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1. Antagonism of BST2/Tetherin, a new restriction factor of respiratory syncytial virus, requires the viral NS1 protein.

Author: Katherine Marougka, Delphine Judith, Tristan Jaouen, Sabine Blouquit-Laye, Gina Cosentino, Clarisse Berlioz-Torrent, Marie-Anne Rameix-Welti, and Delphine Sitterlin
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Human respiratory syncytial virus (RSV) is an enveloped RNA virus and the leading viral agent responsible for severe pediatric respiratory infections worldwide. Identification of cellular factors able to restrict viral infection is one of the key strategies used to design new drugs against infection. Here, we report for the first time that the cellular protein BST2/Tetherin (a widely known host antiviral molecule) behaves as a restriction factor of RSV infection. We showed that BST2 silencing resulted in a significant rise in viral production during multi-cycle infection, suggesting an inhibitory role during the late steps of RSV's multiplication cycle. Conversely, BST2 overexpression resulted in the decrease of the viral production. Furthermore, BST2 was found associated with envelope proteins and co-localized with viral filaments, suggesting that BST2 tethers RSV particles. Interestingly, RSV naturally downregulates cell surface and global BST2 expression, possibly through a mechanism dependent on ubiquitin. RSV's ability to enhance BST2 degradation was also validated in a model of differentiated cells infected by RSV. Additionally, we found that a virus deleted of NS1 is unable to downregulate BST2 and is significantly more susceptible to BST2 restriction compared to the wild type virus. Moreover, NS1 and BST2 interact in a co- immunoprecipitation experiment. Overall, our data support a model in which BST2 is a restriction factor against RSV infection and that the virus counteracts this effect by limiting the cellular factor's expression through a mechanism involving the viral protein NS1.
Published: 2024
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2. Escape of SARS-CoV-2 Variants KP.1.1, LB.1, and KP.3.3 From Approved Monoclonal Antibodies

Author: Delphine Planas, Isabelle Staropoli, Cyril Planchais, Emilie Yab, Banujaa Jeyarajah, Yannis Rahou, Matthieu Prot, Florence Guivel-Benhassine, Frederic Lemoine, Vincent Enouf, Etienne Simon-Loriere, Hugo Mouquet, Marie-Anne Rameix-Welti, and Olivier Schwartz
Subjects: Pathology, RB1-214, Immunologic diseases. Allergy, RC581-607
Abstract: Background: First-generation anti-SARS-CoV-2 monoclonal antibodies (mAbs) used for prophylaxis or therapeutic purposes in immunocompromised patients have been withdrawn because of the emergence of resistant Omicron variants. In 2024, 2 novel mAbs, VYD222/Pemivibart and AZD3152/Sipavibart, were approved by health authorities, but their activity against contemporary JN.1 sublineages is poorly characterized. Methods: We isolated authentic JN.1.1, KP.1.1, LB.1, and KP.3.3 viruses and evaluated their sensitivity to neutralization by these mAbs in 2 target cell lines. Results: Compared to ancestral strains, VYD222/Pemivibart remained moderately active against JN.1 subvariants, with a strong increase of 50% Inhibitory Concentration (IC50), reaching up to 3 to 15 µg/mL for KP.3.3. AZD3152/Sipavibart neutralized JN.1.1 but lost antiviral efficacy against KP.1.1, LB.1, and KP.3.3. Conclusions: Our results highlight the need for a close clinical monitoring of VYD222/Pemivibart and raise concerns about the clinical efficacy of AZD3152/Sipavibart.
Published: 2024
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3. Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor

Author: Svenja M. Sake, Xiaoyu Zhang, Manoj Kumar Rajak, Melanie Urbanek-Quaing, Arnaud Carpentier, Antonia P. Gunesch, Christina Grethe, Alina Matthaei, Jessica Rückert, Marie Galloux, Thibaut Larcher, Ronan Le Goffic, Fortune Hontonnou, Arnab K. Chatterjee, Kristen Johnson, Kaycie Morwood, Katharina Rox, Walid A. M. Elgaher, Jiabin Huang, Martin Wetzke, Gesine Hansen, Nicole Fischer, Jean-Francois Eléouët, Marie-Anne Rameix-Welti, Anna K. H. Hirsch, Elisabeth Herold, Martin Empting, Chris Lauber, Thomas F. Schulz, Thomas Krey, Sibylle Haid, and Thomas Pietschmann
Subjects: Science
Abstract: Abstract Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.
Published: 2024
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4. Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus

Author: Ana Vieira Antão, Friederike Oltmanns, Anna Schmidt, Vera Viherlehto, Pascal Irrgang, Marie-Anne Rameix-Welti, Wibke Bayer, Dennis Lapuente, and Matthias Tenbusch
Subjects: respiratory viruses, mucosal immunity, combinatory vaccine, adjuvant, influenza A virus (IAV), respiratory syncytial virus (RSV), Immunologic diseases. Allergy, RC581-607
Abstract: Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFβ or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.
Published: 2024
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5. Respiratory syncytial virus ribonucleoproteins hijack microtubule Rab11 dependent transport for intracellular trafficking.

Author: Gina Cosentino, Katherine Marougka, Aurore Desquesnes, Nicolas Welti, Delphine Sitterlin, Elyanne Gault, and Marie-Anne Rameix-Welti
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.
Published: 2022
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6. Molecular investigation of a RSV outbreak in a geriatric hospital

Author: Yohan Hababou, Assia Taleb, Amélie Recoing, Frédérique Moreau, Isabelle Simon, Florence Muller de Schongor, Elyanne Gault, and Marie-Anne Rameix-Welti
Subjects: Geriatrics, RC952-954.6
Abstract: Abstract Background Acquired infections in hospitalized elderly people are a growing concern. In long-term care facilities with multiple staff and visitor contacts, virus outbreaks are a common challenge for infection prevention teams. Although several studies have reported nosocomial RSV outbreaks in long term care facilities, molecular epidemiology data are scarce. Methods RSV RNA was detected in respiratory samples from 19 patients in a long-term care hospital for elderly in Paris in March 2019 over a 3 weeks period. Genotyping was performed using nucleotide sequencing. Sociodemographic and clinical characteristics of cases part of a unique cluster, were retrospectively reviewed. Results Molecular investigation of theses RSV cases, revealed a unique cluster of 12 nosocomial cases in 2 adjacent wards. Mean age of these outbreak’s cases was 89. All patients had underlying medical conditions. Seven exhibited lower respiratory symptoms and three experienced decompensation of underlying chronic heart condition. Two patients died. Conclusions This case report highlights the importance of RSV in causing substantial disease in elderly in case of nosocomial outbreak and the contributions of molecular epidemiology in investigation and management of such outbreak.
Published: 2021
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7. Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection.

