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DC-SIGN is a C-type lectin expressed in myeloid cells such as immature dendritic cells and macrophages. Through glycan recognition in viral envelope glycoproteins, DC-SIGN has been shown to act as a receptor for a number of viral agents such as HIV, Ebola virus, SARS-CoV, and SARS-CoV-2. Using a system of Vesicular Stomatitis Virus pseudotyped with MERS-CoV spike protein, here, we show that DC-SIGN is partially responsible for MERS-CoV infection of dendritic cells and that DC-SIGN efficiently mediates trans-infection of MERS-CoV from dendritic cells to susceptible cells, indicating a potential role of DC-SIGN in MERS-CoV dissemination and pathogenesis. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed, viral, tickborne disease. In Europe, cases have been reported only in the southeastern part of the continent. We report two autochthonous cases in Spain. The index patient acquired the disease through a tick bite in the province of Ávila - 300 km away from the province of Cáceres, where viral RNA from ticks was amplified in 2010. The second patient was a nurse who became infected while caring for the index patient. Both were infected with the African 3 lineage of this virus. (Funded by Red de Investigación Cooperativa en Enfermedades Tropicales [RICET] and Efficient Response to Highly Dangerous and Emerging Pathogens at EU [European Union] Level [EMERGE].). [ABSTRACT FROM AUTHOR]

Nucleotide incorporation and lacZ-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg 2+ ) using an in vitro-transcribed RNA were around 7.3 × 10 -5 . In agreement with previous reports, an 8-fold increase in RT's accuracy was observed after reducing Mg 2+ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10 -5 ). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg 2+ (7.4 × 10 -5 ) were similar to those determined with the in vitro-transcribed RNA, and were reduced to 1.8 × 10 -5 in the presence of 0.5 mM Mg 2+ . Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The endemic nature of the Ebola virus disease in Africa underscores the need for prophylactic and therapeutic drugs that are affordable and easy to administer. Through a phenotypic screening employing viral pseudotypes and our in-house chemical library, we identified a promising hit featuring a thiophene scaffold, exhibiting antiviral activity in the micromolar range. Following up on this thiophene hit, a new series of compounds that retain the five-membered heterocyclic scaffold while modifying several substituents was synthesized. Initial screening using a pseudotype viral system and validation assays employing authentic Ebola virus demonstrated the potential of this new chemical class as viral entry inhibitors. Subsequent investigations elucidated the mechanism of action through site-directed mutagenesis. Furthermore, we conducted studies to assess the pharmacokinetic profile of selected compounds to confirm its pharmacological and therapeutic potential.

DC-SIGN (CD209) and L-SIGN (CD209L) are two C-type lectin receptors (CLRs) that facilitate SARS-CoV-2 infections as viral co-receptors. SARS-CoV-2 manipulates both DC-SIGN and L-SIGN for enhanced infection, leading to interest in developing receptor antagonists. Despite their structural similarity (82% sequence identity), they function differently. DC-SIGN, found in dendritic cells, shapes the immune response by recognizing pathogen-associated carbohydrate patterns. In contrast, L-SIGN, expressed in airway epithelial endothelial cells, is not directly involved in immunity. COVID-19's primary threat is the hyperactivation of the immune system, potentially reinforced if DC-SIGN engages with exogenous ligands. Therefore, L-SIGN, co-localized with ACE2-expressing cells in the respiratory tract, is a more suitable target for anti-adhesion therapy. However, designing a selective ligand for L-SIGN is challenging due to the high sequence identity of the Carbohydrate Recognition Domains (CRDs) of the two lectins. We here present Man84, a mannose ring modified with a methylene guanidine triazole at position 2. It binds L-SIGN with a K D of 12.7μM ± 1 μM (ITC) and is the first known L-SIGN selective ligand, showing 50-fold selectivity over DC-SIGN (SPR). The X-ray structure of the L-SIGN CRD/Man84 complex reveals the guanidinium group's role in achieving steric and electrostatic complementarity with L-SIGN. This allows us to trace the source of selectivity to a single amino acid difference between the two CRDs. NMR analysis confirms the binding mode in solution, highlighting Man84's conformational selection upon complex formation. Dimeric versions of Man84 achieve additional selectivity and avidity in the low nanomolar range. These compounds selectively inhibit L-SIGN dependent trans-infection by SARS-CoV-2 and Ebola virus. Man84 and its dimeric constructs display the best affinity and avidity reported to date for low-valency glycomimetics targeting CLRs. They are promising tools for competing with SARS-CoV-2 anchoring in the respiratory tract and have potential for other medical applications., Competing Interests: The authors declare the following competing financial interest(s): S. P., C. D., M. T., F. F., and A. B. declare the filing of a patent covering the use of glycomimetic L-SIGN ligands as antagonist, anti-viral adhesion, and for targeting human L-SIGN-expressing cells., (This journal is © The Royal Society of Chemistry.)

