπογενείς δραστηριότητες και δεσμεύουν αυστηρά τα συμβαλλόμενα κράτη μέλη. Η προστασία του περιβάλλοντος και η βελτίωση των χαρακτηριστικών του, όπως ο ατμοσφαιρικός αέρας, ο υδροφόρος ορίζοντας, τα θαλάσσια οικοσυστήματα, το μικροκλίμα, κλπ., μέσα από συγκεκριμένες πολιτικές και λύσεις, συνδέεται άμεσα με την παγκόσμια βιωσιμότητα. Η δημόσια και παγκόσμια υγεία, η αειφορία των πόλεων, οι επιπτώσεις των υποδομών, η περιβαλλοντική ανάπτυξη και η βελτίωση της ποιότητας ζωής στο ανθρωπογενές αλλά και το φυσικό περιβάλλον καθορίζεται και είναι πλέον μετρήσιμη με συγκεκριμένες προδιαγραφές που αποτελούν τους περιβαλλοντικούς δείκτες. Η έννοια της βιοοικονομίας, από την άλλη πλευρά, είναι η δημιουργία οικονομίας η οποία στηρίζεται στην αξιοποίηση των ανανεώσιμων φυσικών πόρων, επομένως είναι άρρηκτα συνδεδεμένη με την αειφόρο ανάπτυξη το βασικό πλαίσιο της οποίας συνδέεται με την υποχρέωση των ανθρώπων να κληροδοτήσουν στις επόμενες γενιές έναν καλύτερο κόσμο από τον σημερινό ο οποίος να μπορεί με ασφάλεια να καλύπτει τις μελλοντικές ανάγκες του πλανήτη. Το αντικείμενο της εργασίας είναι να διερευνήσει τη διασύνδεση ανάμεσα στην περιβαλλοντική υγεία, την αειφορία και τη σύγχρονη βιοοικονομία. Ειδικότερα, καταρχάς θα μελετηθεί το πρόβλημα της περιβαλλοντικής υποβάθμισης και οι συνέπειες του, όπως και οι προκρινόμενες λύσεις με κύρια την αειφόρο οικονομία. Ακολούθως, θα δοθεί βαρύτητα στις παραμέτρους της περιβαλλοντικής υγείας και τον τρόπο που η κυκλική οικονομία μπορεί να συμβάλλει σε μία αειφόρο περιβαλλοντική και αστική ανάπτυξη, αλλά και στη μετρίαση της κλιματικής αλλαγής.In the second half of the 20th century, public health suffered a significant burden, because of the pollution and damage of the environment, the climate change and the uncontrolled waste of non-renewable natural resources. The above in combination with other problems, such as indicative new diseases that afflict the planet, have contributed to the increase of specific health problems, both in the western world and in developing countries. Since the 1990s, there has been a worldwide shift in the regulation of environmental impacts caused by business and other human activities, there has been a public outcry to reduce greenhouse gas emissions and climate change, and many development sectors, in particular construction, transport and energy are now oriented towards the exploitation of renewable sources. The above have been determined through international conventions initiated by the UN, but also the European Union. Sustainable development, which is about achieving a harmonious balance between the economy, society and the environment, is the important bet of the planet for the 21st century and significant steps of change are already taken to achieve sustainability. In addition to the above three pillars of sustainable development, the UN has explicitly set 17 specific goals which concern the general sustainability of people, the economy and the earth. In this context, the European Union has developed a number of specific environmental protection policies, which regulate potentially aggravating anthropogenic activities and are strictly committed to the Contracting Member States. The protection of the environment and the improvement of its characteristics, such as atmospheric air, aquifer, marine ecosystems, microclimate, etc., through specific policies and solutions directly linked to global sustainability. Public and global health, the sustainability of cities, the impact of infrastructure, environmental development and the improvement of the quality of life in both manufactured and natural environments are defined and are now measurable with specific specifications that constitute environmental indicators. The concept of bioeconomy, on the other hand, is the creation of an economy based on the use of renewable natural resources. Therefore, it is inextricably connected to sustainable development, the basic framework of which has to do with the obligation of people to inherit in next generations a better world than today that can safely meet the future needs of the planet. The object of the work is to explore the link between environmental health, sustainability and the modern bio-economy. In particular, the problem of environmental degradation and its consequences is studied firstly, as well as the preferred solutions, mainly the sustainable economy. Next, we put emphasis on environmental health parameters and on how the circular economy can contribute to sustainable environmental and urban development, as well as on mitigating climate change

Andes Virus Disrupts the Endothelial Cell Barrier by Induction of Vascular Endothelial Growth Factor and Downregulation of VE-Cadherin▿ †

Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) are severe diseases associated with hantavirus infection. High levels of virus replication occur in microvascular endothelial cells but without a virus-induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of these diseases. VE-cadherin is a major component of adherens junctions, and its interaction with the vascular endothelial growth factor (VEGF) receptor, VEGF-R2, is important for maintaining the integrity of the endothelial barrier. Here we report that increased secreted VEGF and concomitant decreased VE-cadherin are seen at early times postinfection of human primary lung endothelial cells with an HPS-associated hantavirus, Andes virus. Furthermore, active virus replication results in increased permeability and loss of the integrity of the endothelial cell barrier. VEGF binding to VEGF-R2 is known to result in dissociation of VEGF-R2 from VE-cadherin and in VE-cadherin activation, internalization, and degradation. Consistent with this, we showed that an antibody which blocks VEGF-R2 activation resulted in inhibition of the Andes virus-induced VE-cadherin reduction. These data implicate virus induction of VEGF and reduction in VE-cadherin in the endothelial cell permeability seen in HPS and suggest potential immunotherapeutic targets for the treatment of the disease

Endocytic Pathways Used by Andes Virus to Enter Primary Human Lung Endothelial Cells.

Andes virus (ANDV) is the major cause of hantavirus pulmonary syndrome (HPS) in South America. Despite a high fatality rate (up to 40%), no vaccines or antiviral therapies are approved to treat ANDV infection. To understand the role of endocytic pathways in ANDV infection, we used 3 complementary approaches to identify cellular factors required for ANDV entry into human lung microvascular endothelial cells. We screened an siRNA library targeting 140 genes involved in membrane trafficking, and identified 55 genes required for ANDV infection. These genes control the major endocytic pathways, endosomal transport, cell signaling, and cytoskeleton rearrangement. We then used infectious ANDV and retroviral pseudovirions to further characterize the possible involvement of 9 of these genes in the early steps of ANDV entry. In addition, we used markers of cellular endocytosis along with chemical inhibitors of known endocytic pathways to show that ANDV uses multiple routes of entry to infect target cells. These entry mechanisms are mainly clathrin-, dynamin-, and cholesterol-dependent, but can also occur via a clathrin-independent manner