Author: Melanie Brügger, Thomas Démoulins, G Tuba Barut, Beatrice Zumkehr, Blandina I Oliveira Esteves, Kemal Mehinagic, Quentin Haas, Aline Schögler, Marie-Anne Rameix-Welti, Jean-François Eléouët, Ueli Moehrlen, Thomas M Marti, Ralph A Schmid, Artur Summerfield, Horst Posthaus, Nicolas Ruggli, Sean R R Hall, and Marco P Alves
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.
Published: 2021
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8. Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis

Author: Emilie Jacque, Claire Chottin, Daphné Laubreton, Michel Nogre, Cécile Ferret, Sandrine de Marcos, Linda Baptista, Carole Drajac, Philippe Mondon, Christophe De Romeuf, Marie-Anne Rameix-Welti, Jean-François Eléouët, Sami Chtourou, Sabine Riffault, Gérald Perret, and Delphyne Descamps
Subjects: concentration, nasal administration, bioprocess, human plasma, hyper-immune immunoglobulins, prophylactic strategy, Immunologic diseases. Allergy, RC581-607
Abstract: Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases.ImportanceRespiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.
Published: 2021
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9. Single-Stranded Oligonucleotide-Mediated Inhibition of Respiratory Syncytial Virus Infection

Author: Sandra Axberg Pålsson, Aleksandra Dondalska, Joseph Bergenstråhle, Caroline Rolfes, Albin Björk, Laura Sedano, Ultan F. Power, Marie-Anne Rameix-Welti, Joakim Lundeberg, Marie Wahren-Herlenius, Peter Mastrangelo, Jean-Francois Eleouet, Ronan Le Goffic, Marie Galloux, and Anna-Lena Spetz
Subjects: respiratory syncytial virus (RSV), oligonucleotides, single-stranded oligonucleotides, ssON, antiviral, nucleolin, Immunologic diseases. Allergy, RC581-607
Abstract: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON’s antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro. Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1, Stat2, Cxcl10, and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro, likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs.
Published: 2020
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10. Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

Author: Marie Galloux, Jennifer Risso-Ballester, Charles-Adrien Richard, Jenna Fix, Marie-Anne Rameix-Welti, and Jean-François Eléouët
Subjects: RSV, inclusion bodies, liquid-liquid phase, nucleoprotein, phosphoprotein, protein-protein interactions, Microbiology, QR1-502
Abstract: ABSTRACT Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.
Published: 2020
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11. Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus

Author: Vincent Rincheval, Mickael Lelek, Elyanne Gault, Camille Bouillier, Delphine Sitterlin, Sabine Blouquit-Laye, Marie Galloux, Christophe Zimmer, Jean-François Eleouet, and Marie-Anne Rameix-Welti
Subjects: Science
Abstract: Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.
Published: 2017
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12. No Incongruity in Respiratory Syncytial Virus M2-1 Protein Remaining Bound to Viral mRNAs during Their Entire Life Time

Author: Marie-Anne Rameix-Welti
Subjects: M2-1, respiratory syncytial virus, transcription, Microbiology, QR1-502
Published: 2019
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13. Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen.

Author: Fabian Sesterhenn, Marie Galloux, Sabrina S Vollers, Lucia Csepregi, Che Yang, Delphyne Descamps, Jaume Bonet, Simon Friedensohn, Pablo Gainza, Patricia Corthésy, Man Chen, Stéphane Rosset, Marie-Anne Rameix-Welti, Jean-François Éléouët, Sai T Reddy, Barney S Graham, Sabine Riffault, and Bruno E Correia
Subjects: Biology (General), QH301-705.5
Abstract: Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.
Published: 2019
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14. Evaluation of the Antiviral Activity of Sephin1 Treatment and Its Consequences on eIF2α Phosphorylation in Response to Viral Infections

Author: Maxime Fusade-Boyer, Gabriel Dupré, Pierre Bessière, Samira Khiar, Charlotte Quentin-Froignant, Cécile Beck, Sylvie Lecollinet, Marie-Anne Rameix-Welti, Jean-François Eléouët, Frédéric Tangy, Barbora Lajoie, Stéphane Bertagnoli, Pierre-Olivier Vidalain, Franck Gallardo, and Romain Volmer
Subjects: PKR, GADD34, PPP1R15A, virus, antiviral, eIF2α, Immunologic diseases. Allergy, RC581-607
Abstract: The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1—GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.
Published: 2019
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15. New Look at RSV Infection: Tissue Clearing and 3D Imaging of the Entire Mouse Lung at Cellular Resolution

Author: Maxence Frétaud, Delphyne Descamps, Daphné Laubreton, Marie-Anne Rameix-Welti, Jean-François Eléouët, Thibaut Larcher, Marie Galloux, and Christelle Langevin
Subjects: RSV infection, tissue clearing, 3D imaging of lungs, inclusion bodies, viral pathophysiology, RSV tropism, Microbiology, QR1-502
Abstract: Background: Respiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis has mostly relied on chest X-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus–host interactions and related acute respiratory disease, at the level of the entire lung. Methods: By combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs. Results: Whole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection. Conclusions: Whole lung clearing and 3D deep imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses.
Published: 2021
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16. Regulatory B Lymphocytes Colonize the Respiratory Tract of Neonatal Mice and Modulate Immune Responses of Alveolar Macrophages to RSV Infection in IL-10-Dependant Manner

Author: Daphné Laubreton, Carole Drajac, Jean-François Eléouët, Marie-Anne Rameix-Welti, Richard Lo-Man, Sabine Riffault, and Delphyne Descamps
Subjects: respiratory syncytial virus, neonates, age-dependent replication, lungs, innate immunity, immunoregulation, Microbiology, QR1-502
Abstract: Respiratory syncytial virus (RSV) is the prevalent pathogen of lower respiratory tract infections in children. The presence of neonatal regulatory B lymphocytes (nBreg) has been associated with a poor control of RSV infection in human newborns and with bronchiolitis severity. So far, little is known about how nBreg may contribute to neonatal immunopathology to RSV. We tracked nBreg in neonatal BALB/c mice and we investigated their impact on lung innate immunity, especially their crosstalk with alveolar macrophages (AMs) upon RSV infection. We showed that the colonization by nBreg during the first week of life is a hallmark of neonatal lung whereas this population is almost absent in adult lung. This particular period of age when nBreg are abundant corresponds to the same period when RSV replication in lungs fails to generate a type-I interferons (IFN-I) response and is not contained. When neonatal AMs are exposed to RSV in vitro, they produce IFN-I that in turn enhances IL-10 production by nBreg. IL-10 reciprocally can decrease IFN-I secretion by AMs. Thus, our work identified nBreg as an important component of neonatal lungs and pointed out new immunoregulatory interactions with AMs in the context of RSV infection.
Published: 2020
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17. RSV hijacks cellular protein phosphatase 1 to regulate M2-1 phosphorylation and viral transcription.

Author: Charles-Adrien Richard, Vincent Rincheval, Safa Lassoued, Jenna Fix, Christophe Cardone, Camille Esneau, Sergei Nekhai, Marie Galloux, Marie-Anne Rameix-Welti, Christina Sizun, and Jean-François Eléouët
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Respiratory syncytial virus (RSV) RNA synthesis occurs in cytoplasmic inclusion bodies (IBs) in which all the components of the viral RNA polymerase are concentrated. In this work, we show that RSV P protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M2-1. We also show that M2-1 dephosphorylation is achieved by a complex formed between P and the cellular phosphatase PP1. We identified the PP1 binding site of P, which is an RVxF-like motif located nearby and upstream of the M2-1 binding region. NMR confirmed both P-M2-1 and P-PP1 interaction regions in P. When the P-PP1 interaction was disrupted, M2-1 remained phosphorylated and viral transcription was impaired, showing that M2-1 dephosphorylation is required, in a cyclic manner, for efficient viral transcription. IBs contain substructures called inclusion bodies associated granules (IBAGs), where M2-1 and neo-synthesized viral mRNAs concentrate. Disruption of the P-PP1 interaction was correlated with M2-1 exclusion from IBAGs, indicating that only dephosphorylated M2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function.
Published: 2018
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18. Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting.