Since WHO has declared the COVID-19 outbreak a global pandemic, nearly seven million deaths have been reported. This efficient spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is facilitated by the ability of the spike glycoprotein to bind multiple cell membrane receptors. Although ACE2 is identified as the main receptor for SARS-CoV-2, other receptors could play a role in viral entry. Among others, C-type lectins such as DC-SIGN are identified as efficient trans-receptor for SARS-CoV-2 infection, so the use of glycomimetics to inhibit the infection through the DC-SIGN blockade is an encouraging approach. In this regard, multivalent nanostructures based on glycosylated [60]fullerenes linked to a central porphyrin scaffold have been designed and tested against DC-SIGN-mediated SARS-CoV-2 infection. First results show an outstanding inhibition of the trans-infection up to 90%. In addition, a deeper understanding of nanostructure-receptor binding is achieved through microscopy techniques, high-resolution NMR experiments, Quartz Crystal Microbalance experiments, and molecular dynamic simulations., (© 2023 The Authors. Small published by Wiley‐VCH GmbH.)

Monocyte-derived macrophages, the major source of pathogenic macrophages in COVID-19, are oppositely instructed by macrophage CSF (M-CSF) or granulocyte macrophage CSF (GM-CSF), which promote the generation of antiinflammatory/immunosuppressive MAFB+ (M-MØ) or proinflammatory macrophages (GM-MØ), respectively. The transcriptional profile of prevailing macrophage subsets in severe COVID-19 led us to hypothesize that MAFB shapes the transcriptome of pulmonary macrophages driving severe COVID-19 pathogenesis. We have now assessed the role of MAFB in the response of monocyte-derived macrophages to SARS-CoV-2 through genetic and pharmacological approaches, and we demonstrate that MAFB regulated the expression of the genes that define pulmonary pathogenic macrophages in severe COVID-19. Indeed, SARS-CoV-2 potentiated the expression of MAFB and MAFB-regulated genes in M-MØ and GM-MØ, where MAFB upregulated the expression of profibrotic and neutrophil-attracting factors. Thus, MAFB determines the transcriptome and functions of the monocyte-derived macrophage subsets that underlie pulmonary pathogenesis in severe COVID-19 and controls the expression of potentially useful biomarkers for COVID-19 severity.

Currently, the majority of the population has been vaccinated against COVID-19 and/or has experienced SARS-CoV-2 infection either before or after vaccination. The immunological response to repeated episodes of infections is not completely clear. We measured SARS-CoV-2 specific neutralization titers by a pseudovirus assay after BA.1 infection and RBD-specific immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) in a cohort of COVID-19 uninfected and triple vaccinated individuals (breakthrough infection group, BTI) as compared with those previously infected by SARS-CoV-2 (reinfection group, REI) who underwent identical vaccination schedule. SARS-CoV-2 specific neutralizing response after BA.1 infection was significantly higher in the BTI group as compared with the REI. Furthermore, neutralization titers in REI were not significant different from convalescent non reinfected controls. RBD-specific IgG and IgA, but not IgM, were also significantly higher in BTI as compared with REI. Our results show that the first episode of SARS-CoV-2 infection induces a significant increase in neutralizing titers in triple vaccinated individuals and that previous SARS-CoV-2 infection compromise significantly the neutralization response induced by reinfection, even by divergent SARS-CoV-2 variants and at least up to 2 years postinfection, suggesting a fundamental limitation in inducing effective booster through the intranasal route in previously infected individuals., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Ebola virus (EBOV) is a single-strand RNA virus belonging to the Filoviridae family, which has been associated to most Ebola virus disease outbreaks to date, including the West African and the North Kivu epidemics between 2013 and 2022. This unprecedented health emergency prompted the search for effective medical countermeasures. Following up on the carbazole hit identified in our previous studies, we synthetized a new series of compounds, which demonstrated to prevent EBOV infection in cells by acting as virus entry inhibitors. The in vitro inhibitory activity was evaluated through the screening against surrogate models based on viral pseudotypes and further confirmed using replicative EBOV. Docking and molecular dynamics simulations joined to saturation transfer difference-nuclear magnetic resonance (STD-NMR) and mutagenesis experiments to elucidate the biological target of the most potent compounds. Finally, in vitro metabolic stability and in vivo pharmacokinetic studies were performed to confirm their therapeutic potential.