Author: Geoffrey P Chase, Marie-Anne Rameix-Welti, Aurelija Zvirbliene, Gintautas Zvirblis, Veronika Götz, Thorsten Wolff, Nadia Naffakh, and Martin Schwemmle
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration.
Published: 2011
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19. Influenza virus infection induces the nuclear relocalization of the Hsp90 co-chaperone p23 and inhibits the glucocorticoid receptor response.

Author: Xingyi Ge, Marie-Anne Rameix-Welti, Elyanne Gault, Geoffrey Chase, Emmanuel dos Santos Afonso, Didier Picard, Martin Schwemmle, and Nadia Naffakh
Subjects: Medicine, Science
Abstract: The genomic RNAs of influenza A viruses are associated with the viral polymerase subunits (PB1, PB2, PA) and nucleoprotein (NP), forming ribonucleoprotein complexes (RNPs). Transcription/replication of the viral genome occurs in the nucleus of infected cells. A role for Hsp90 in nuclear import and assembly of newly synthetized RNA-polymerase subunits has been proposed. Here we report that the p23 cochaperone of Hsp90, which plays a major role in glucocorticoid receptor folding and function, associates with influenza virus polymerase. We show that p23 is not essential for viral multiplication in cultured cells but relocalizes to the nucleus in influenza virus-infected cells, which may alter some functions of p23 and Hsp90. Moreover, we show that influenza virus infection inhibits glucocorticoid receptor-mediated gene transactivation, and that this negative effect can occur through a p23-independent pathway. Viral-induced inhibition of the glucocorticoid receptor response might be of significant importance regarding the physiopathology of influenza infections in vivo.
Published: 2011
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20. Enhancement of the influenza A hemagglutinin (HA)-mediated cell-cell fusion and virus entry by the viral neuraminidase (NA).

Author: Bin Su, Sébastien Wurtzer, Marie-Anne Rameix-Welti, Dominic Dwyer, Sylvie van der Werf, Nadia Naffakh, François Clavel, and Béatrice Labrosse
Subjects: Medicine, Science
Abstract: BACKGROUND:The major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutinin (HA) protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity. METHODOLOGY/PRINCIPAL FINDINGS:The role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogenous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA. CONCLUSION/SIGNIFICANCE:The NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion.
Published: 2009
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21. Enzymatic properties of the neuraminidase of seasonal H1N1 influenza viruses provide insights for the emergence of natural resistance to oseltamivir.

Author: Marie-Anne Rameix-Welti, Vincent Enouf, Frédérique Cuvelier, Patricia Jeannin, and Sylvie van der Werf
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Published: 2008
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22. Retinoic Acid–Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection

Author: Lara S U Schwab, Rubaiyea Farrukee, Jean-François Eléouët, Marie-Anne Rameix-Welti, Sarah L Londrigan, Andrew G Brooks, Aeron C Hurt, Christoph Coch, Thomas Zillinger, Gunther Hartmann, and Patrick C Reading
Subjects: Infectious Diseases, Immunology and Allergy
Abstract: Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid–inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an “antiviral state.” In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.
Published: 2022
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23. Respiratory Syncytial Virus Two-Step Infection Screen Reveals Inhibitors of Early and Late Life Cycle Stages

Author: Svenja M. Sake, Christina Kosch, Sebastian Blockus, Sibylle Haid, Antonia P. Gunesch, Xiaoyu Zhang, Martina Friesland, Sofie B. Trummer, Christina Grethe, Anne Kühnel, Jessica Rückert, W. Paul Duprex, Jiabin Huang, Marie-Anne Rameix-Welti, Martin Empting, Nicole Fischer, Anna K. H. Hirsch, Thomas F. Schulz, Thomas Pietschmann, HAL UVSQ, Équipe, Institute for Experimental Virology [Hanovre, Allemagne], Centre for Experimental and Clinical Infection Research (TWINCORE), Helmholtz Centre for Infection Research (HZI)-Medizinische Hochschule Hannover (MHH)-Helmholtz Centre for Infection Research (HZI)-Medizinische Hochschule Hannover (MHH), Helmholtz Centre for Infection Research (HZI), Saarland University [Saarbrücken], Medizinische Hochschule Hannover (MHH), German Centre for Infection Research (DZIF), University of Pittsburgh (PITT), Pennsylvania Commonwealth System of Higher Education (PCSHE), Universitaetsklinikum Hamburg-Eppendorf = University Medical Center Hamburg-Eppendorf [Hamburg] (UKE), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Ambroise Paré [AP-HP], 11-76251-99-6/19, 14-76103-184 CORONA-13/ 20, ZN3437, Deutsches Zentrum für Infektionsforschung, DZIF, Deutsche Forschungsgemeinschaft, DFG: 390874280, EXC 2155, The purchase of the compound library and screening platform was made possible through financial support from the German Centre for Infection Research (DZIF) within the framework of the Translational Infrastructure ‘Novel Antivirals.’, and A.K.H.H., T.P., and T.F.S. are funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under the Germany’s Excellence Strategy EXC 2155 'RESIST' (Project ID 390874280) T.P. and T.F.S. are funded by the SARS-CoV-2 Immediate Response Initiative funded by the State of Lower Saxony (14-76103-184 CORONA-13/ 20). T.P. is also funded by the INDIRA Project (11-76251-99-6/19 (ZN3437). T.P. and A.K.H.H. also received funds from the Helmholtz International Lab for anti-infectives, and T.F.S. and T.P. are funded by the German Centre for Infection Research (DZIF).
Subjects: [SDV] Life Sciences [q-bio], Pharmacology, antivirals, Infectious Diseases, [SDV]Life Sciences [q-bio], RSV, Pharmacology (medical), drug screen, drug discovery
Abstract: International audience; Human respiratory syncytial virus (hRSV) infection is a leading cause of severe respiratory tract infections. Effective, directly acting antivirals against hRSV are not available. We aimed to discover new and chemically diverse candidates to enrich the hRSV drug development pipeline. We used a two-step screen that interrogates compound efficacy after primary infection and a consecutive virus passaging. We resynthesized selected hit molecules and profiled their activities with hRSV lentiviral pseudotype cell entry, replicon, and time-of-addition assays. The breadth of antiviral activity was tested against recent RSV clinical strains and human coronavirus (hCoV-229E), and in pseudotype-based entry assays with non-RSV viruses. Screening 6,048 molecules, we identified 23 primary candidates, of which 13 preferentially scored in the first and 10 in the second rounds of infection, respectively. Two of these molecules inhibited hRSV cell entry and selected for F protein resistance within the fusion peptide. One molecule inhibited transcription/replication in hRSV replicon assays, did not select for phenotypic hRSV resistance and was active against non-hRSV viruses, including hCoV-229E. One compound, identified in the second round of infection, did not measurably inhibit hRSV cell entry or replication/transcription. It selected for two coding mutations in the G protein and was highly active in differentiated BCi-NS1.1 lung cells. In conclusion, we identified four new hRSV inhibitor candidates with different modes of action. Our findings build an interesting platform for medicinal chemistry-guided derivatization approaches followed by deeper phenotypical characterization in vitro and in vivo with the aim of developing highly potent hRSV drugs. Copyright
Published: 2022
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24. Monocyte distribution width as a biomarker of resistance to corticosteroids in patients with sepsis: the MOCORSEP observational study

Author: Heming Nicholas, François Bruno, Jean-Claude Alvarez, Sylvie Chevret, Djillali Annane, François Fay, Murg Eleonora, Abdeladim Lilia, Zaineb Chelly, U. Richly Maximilian, Fayssoil Abdallah, Véronique Godot, Jean Sébastien Blanchet, Grassin-Delyle Stanislas, Clair Bernard, Fleuriet Jérome, Loïc Josseran, Grimaldi Lamiae, Devillier Philippe, Kedad Zoubida, Blanchet Jean-Sébastien, Pierre Moine, Roux Anne-Laure, Godot Véronique, Garchon Henri-Jean, Talini Luc, Rottman Martin, Antoine Vieillard-Baron, Maxime Virginie, Bouneb Rania, Moine Pierre, Jérôme Fleuriet, Bruno Mégarbane, Karine Zeitouni, Nicholas Heming, Chevret Sylvie, Annane Djillali, Guillaume Geri, Laurent Dumas, Valérie Bardet, Martin Rottman, Paris Seine Nord Endeavour for Personalized Interventions for Sepsis, Anne-Laure Roux, Henri-Jean Garchon, Lassalle Philippe, Lamiae Grimaldi, Pierre Tissières, Ziad A. Massy, Zeitouni Karine, Marie-Anne Rameix-Welti, Stanislas Grassin-Delyle, Mahmoud Zureik, Xavier Monnet, Orlikowski David, Amine Larabi, Daniel Christel, and Marie Essig
Subjects: medicine.medical_specialty, business.industry, Monocyte, Critical Care and Intensive Care Medicine, medicine.disease, Gastroenterology, Sepsis, medicine.anatomical_structure, Internal medicine, medicine, Biomarker (medicine), Distribution (pharmacology), In patient, Observational study, business
Published: 2021
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25. Ribonucleoprotein transport in Negative Strand RNA viruses

Author: Cédric Diot, Gina Cosentino, and Marie‐Anne Rameix‐Welti
Subjects: Cell Biology, General Medicine
Abstract: Negative-sense, single-stranded RNA (-ssRNA) viruses comprise some of the deadliest human pathogens (Ebola, rabies, influenza A viruses etc.). Developing therapeutic tools relies on a better understanding of their multiplication cycle. For these viruses, the genome replication and transcription activities most-often segregate in membrane-less environments called inclusion bodies (IBs) or viral factories. These "organelles" usually locate far from the cell surface from where new virions are released, and -ssRNA viruses do not encode for transport factors. The efficient trafficking of the genome progeny toward the cell surface is most often ensured by mechanisms co-opting the cellular machineries. In this review, for each -ssRNA viral family, we cover the methods employed to characterize these host-virus interactions, the strategies used by the viruses to promote the virus genome transport, and the current gaps in the literature. Finally, we highlight how Rab11 has emerged as a target of choice for the intracellular transport of -ssRNA virus genomes.
Published: 2022

26. A condensate-hardening drug blocks RSV replication in vivo

Author: Zhaoguo Wang, Ronan Le Goffic, Jean-François Eléouët, Ralf Altmeyer, Jennifer Risso-Ballester, Cao Jingjing, Miaomiao Du, Sibylle Haid, Marie Galloux, Huanyun Zhang, Aurore Desquesnes, Youming Zhang, Thomas Pietschmann, Fortune Hontonnou, Vincent Rincheval, Aude Jobart-Malfait, Svenja M. Sake, Marie-Anne Rameix-Welti, Xiumei Zhang, Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Shandong University, Centre for Experimental and Clinical Infection Research (TWINCORE), Helmholtz Centre for Infection Research (HZI)-Medizinische Hochschule Hannover (MHH), Qingdao Municipal Center for Disease Control and Prevention, Partenaires INRAE, Hôpital Ambroise Paré [AP-HP], The University of Hong Kong (HKU), Fondation Air Liquide, China - 111 Project B16030, ATIP-AVENIR INSERM and Fondation Del Duca-Institut de France, People’s Livelihood Technology Project of Qingdao City (17-3-3-2-nsh), Ile de France region (SESAME), Natural Science Foundation of China Youth Project (31900147), Sino-German Helmholtz International Lab grant (10000089395401), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Région Île-de-France, Natural Science Foundation of China Youth Project: 31900147, Fondation Air Liquide, Ministry of Education, China - 111 Project: B16030, ATIP-AVENIR INSERM program, Fondation Del Duca-Institut de France grant, Sino-German Helmholtz International Lab grant: 10000089395401, People's Livelihood Technology Project of Qingdao City: 17-3-3-2-nsh, and Le Goffic, Ronan
Subjects: 0303 health sciences, Multidisciplinary, Cyclopamine, Point mutation, viruses, RNA, Inclusion bodies, Virus, 3. Good health, Cell biology, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Viral replication, In vivo, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Transcription factor, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6. Cyclopamine and its chemical analogue A3E inhibit replication of human respiratory syncytial virus (RSV) by hardening the liquid–liquid phase-separated inclusion bodies, resulting in the inhibition of virus replication in the lungs of RSV-infected mice.
Published: 2021
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27. Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection

Author: Jean-François Eléouët, Ralph A. Schmid, Quentin Haas, Artur Summerfield, Kemal Mehinagic, Beatrice Zumkehr, Sean R R Hall, Nicolas Ruggli, B. Oliveira Esteves, Melanie Brügger, Marie-Anne Rameix-Welti, Thomas Démoulins, Thomas M. Marti, Ueli Moehrlen, Horst Posthaus, A. Schogler, G. T. Barut, Marco P. Alves, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Bern, Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University Children’s Hospital Zurich, Bern University Hospital [Berne] (Inselspital), Gillies McIndoe Research Institute (GMRI), Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, SNF: 310030_172895 Gottfried und Julia Bangerter-Rhyner-Stiftung, This work acknowledges support from the Swiss National Science Foundation to MPA (www.snf.ch, project 310030_172895), that included partial salary support for MB and TD. This work was also supported by a grant from the Gottfried and Julia Bangerter-Rhyner Foundation to MPA (www.bangerter-stiftung.ch), that included partial salary support to MB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., HAL UVSQ, Équipe, University of Zurich, and Alves, Marco P
Subjects: Pulmonology, Angiogenesis, Physiology, [SDV]Life Sciences [q-bio], 2405 Parasitology, Pediatrics, 0302 clinical medicine, Medical Conditions, Animal Cells, Immune Physiology, Medicine and Health Sciences, Respiratory system, Biology (General), Lung, 0303 health sciences, Innate Immune System, 630 Agriculture, Stem Cells, 2404 Microbiology, Cell Differentiation, respiratory system, 3. Good health, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Infectious Diseases, 030220 oncology & carcinogenesis, Cytokines, Cellular Types, Pediatric Infections, Research Article, Stromal cell, QH301-705.5, T cell, Immunology, Acute Lung Injury, 610 Medicine & health, Respiratory Syncytial Virus Infections, Lung injury, Biology, Virus, 03 medical and health sciences, Respiratory Disorders, 1311 Genetics, medicine, 1312 Molecular Biology, Animals, Humans, 10220 Clinic for Surgery, Respiratory Physiology, 030304 developmental biology, 2403 Immunology, Sheep, Regeneration (biology), Mesenchymal stem cell, Biology and Life Sciences, Mesenchymal Stem Cells, Convalescence, Cell Biology, RC581-607, Molecular Development, Health Care, Immune System, Respiratory Syncytial Virus, Human, Respiratory Infections, 2406 Virology, Immunologic diseases. Allergy, Developmental Biology
Abstract: Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications., Author summary This work identifies a novel function of lung-resident MSCs during virus-induced acute lung injury. These findings contribute to the understanding of host response and lung repair mechanisms during a highly prevalent clinical situation and may have therapeutic implications.
Published: 2021
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28. Performance of 30 commercial SARS-CoV-2 serology assays in testing symptomatic COVID-19 patients