We have measured the humoral response to messenger RNA (mRNA) vaccines in COVID-19 naïve and convalescent individuals. Third doses of mRNA COVID-19 vaccines induced a significant increase in potency and breadth of neutralization against SARS-CoV-2 variants of concern (VoC) including Omicron subvariants BA.1, BA.2, and BA.2.12.1, that were cross-neutralized at comparable levels and less for BA.4/5. This booster effect was especially important in naïve individuals that only after the third dose achieved a level that was comparable with that of vaccinated COVID-19 convalescents except for BA.4/5. Avidity of RBD-binding antibodies was also significantly increased in naïve individuals after the third dose, indicating an association between affinity maturation and cross neutralization of VoC. These results suggest that at least three antigenic stimuli by infection or vaccination with ancestral SARS-CoV-2 sequences are required to induce high avidity cross-neutralizing antibodies. Nevertheless, the circulation of new subvariants such as BA.4/5 with partial resistance to neutralization will have to be closely monitored and eventually consider for future vaccine developments., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Toll-like receptor 7 (TLR7) is an endosomal pathogen-associated molecular pattern (PAMP) receptor that senses single-stranded RNA (ssRNA) and whose engagement results in the production of type I IFN and pro-inflammatory cytokines upon viral exposure. Recent genetic studies have established that a dysfunctional TLR7-initiated signaling is directly linked to the development of inflammatory responses. We present evidence that TLR7 is preferentially expressed by monocyte-derived macrophages generated in the presence of M-CSF (M-MØ). We now show that TLR7 activation in M-MØ triggers a weak MAPK, NFκB, and STAT1 activation and results in low production of type I IFN. Of note, TLR7 engagement reprograms MAFB+ M-MØ towards a pro-inflammatory transcriptional profile characterized by the expression of neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8), whose expression is dependent on the transcription factors MAFB and AhR. Moreover, TLR7-activated M-MØ display enhanced pro-inflammatory responses and a stronger production of neutrophil-attracting chemokines upon secondary stimulation. As aberrant TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with impaired resolution of virus-induced inflammatory responses, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Several anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs) have received emergency authorization for coronavirus disease 2019 (COVID-19) treatment. However, most of these mAbs are not active against the highly mutated Omicron SARS-CoV-2 subvariants. We have tested a polyclonal approach of equine anti-SARS-CoV-2 F(ab')2 antibodies that achieved a high level of neutralizing potency against all SARS-CoV-2 variants of concern tested including Omicron BA.1, BA.2, BA.2.12 and BA.4/5. A repertoire of antibodies targeting conserved epitopes in different regions of the spike protein could plausibly account for this remarkable breadth of neutralization. These results warrant the clinical investigation of equine polyclonal F(ab')2 antibodies as a novel therapeutic strategy against COVID-19., Competing Interests: Potential conflicts of interest. P. R., L. N., and C. H. H.) are employees of Fab’entech, the producer of the equine antibodies. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

We have investigated the evolution of the neutralizing response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants at 8 months after Pfizer-BNT162b2 vaccination in coronavirus disease 2019 (COVID-19)-naive (n = 21) and COVID-19-convalescent (n = 21) individuals. Neutralizing levels declined for all variants (range 2- to 3.7-fold). Eight months after vaccination, a significant proportion (4/21) of naive individuals lacked detectable neutralizing activity against the highly transmissible SARS-CoV-2 delta variant. In the convalescent group, the impressive high initial humoral response resulted in detectable neutralizing antibody levels against all variants throughout this period., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

The synthesis of new biocompatible antiviral materials to fight against the development of multidrug resistance is being widely explored. Due to their unique globular structure and excellent properties, [60]fullerene-based antivirals are very promising bioconjugates. In this work, fullerene derivatives with different topologies and number of glycofullerene units were synthesized by using a SPAAC copper free strategy. This procedure allowed the synthesis of compounds 1 - 3 , containing from 20 to 40 mannose units, in a very efficient manner and in short reaction times under MW irradiation. The glycoderivatives were studied in an infection assay by a pseudotyped viral particle with Ebola virus GP1. The results obtained show that these glycofullerene oligomers are efficient inhibitors of EBOV infection with IC 50 s in the nanomolar range. In particular, compound 3 , with four glycofullerene moieties, presents an outstanding relative inhibitory potency (RIP). We propose that this high RIP value stems from the appropriate topological features that efficiently interact with DC-SIGN.