Author: Agnès Carol, Narjis Boukli, Elyanne Gault, Juliette Villemonteix, Tiffany Guilleminot, Héloïse Petit, Guislaine Carcelain, Dominique Challine, Diane Descamps, Sarah Maylin, Jean-François Meritet, Claire Périllaud-Dubois, Laurence Morand-Joubert, Sonia Burrel, Marie-Anne Rameix-Welti, Frédérique Moreau, Charlotte Charpentier, Jérôme LeGoff, Jean-Michel Pawlotsky, Anne-Geneviève Marcelin, Emmanuel Gordien, Christelle Vauloup-Fellous, Samuel Lepape, Isabelle Podglajen, Slim Fourati, Sepideh Akhavan, Nadhira Houhou-Fidouh, Ana-Maria Roque-Afonso, Marianne Leruez-Ville, Vincent Mackiewicz, Florence Damond, Véronique Avettand-Fenoel, Jacques Fourgeaud, Jean-Baptiste Ronat, Chakib Alloui, Ségolène Brichler, Joël Gozlan, Thierry Naas, Elise Gardiennet, Marie-Laure Chaix, Stéphane Bonacorsi, Constance Delaugerre, Flore Rozenberg, Laurent Dortet, Stéphane Marot, Service de Virologie [Hôpital Paul-Brousse - APHP] (World Health Organization Rubella National Reference Laboratory), Hôpital Paul Brousse, Génomes, biologie cellulaire et thérapeutiques (GenCellDi (UMR_S_944)), Collège de France (CdF (institution))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Service de Microbiologie Clinique [Hôpital Avicenne - APHP], Hôpital Avicenne [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Service de Microbiologie et Hygiène [AP-HP Hôpital Ambroise-Paré], Hôpital Ambroise Paré [AP-HP], Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Infection, Anti-microbiens, Modélisation, Evolution (IAME (UMR_S_1137 / U1137)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)-Université Sorbonne Paris Nord, Service de Virologie [CHU Cochin], Hôpital Cochin [AP-HP], Laboratoire de Virologie [Hôpital Européen Georges Pompidou - APHP], Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Institut Pierre Louis d'Epidémiologie et de Santé Publique (iPLESP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Service de Microbiologie [Hôpital Robert Debré - APHP], Centre National de Référence associé Escherichia coli [Hôpital Robert Debré - APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Hôpital Robert Debré, Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Comets, Emmanuelle, Génomes, biologie cellulaire et thérapeutiques (GenCellDi (U944 / UMR7212)), Collège de France (CdF (institution))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Université Sorbonne Paris Nord, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Cochin [AP-HP], Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de Référence associé Escherichia coli [Hôpital Robert Debré - APHP], and Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Microbiology (medical), 2019-20 coronavirus outbreak, medicine.medical_specialty, Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 030106 microbiology, Antibodies, Viral, Sensitivity and Specificity, COVID-19 Serological Testing, Serology, 03 medical and health sciences, 0302 clinical medicine, Internal medicine, medicine, Humans, 030212 general & internal medicine, Immunoassay, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], business.industry, SARS-CoV-2, Brief Report, COVID-19, General Medicine, Report evaluation, Serum samples, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], 3. Good health, Infectious Diseases, Immunoglobulin M, Reagent Kits, Diagnostic, business
Abstract: We report evaluation of 30 assays’ (17 rapid tests (RDTs) and 13 automated/manual ELISA/CLIA assay (IAs)) clinical performances with 2594 sera collected from symptomatic patients with positive SARS-CoV-2 rRT-PCR on a respiratory sample, and 1996 pre-epidemic serum samples expected to be negative. Only 4 RDT and 3 IAs fitted both specificity (> 98%) and sensitivity (> 90%) criteria according to French recommendations. Serology may offer valuable information during COVID-19 pandemic, but inconsistent performances observed among the 30 commercial assays evaluated, which underlines the importance of independent evaluation before clinical implementation. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-021-04232-3.
Published: 2021
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29. Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

Author: Pierre Isnard, Giovanna Barba-Spaeth, Gérard Pierron, Christian Weber, Etienne Simon-Loriere, Sylvie Souquere, Christophe Zimmer, Jean-Baptiste Gibier, Stefano Pietropaoli, Marie-Anne Rameix-Welti, Marion Rabant, Florian Mueller, Elena Rensen, Imagerie et Modélisation - Imaging and Modeling, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Virologie Structurale - Structural Virology, Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Anatomie et Cytologie Pathologiques, CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Necker-Enfants Malades, Service d'anatomopathologie, Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Institut Pasteur [Paris], Université Paris-Saclay, Hôpital Ambroise Paré [AP-HP], Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiologie et physiopathologie des rétrovirus endogènes et infectieux (RETRO-ENDO), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), ESL acknowledges funding from the French Government’s Investissement d’Avenir program, ‘INCEPTION’ (ANR-16-CONV-0005). GBS acknowledges funding by the Institut Pasteur Coronavirus task force (don AXA COVID-19 project COVID-SPREAD). This work was funded by Institut Pasteur., and ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016)
Subjects: 0303 health sciences, medicine.diagnostic_test, biology, Coronavirus disease 2019 (COVID-19), Oligonucleotide, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), viruses, RNA, RNA virus, biology.organism_classification, Virology, 3. Good health, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), medicine, In patient, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, 030217 neurology & neurosurgery, 030304 developmental biology, Fluorescence in situ hybridization
Abstract: The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescencein situhybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.
Published: 2021

30. New Look at RSV Infection: Tissue Clearing and 3D Imaging of the Entire Mouse Lung at Cellular Resolution

Author: Jean-François Eléouët, Delphyne Descamps, Christelle Langevin, Thibaut Larcher, Marie Galloux, Maxence Frétaud, Marie-Anne Rameix-Welti, Daphné Laubreton, Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), and ANR-19-CE11-0017,DecRisP,Décrypter les synthèses d'ARN par les pneumovirus(2019)
Subjects: Larcher, 0301 basic medicine, [SDV]Life Sciences [q-bio], lcsh:QR1-502, inclusion bodies, Disease, viral pathophysiology, Virus Replication, lcsh:Microbiology, Inclusion bodies, M, Inclusion Bodies, Viral, Pathogenesis, Mice, 0302 clinical medicine, Eléouët, Respiratory system, Descamps, Pathogen, Lung, ComputingMilieux_MISCELLANEOUS, C. New Look at RSV RSV infection, Brief Report, T, respiratory system, 3. Good health, M.-A, Infectious Diseases, medicine.anatomical_structure, Laubreton, D, tissue clearing, Frétaud, Respiratory Syncytial Virus Infections, Virus, 03 medical and health sciences, Imaging, Three-Dimensional, Virology, medicine, Animals, RSV infection, J.-F, business.industry, RSV tropism, Epithelial Cells, Disease Models, Animal, 030104 developmental biology, Rameix-Welti, Respiratory Syncytial Virus, Human, Immunology, Galloux, Langevin, 3D imaging of lungs, business, 030217 neurology & neurosurgery, Respiratory tract
Abstract: Background: Respiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis has mostly relied on chest X-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus–host interactions and related acute respiratory disease, at the level of the entire lung. Methods: By combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs. Results: Whole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection. Conclusions: Whole lung clearing and 3D deep imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses.
Published: 2021
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31. New look at RSV infection : tissue clearing and 3D imaging of the entire mouse lung at cellular resolution

Author: Jean-François Eléouët, Thibaut Larcher, Christelle Langevin, Marie Galloux, Marie-Anne Rameix-Welti, Maxence Frétaud, Delphyne Descamps, and Daphné Laubreton
Subjects: Pathogenesis, Lung, medicine.anatomical_structure, business.industry, Immunology, medicine, Disease, Respiratory system, business, Pathogen, Pathophysiology, Virus, Respiratory tract
Abstract: BackgroundRespiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis mostly relied on chest x-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus-host interactions and related acute respiratory disease, at the level of the entire lung.MethodsBy combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs.ResultsWhole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection.ConclusionsWhole lung clearing and 3D deep-imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses.
Published: 2020
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32. Clinical Cutaneous Features of Patients Infected With SARS-CoV-2 Hospitalized for Pneumonia: A Cross-sectional Study

Author: Jean-François Emile, Stephanie Landowski, Elyanne Gault, Julia N'guyen Van Thanh, Pierre de Truchis, Isabelle Bourgault-Villada, Louis Jacob, Aurélie Le Gal, Jean-Louis Gaillard, Véronique Perronne, Aymeric Lanore, Nicolas Kiavue, Simon Bessis, Hélène Mascitti, Frédérique Bouchand, Aurélien Dinh, Faten El Sayed, Morgan Matt, Benjamin Davido, Valérie Sivadon-Tardy, Marie-Anne Rameix-Welti, Anne-Laure Roux, Elsa Salomon, Benjamin Bonsang, Christian Perronne, Soline Siméon, Clara Duran, Robert Carlier, Thibault Leblanc, Frédérique Moreau, and Florence Assan
Subjects: Livedo, medicine.medical_specialty, pandemics, Major Articles, Capillary leak, 030207 dermatology & venereal diseases, 03 medical and health sciences, 0302 clinical medicine, Edema, Biopsy, medicine, pneumonia, 030212 general & internal medicine, medicine.diagnostic_test, business.industry, COVID-19, medicine.disease, Dermatology, dermatology, Editor's Choice, Purpura, Pneumonia, AcademicSubjects/MED00290, Infectious Diseases, Oncology, Macular Rash, exanthema, medicine.symptom, business, Cytokine storm
Abstract: Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a current pandemic worldwide. This virus can reach all organs and disturbs the immune system, leading to a cytokine storm in severe forms. We aimed to report cutaneous features among coronavirus disease 2019 (COVID-19) hospitalized patients. Methods We performed a cross-sectional study on 1 given day among all patients hospitalized in acute care for COVID-19 and included all patients with cutaneous features. Follow-up 48 hours later was obtained. Results Among 59 adult patients hospitalized on the day of the study in an infectious diseases ward for SARS-CoV-2 infection who were confirmed by molecular assay and/or radiological findings (computed tomography scan), 40 were included. Several cutaneous manifestations were found: macular exanthema (80%), face edema (32%), livedo (13%), urticarial rash (8%), purpura (5%), oral lichenoid lesions (33%), and conjunctivitis (18%). Cutaneous biopsy was performed in 17 patients. Histological findings showed mast cell hyperplasia (100%), superficial perivascular infiltrate of lymphocytes (94%), and superficial edema (47%) consistent with capillary leak. Conclusions Various dermatological signs can be encountered during COVID-19. A macular rash was the most frequent. All cutaneous features could be related to a vascular leak process., This cross-sectional study showed that cutaneous features are various among hospitalized patients with COVID-19. Macular rash was the most frequent and could be predictive of worsening. They seem related to an immune system disturbance, leading to a vascular leak process.
Published: 2020
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33. A condensate-hardening drug blocks RSV replication in vivo

Author: Jennifer, Risso-Ballester, Marie, Galloux, Jingjing, Cao, Ronan, Le Goffic, Fortune, Hontonnou, Aude, Jobart-Malfait, Aurore, Desquesnes, Svenja M, Sake, Sibylle, Haid, Miaomiao, Du, Xiumei, Zhang, Huanyun, Zhang, Zhaoguo, Wang, Vincent, Rincheval, Youming, Zhang, Thomas, Pietschmann, Jean-François, Eléouët, Marie-Anne, Rameix-Welti, and Ralf, Altmeyer
Subjects: Biomolecular Condensates, Inclusion Bodies, Mice, Inbred BALB C, SARS-CoV-2, viruses, fungi, Veratrum Alkaloids, food and beverages, virus diseases, respiratory system, Virus Replication, Antiviral Agents, Research Highlight, Cell Line, Mice, Viral Proteins, Viral infection, Respiratory Syncytial Virus, Human, Animals, Humans, Female, Lung, Transcription Factors
Abstract: The pathogenicity of SARS-CoV-2 and respiratory syncytial virus (RSV) involves mechanisms of liquid–liquid phase separation, which can be explored as targets for new antiviral therapeutics.
Published: 2020

34. Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry

Author: Gesine Hansen, Jean-François Eléouët, Marie Galloux, Hans Prochnow, Sebastian Blockus, Mark Brönstrup, Thomas Pietschmann, Theresa Graalmann, Sibylle Haid, Zeljka Rupcic, Martin Wetzke, Marina Pils, Ronald Dijkman, Ronan Le Goffic, Bettina Wiegmann, Katharina Rox, Stephan Hüttel, Svenja M. Sake, W. Paul Duprex, Volker Thiel, Marie-Anne Rameix-Welti, Christina Grethe, TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany., Centre for Experimental and Clinical Infection Research (TWINCORE), Helmholtz Centre for Infection Research (HZI)-Medizinische Hochschule Hannover (MHH), Institute for Experimental Virology [Hanovre, Allemagne], Helmholtz Centre for Infection Research (HZI)-Medizinische Hochschule Hannover (MHH)-Helmholtz Centre for Infection Research (HZI)-Medizinische Hochschule Hannover (MHH), Hannover Medical School [Hannover] (MHH), German Center for Infection Research - partner site Hannover-Braunschweig (DZIF), Institute for Experimental Infection Research, Centre for Experimental and Clinical Infection Research [Hanover] (TWINCORE), Helmholtz Centre for Infection Research (HZI), Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Department of Chemical Biology, German Research Centre for Biotechnology, Institute of Virology and Immunology [Mittelhäusern] (IVI), University of Bern, Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), National Emerging Infectious Diseases Laboratories (NEIDL), Boston University [Boston] (BU), Supported by the Innovation Fonds of the Helmholtz Association, by the Pre-4D-Fonds of the Helmholtz Centre for Infection Research, and by the Helmholtz-Alberta Initiative for Infectious Disease Research (HAI-IDR), Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy – EXC 2155 'RESIST' – Project ID 39087428., Clinical leave scholarship from the German Centre for Infection Research (DZIF), Scholarship from the Young Academy of Hannover Medical School, and Swiss National Science Foundation (grants 310030_179260 and 310030_173085)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], 030106 microbiology, Cell, 610 Medicine & health, Respiratory Syncytial Virus Infections, Antiviral Agents, Virus, Virolytic, Cell Line, Labyrinthopeptin, Lanthipeptide, 03 medical and health sciences, Virus entry, Mice, Therapeutic index, Bacteriocins, In vivo, Viral entry, Virology, medicine, Animals, Humans, Antiviral activity, Lung, Cells, Cultured, Pharmacology, Mice, Inbred BALB C, Respiratory tract infections, 630 Agriculture, business.industry, Viral membrane, Virus Internalization, 3. Good health, Human respiratory syncytial virus (hRSV), 030104 developmental biology, medicine.anatomical_structure, Respiratory Syncytial Virus, Human, 570 Life sciences, biology, Female, business, Ex vivo
Abstract: International audience; Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 μM and 4.97 μM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.
Published: 2020
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35. Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

Author: Jennifer Risso-Ballester, Jenna Fix, Marie-Anne Rameix-Welti, Jean-François Eléouët, Charles-Adrien Richard, Marie Galloux, HAL UVSQ, Équipe, Décrypter les synthèses d'ARN par les pneumovirus - - DecRisP2019 - ANR-19-CE11-0017 - AAPG2019 - VALID, Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Ambroise Paré [AP-HP], and ANR-19-CE11-0017,DecRisP,Décrypter les synthèses d'ARN par les pneumovirus(2019)
Subjects: viruses, inprotein-protein interactions, protein-protein interactions, liquid-liquid phase, inclusion bodies, Microbiology, Virus, Inclusion bodies, 03 medical and health sciences, Stress granule, Transcription (biology), [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Virology, Polymerase, 030304 developmental biology, nucleoprotein, Inclusion, Bodiesliquid-liquid phase, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0303 health sciences, biology, Chemistry, 030302 biochemistry & molecular biology, RNA, RSV, phosphoprotein, QR1-502, 3. Good health, Cell biology, Nucleoprotein, phosphoprote, Phosphoprotein, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, biology.protein, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases
Abstract: Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.
Published: 2020
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36. De novo protein design enables the precise induction of RSV-neutralizing antibodies

Author: Sean Ervin, Jean-François Eléouët, Sabrina Vollers, Marie-Anne Rameix-Welti, Marie Galloux, Stéphane Rosset, Jean-Philippe Julien, Xiaolin Wen, Yuxing Li, Johannes T. Cramer, Che Yang, Jaume Bonet, Thomas Krey, Theodore S. Jardetzky, Patricia Corthésy, Yimeng Wang, Sabine Riffault, Chi-I Chiang, Iga Kucharska, Delphyne Descamps, Elie Dheilly, Sandrine Georgeon, Giacomo Castoro, Mélanie Villard, Charles-Adrien Richard, Teresa C. Delgado, Fabian Sesterhenn, Elisa Oricchio, Vicente Mas, Luciano A. Abriata, Bruno E. Correia, John T. Bates, Instituto de Salud Carlos III, European Research Council, Swiss National Science Foundation, Stavros Niarchos Foundation, EPFL Postdoctoral Fellows, German Center for Infection Research (Alemania), Deutsche Forschungsgemeinschaft (Alemania), Federal Ministry of Education & Research (Alemania), Canada Research Chairs, NIH - National Institute of Allergy and Infectious Diseases (NIAID) (Estados Unidos), Ecole Polytechnique Fédérale de Lausanne (EPFL), Swiss Institute of Bioinformatics [Lausanne] (SIB), Université de Lausanne (UNIL), Hannover Medical School [Hannover] (MHH), Stanford School of Medicine [Stanford], Stanford Medicine, Stanford University-Stanford University, University of Maryland [Baltimore], The Hospital for sick children [Toronto] (SickKids), Department of Biochemistry [University of Toronto], University of Toronto, Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Swiss Institute for Experimental Cancer Research - Lausanne (ISREC), Swiss Institute for Experimental Cancer Research, Centro Nacional de Microbiología [ISCIII, Madrid, Spain] (CNM), Instituto de Salud Carlos III [Madrid] (ISC), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Wake Forest Baptist Medical Center, University of Mississippi Medical Center (UMMC), University of Maryland School of Medicine, University of Maryland System, German Center for Infection Research - partner site Hannover-Braunschweig (DZIF), University of Luebeck, INSTITUT FUR ANGEWANDTE BODENBIOLOGIE GMBH HAMBURG DEU, Partenaires IRSTEA, Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA)-Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA), European Project: 716058,DeNovoImmunoDesign, Université de Lausanne = University of Lausanne (UNIL), and European Project: 716058,EXCELLENT SCIENCE - European Research Council (ERC),ERC-2016-STG - ERC Starting Grant,DeNovoImmunoDesign(2017)
Subjects: Immunogen, medicine.drug_class, Protein Conformation, Recombinant Fusion Proteins, Protein design, Amino Acid Motifs, computational design, Computational biology, Biology, Monoclonal antibody, Protein Engineering, Epitope, Article, Affinity maturation, 03 medical and health sciences, 0302 clinical medicine, Antigen, backbone, medicine, Respiratory Syncytial Virus Vaccines, Humans, b-cells, 030304 developmental biology, affinity maturation, 0303 health sciences, epitope, Multidisciplinary, dengue virus, Immunodominant Epitopes, Computational Biology, fusion-glycoprotein vaccine, Single-Domain Antibodies, [SHS.ECO]Humanities and Social Sciences/Economics and Finance, Fusion protein, Antibodies, Neutralizing, 3. Good health, nmr structure determination, Respiratory Syncytial Virus, Human, biology.protein, potent, Antibody, influenza, 030217 neurology & neurosurgery
Abstract: De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs. This work was supported by the swiss initiative for systems biology (SystemsX.ch), the European Research Council (Starting grant - 716058), the Swiss National Science Foundation (310030_163139) and the EPFL’s Catalyze4Life initiative. F.S. was supported by an SNF/Innosuisse BRIDGE Proof-of-Concept grant. J.B. was supported by the EPFL Fellows postdoctoral fellowship. T.K. received funding from the German Center of Infection Research (DZIF) and the Cluster of Excellence RESIST (EXC 2155) of the German Research foundation. J.T.C. was funded by the ERA-Net PrionImmunity project 01GM1503 (Federal Ministry of Education and Research, Germany). V.M. received funding from “AESI-18” (Instituto de Salud Carlos III), grant MPY 375/18. J.PJ. was funded by the Canada Research Chairs program (J.P.J.), T.J. and X.W. were funded by the NIH NIAID (R01 AI137523). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí
Published: 2020
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37. Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