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection., Competing Interests: The authors have declared that no competing interests exist.

Background: The objective of this study was to investigate the neutralizing response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoC) during coronavirus disease 2019 (COVID-19) convalescence and after vaccination., Methods: COVID-19-convalescent and -naïve individuals were tested for neutralizing activity against SARS-CoV-2 VoC Alpha, Beta, Gamma, and Delta at 1 and 7 months postinfection and 4-6 weeks after BNT162b2 vaccination., Results: Vaccination induced a high neutralizing response in naïve individuals. Interestingly, vaccination of convalescent patients induced a boosted response that was able to neutralize all VoC at high titers., Conclusions: Vaccination with BNT162b2 induced high levels of neutralization against SARS-CoV-2 VoC in most patients; this is especially beneficial in COVID-19-convalescent individuals., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Objectives: This study compares the infectivity of SARS-CoV-2 in respiratory samples from patients with mild COVID-19 with those from hospitalized patients with severe bilateral pneumonia. In severe COVID-19, we also analysed the presence of neutralizing activity in paired sera., Methods: We performed cell cultures on 193 real-time reverse transcription polymerase chain reaction respiratory samples, positive for SARS-CoV-2, obtained from 189 patients at various times, from clinical diagnosis to follow-up. Eleven samples were obtained from asymptomatic individuals, 91 samples from 91 outpatients with mild forms of COVID-19 and 91 samples from 87 inpatients with severe pneumonia. In these patients, neutralizing activity was analysed in 30 paired sera collected after symptom onset >10 days., Results: We detected a cytopathic effect (CPE) in 91/193 (47%) samples. Viral viability was maintained for up to 10 days in patients with mild COVID-19. In patients with severe COVID-19, the virus remained viable for up to 32 days after the onset of symptoms. Patients with severe COVID-19 presented infectious virus at a significantly higher rate in the samples with moderate to low viral load (cycle threshold value ≥ 26): 32/75 (43%) versus 14/63 (22%) for mild cases (p < 0.01). We observed a positive CPE despite the presence of clear neutralizing activity (NT50 > 1:1024 in 10% (3/30) of samples., Discussion: Patients with severe COVID-19 might shed viable virus during prolonged periods of up to 4 weeks after symptom onset, even when presenting high cycle threshold values in their respiratory samples and despite having developed high neutralizing antibody titres., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection., Competing Interests: The authors have declared that no competing interests exist.

Despite the efforts to develop new treatments against Ebola virus (EBOV) there is currently no antiviral drug licensed to treat patients with Ebola virus disease (EVD). Therefore, there is still an urgent need to find new drugs to fight against EBOV. In order to do this, a virtual screening was done on the druggable interaction between the EBOV glycoprotein (GP) and the host receptor NPC1 with a subsequent selection of compounds for further validation. This screening led to the identification of new small organic molecules with potent inhibitory action against EBOV infection using lentiviral EBOV-GP-pseudotype viruses. Moreover, some of these compounds have shown their ability to interfere with the intracellular cholesterol transport receptor NPC1 using an ELISA-based assay. These preliminary results pave the way to hit to lead optimization programs that lead to successful candidates., (Copyright © 2021 Elsevier B.V. All rights reserved.)

After the last epidemic of the Zika virus (ZIKV) in Brazil that peaked in 2016, growing evidence has been demonstrated of the link between this teratogenic flavivirus and microcephaly cases. However, no vaccine or antiviral drug has been approved yet. ZIKV and Dengue viruses (DENV) entry to the host cell takes place through several receptors, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), so that the blockade of this receptor through multivalent glycoconjugates supposes a promising biological target to inhibit the infection process. In order to get enhanced multivalency in biocompatible systems, tridecafullerenes appended with up to 360 1,2-mannobiosides have been synthesized using a strain-promoted cycloaddition of azides to alkynes (SPAAC) strategy. These systems have been tested against ZIKV and DENV infection, showing an outstanding activity in the picomolar range.

During 2011-2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome.

Background: In Ebola virus (EBOV) infection, the specific neutralizing activity of convalescent plasma against other members of the Ebolavirus genus has not been extensively analyzed., Methods: We measured the neutralizing activity in plasma from 3 survivors of the recent outbreak due to the Makona variant of EBOV and tested its neutralizing potency against other variants of EBOV (ie, Mayinga and Kikwit) and against Sudan virus (SUDV), Bundibugyo virus (BDBV), and Reston virus (RESTV), using a glycoprotein (GP)-pseudotyped lentiviral system both with full-length GP and in vitro-cleaved GP (GPCL)., Results: Convalescent plasma specimens from survivors of EBOV infection showed low neutralizing activity against full-length GPs of SUDV, BDBV, RESTV, and EBOV variants Mayinga and Kikwit. However, broad and potent neutralizing activity was observed against the GPCL forms of SUDV, BDBV, and RESTV., Discussion: Removal of the mucin-like domain and glycan cap from the GP of members of the Ebolavirus genus presumably exposes conserved epitopes in or in the vicinity of the receptor binding site and internal fusion loop that are readily amenable to neutralization. These types of broad neutralizing antibodies could be induced by using immunogens mimicking GPCL.

SWCNTs, MWCNTs, and SWCNHs have been employed as virus-mimicking nanocarbon platforms for the multivalent presentation of carbohydrates in an artificial Ebola virus infection model assay. These carbon nanoforms have been chemically modified by the covalent attachment of glycodendrons and glycofullerenes using the CuAAC "click chemistry" approach. This modification dramatically increases the water solubility of these structurally different nanocarbons. Their efficiency in blocking DC-SIGN-mediated viral infection by an artificial Ebola virus has been tested in a cellular experimental assay, finding that glycoconjugates based on MWCNTs functionalized with glycofullerenes are potent inhibitors of viral infection.

A series of amphiphilic glycodendro[60]fullerene monoadducts were efficiently synthesized using the CuAAC "click chemistry" approach. These glycodendrofullerenes can self-assemble in aqueous media, in a process favoured through π-π interactions between the [60]fullerene moieties. This aggregation process leads to big and well-defined compact micelles with a uniform size and spherical-shape. The supramolecular aggregates were characterized using electronic microscopy (SEM and TEM), light scattering methods (DLS) and X-ray methodologies (SAXS and XRD). The antiviral efficiency of these aggregates has been tested in an experimental infection assay using Ebola virus glycoprotein (EboGP) pseudotyped viral particles on Jurkat cells overexpressing DC-SIGN and an improvement in the IC 50 value with respect to other systems endowed with a higher number of carbohydrate ligands is observed.

Since the first documented autochthonous transmission of chikungunya virus in the Caribbean island of Saint Martin in 2013, the infection has been reported within the Caribbean region as well as North, Central and South America. The risk of autochthonous transmission of chikungunya virus becoming established in Spain may be elevated due to the large numbers of travellers returning to Spain from countries affected by the 2013 epidemic in the Caribbean and South America, as well as the existence of the Aedes albopictus vector in certain parts of Spain. We retrospectively analysed the laboratory diagnostic database of the National Centre for Microbiology, Institute of Health Carlos III (CNM-ISCIII) from 2008 to 2014. During the study period, 264 confirmed cases, of 1,371 suspected cases, were diagnosed at the CNM-ISCIII. In 2014 alone, there were 234 confirmed cases. The highest number of confirmed cases were reported from the Dominican Republic (n = 136), Venezuela (n = 30) and Haiti (n = 11). Six cases were viraemic in areas of Spain where the vector is present. This report highlights the need for integrated active case and vector surveillance in Spain and other parts of Europe where chikungunya virus may be introduced by returning travellers., Competing Interests: Conflicts of Interest: None declared, (This article is copyright of The Authors, 2016.)

Background: In the current epidemic of Ebola virus disease, health-care workers have been transferred to Europe and the USA for optimised supportive care and experimental treatments. We describe the clinical course of the first case of Ebola virus disease contracted outside of Africa, in Madrid, Spain., Methods: Herein we report clinical, laboratory, and virological findings of the treatment of a female nurse assistant aged 44 years who was infected with Ebola virus around Sept 25-26, 2014, while caring for a Spanish missionary with confirmed Ebola virus disease who had been medically evacuated from Sierra Leone to La Paz-Carlos III University Hospital, Madrid. We also describe the use of experimental treatments for Ebola virus disease in this patient., Findings: The patient was symptomatic for 1 week before first hospital admission on Oct 6, 2014. We used supportive treatment with intravenous fluids, broad-spectrum antibiotics, and experimental treatments with convalescent plasma from two survivors of Ebola virus disease and high-dose favipiravir. On day 10 of illness, she had acute respiratory distress syndrome, possibly caused by transfusion-related acute lung injury, which was managed without mechanical ventilation. Discharge was delayed because of the detection of viral RNA in several bodily fluids despite clearance of viraemia. The patient was discharged on day 34 of illness. At the time of discharge, the patient had possible subacute post-viral thyroiditis. None of the people who had contact with the patient before and after admission became infected with Ebola virus., Interpretation: This report emphasises the uncertainties about the efficacy of experimental treatments for Ebola virus disease. Clinicians should be aware of the possibility of transfusion-related acute lung injury when using convalescent plasma for the treatment of Ebola virus disease., Funding: La Paz-Carlos III University Hospital., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The presentation of short viral peptide antigens by human leukocyte antigen (HLA) class I molecules on cell surfaces is a key step in the activation of cytotoxic T lymphocytes, which mediate the killing of pathogen-infected cells or initiate autoimmune tissue damage. HLA-B27 is a well known class I molecule that is used to study both facets of the cellular immune response. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HLA-B*2705(+) cells, we identified 200 naturally processed HLA-B*2705 ligands. Our analyses revealed that a change in the position (P) 2 anchor motif was detected in the 3% of HLA-B*2705 ligands identified. B*2705 class I molecules were able to bind these six GlnP2 peptides, which showed significant homology to pathogenic bacterial sequences, with a broad range of affinities. One of these ligands was able to bind with distinct conformations to HLA-B27 subtypes differentially associated with ankylosing spondylitis. These conformational differences could be sufficient to initiate autoimmune damage in patients with ankylosing spondylitis-associated subtypes. Therefore, these kinds of peptides (short, with GlnP2, and similar low affinity to all HLA-B27 subtypes tested but with unlike conformations in differentially ankylosing spondylitis-associated subtypes) must not be excluded from future researches involving potential arthritogenic peptides.

The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P(1) position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease.

Individuals with nonfunctional transporters associated with antigen processing (TAP) complexes are not particularly susceptible to viral infections or neoplasms. Therefore, their immune system must be reasonably efficient, and the present, though reduced, cytolytic CD8 αβ T subpopulation specific for TAP-independent antigens may be sufficient to establish an immune defense protecting against viral infections in these individuals. The objective of the present study was to identify TAP-independent ligands from HIV gp160 protein. An analysis and comparison of complex human histocompatibility complex (HLA)-bound peptide pools isolated from large quantities of healthy or HIV gp160-expressing human cells was performed using mass spectrometry and bioinformatics tools. A conserved TAP-independent HLA peptide ligand endogenously processed and presented in infected human cells was identified. This ligand originates from the envelope protein bound to the HLA-Cw1 class I molecule with high affinity. It was concluded that HLA class I peptides derived from a large fraction of the N-terminal HIV envelope protein could be presented even in the absence of the TAP complex.

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.

Leishmaniasis is a parasitic disease that courses with cutaneous or visceral clinical manifestations. The amastigote stage of the parasite infects phagocytes and modulates the effector function of the host cells. Our group has described that the interaction between Leishmania and immature monocyte-derived dendritic cells (DCs) takes place through dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin that specifically recognizes fungal, viral and bacterial pathogens. The DC-SIGN-mediated recognition of Leishmania amastigotes does not induce DC maturation, and the DC-SIGN ligand/s on Leishmania parasites is/are still unknown. We have also found that the DC-SIGN-related molecule L-SIGN, specifically expressed in lymph node and liver sinusoidal endothelial cells, acts as a receptor for L. infantum, the parasite responsible for visceral leishmaniasis, but does not recognize L. pifanoi, which causes the cutaneous form of the disease. Therefore, DC-SIGN and L-SIGN differ in their ability to interact with Leishmania species responsible for either visceral or cutaneous leishmaniasis. A deeper knowledge of the parasite-C-type lectin interaction may be helpful for the design of new DC-based therapeutic vaccines against Leishmania infections.

Because previous investigations show that mistreatment during residency is frequent we undertook this research to assess the prevalence of psychological abuse, physical violence and sexual harassment in residency training programs and the professional status of perpetrators. Through a literature review and a previous qualitative study we developed a self-administered questionnaire. Thirteen events that could be experienced by residents and ten possible perpetrators were described in the questionnaire. Residents were asked to record whether they had experienced these events, at least once, and who was responsible. This survey was distribuided among the residents from three hospitals of Buenos Aires city. A total of 322 residents answered the questionnaire (55% women) 72% of whom worked in clinical programs, 22% in surgical programs, and 6% in diagnostic programs. Mistreatment was reported by 89% of the residents. On average each resident recorded 8 different episodes of mistreatment, such as being criticized for not completing administrative work (75%), being shouted at (64%), being humiliated in public (57%), sexual harassment (10%), being exposed to pornographic material without permission (7%), being hit or pushed (15%), suffering racial or religious discrimination (10%). The most common perpetrators were senior residents (26%), chief resident (19%), attending physicians (14%), and nurses (8%). Mistreatment and abuse are commonly experienced by residents in training programs. A multidisciplinary approach is needed to address this problem in the training environment.

Cyanovirin-N can exist in solution in monomeric and domain-swapped dimeric forms, with HIV-antiviral activity being reported for both. Here we present results for CV-N variants that form stable solution dimers: the obligate dimer [DeltaQ50]CV-N and the preferential dimer [S52P]CV-N. These variants exhibit comparable DeltaG values (10.6 +/- 0.5 and 9.4 +/- 0.5 kcal.mol(-1), respectively), similar to that of stabilized, monomeric [P51G]CV-N (9.8 +/- 0.5 kcal.mol(-1)), but significantly higher than wild-type CV-N (4.1 +/- 0.2 kcal.mol(-1)). During folding/unfolding, no stably folded monomer was observed under any condition for the obligate dimer [DeltaQ50]CV-N, whereas two monomeric, metastable species were detected for [S52P]CV-N at low concentrations. This is in contrast to our previous results for [P51G]CV-N and wild-type CV-N, for which the dimeric forms were found to be the metastable species. The dimeric mutants exhibit comparable antiviral activity against HIV and Ebola, similar to that of wild-type CV-N and the stabilized [P51G]CV-N variant., (Copyright 2004 Elsevier Ltd.)

Cyanovirin-N (CV-N) has been shown to inhibit Ebola Zaire virus (EboZV) infection, both in vitro and in vivo, through its ability to bind to oligomannoses-8/9 on the EboZV surface glycoprotein (GP). Here, we report the in vitro potency of CV-N to inhibit EboZV GP- and Marburg virus GP-pseudotyped viruses (EC50 approximately 40-60 nmol/L and approximately 6-25 nmol/L, respectively) from mediating gene transduction into HeLa cells. In addition, we provide evidence that CV-N can effectively inhibit DC-SIGN-mediated EboZV infection. Our data emphasize both the utility of GP-pseudotyped vectors in the assessment of compounds that affect cell entry by filovirus and the use of CV-N as a reagent for the probing of carbohydrate-dependent interactions at viral entry.

We have designed a glycodendritic structure, BH30sucMan, that blocks the interaction between dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and Ebola virus (EBOV) envelope. BH30sucMan inhibits DC-SIGN-mediated EBOV infection at nanomolar concentrations. BH30sucMan may counteract important steps of the infective process of EBOV and, potentially, of microorganisms shown to exploit DC-SIGN for cell entry and infection.

Ebola virus is a highly lethal pathogen responsible for several outbreaks of hemorrhagic fever. Here we show that the primate lentiviral binding C-type lectins DC-SIGN and L-SIGN act as cofactors for cellular entry by Ebola virus. Furthermore, DC-SIGN on the surface of dendritic cells is able to function as a trans receptor, binding Ebola virus-pseudotyped lentiviral particles and transmitting infection to susceptible cells. Our data underscore a role for DC-SIGN and L-SIGN in the infective process and pathogenicity of Ebola virus infection.