Author: Elena Rensen, Stefano Pietropaoli, Florian Mueller, Christian Weber, Sylvie Souquere, Sina Sommer, Pierre Isnard, Marion Rabant, Jean-Baptiste Gibier, Fabiola Terzi, Etienne Simon-Loriere, Marie-Anne Rameix-Welti, Gérard Pierron, Giovanna Barba-Spaeth, Christophe Zimmer, Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), National Cystic Fibrosis Reference Center [CHU Necker] (CNR - INSERM U1151), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Lille, Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Ambroise Paré [AP-HP], Institut Gustave Roussy (IGR), Virologie Structurale - Structural Virology, E Simon-Loriere acknowledges funding from the French Government’s Investissement d’Avenir program, 'INCEPTION’ (ANR-16-CONV-0005). G Barba-Spaeth acknowledges funding by the Institut Pasteur Coronavirus task force (don AXA COVID-19 project COVID-SPREAD). C Zimmer acknowledges funding by Fondation pour la Recherche Médicale (Equipe FRM, DEQ 20150331762), Institut Carnot Pasteur MS, and Institut Pasteur., We would like to thank Edouard Bertrand, who originally developed smiFISH and Hans Johansson for insightful discussions. We also thank Guillaume Dumenil (Ultrastructural Bioimaging UTechS of Institut Pasteur) for having suggested the application of CoronaFISH to EM and for follow-up discussions. We thank Nathalie Jolly and Nathalie Clément (Center for Translational Science, Institut Pasteur) and Cloé Giquel (Legal department, Institut Pasteur) for their help in obtaining authorization to use patient samples., ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016), Barba Spaeth, Giovanna, Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs - - INCEPTION2016 - ANR-16-CONV-0005 - CONV - VALID, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), Département Croissance et Signalisation [Paris], Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Pasteur [Paris], and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)
Subjects: [SDV]Life Sciences [q-bio], viruses, Health, Toxicology and Mutagenesis, Plant Science, Virus Replication, Antiviral Agents, Sensitivity and Specificity, Biochemistry, Genetics and Molecular Biology (miscellaneous), 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, Chlorocebus aethiops, Animals, Humans, Vero Cells, In Situ Hybridization, In Situ Hybridization, Fluorescence, Virus Release, Research Articles, 030304 developmental biology, 0303 health sciences, Alanine, Ecology, SARS-CoV-2, COVID-19, Reproducibility of Results, respiratory system, Adenosine Monophosphate, respiratory tract diseases, COVID-19 Drug Treatment, 3. Good health, [SDV] Life Sciences [q-bio], Microscopy, Electron, RNA, Viral, Caco-2 Cells, 030217 neurology & neurosurgery, Research Article
Abstract: We design an RNA-FISH method to target the plus and minus strands of SARS-CoV-2 RNA, and apply it to image the virus in cell lines, lung tissue, and nasal swabs, by fluorescence and electron microscopy., The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.
Published: 2022
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38. RSV N-nanorings fused to palivizumab-targeted neutralizing epitope as a nanoparticle RSV vaccine

Author: Charlotte Deloizy, Jenna Fix, Sabine Riffault, Jean-François Eléouët, Marie-Anne Rameix-Welti, Delphyne Descamps, Pierre-Louis Hervé, Jason S. McLellan, Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Université Paris Saclay (COmUE), DBV Technologies, GENOSAFE, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Laboratoire de Microbiologie, Hôpital Raymond Poincareé, Geisel School of Medicine, Partenaires INRAE, Hervé, Pierre Louis, Eléouët, Jean Francois, and Riffault, Sabine
Subjects: 0301 basic medicine, Cellular immunity, respiratory syncytial virus, viruses, Pharmaceutical Science, Medicine (miscellaneous), Antibodies, Viral, epitobe-focused vaccine, Epitope, Epitopes, Mice, 0302 clinical medicine, Medicine, General Materials Science, Neutralizing antibody, biology, Respiratory Syncytial Viruses, 3. Good health, Vaccinology, 030220 oncology & carcinogenesis, Molecular Medicine, medicine.drug, Palivizumab, Biomedical Engineering, Bioengineering, Respiratory Syncytial Virus Infections, Article, Virus, 03 medical and health sciences, Respiratory Syncytial Virus Vaccines, Animals, Humans, nucleoprotein nanoparticles, mucosal vaccine, F glycoprotein, Sigmodontinae, business.industry, Antibodies, Neutralizing, Virology, 030104 developmental biology, Immunization, Vaccinologie, Immunology, biology.protein, Nanoparticles, [SDV.IMM.VAC]Life Sciences [q-bio]/Immunology/Vaccinology, business, Viral Fusion Proteins, Conformational epitope
Abstract: Respiratory syncytial virus (RSV) is the leading cause of acute respiratory infections in children, yet no vaccine is available. The sole licensed preventive treatment against RSV is composed of a monoclonal neutralizing antibody (palivizumab), which targets a conformational epitope located on the fusion protein (F). Palivizumab reduces the burden of bronchiolitis but does not prevent infection. Thus, the development of RSV vaccines remains a priority.We previously evaluated nanorings formed by RSV nucleoprotein (N) as an RSV vaccine, as well as an immunostimulatory carrier for heterologous antigens. Here we linked the palivizumab-targeted epitope (called FsII) to N, to generate N-FsII-nanorings. Intranasal N-FsII immunization elicited anti-F antibodies in mice that were non-neutralizing in vitro. Nevertheless, RSV-challenged animals were better protected against virus replication than mice immunized with N-nanorings, especially in the upper airways. In conclusion, an N-FsII-focused vaccine is an attractive candidate combining N-specific cellular immunity and F-specific antibodies for protection
Published: 2017
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39. A small fragmented P protein of respiratory syncytial virus inhibits virus infection by targeting P protein

Author: Hiroshi Watanabe, Jean-François Eléouët, Takahito Kashiwagi, Kenichiro Yaita, Kattareeya Kumthip, Marie-Anne Rameix-Welti, Koyu Hara, Pattara Khamrin, Serva, Camille, Kurume University School of Medicine, Kurume University, Chidoribashi General Hospital, Chiang Mai University (CMU), Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Ambroise Paré [AP-HP], Promotion of Science and the Ministry of Education, Science, Sports and Culture, Japan, Grant- in- Aid for Scientific Research (C), 15K09672 (2015–2017) and 18K07834 (2018–2020), Ishibashi Foundation for the promotion of Science, Astellas Pharma, Daiichi Sankyo, SD K. K., Pfizer Japan, and Takeda Pharmaceutical
Subjects: 0301 basic medicine, viruses, respiratory syncytial virus, 030106 microbiology, Peptide, Biology, Antiviral Agents, Virus, law.invention, Cell Line, 03 medical and health sciences, Viral Proteins, law, Virology, Cricetinae, Animals, Humans, Amino Acid Sequence, Nucleocapsid, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Polymerase, Ribonucleoprotein, chemistry.chemical_classification, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Viral matrix protein, peptide inhibitors, phosphoprotein, Phosphoproteins, Nucleoprotein, polymerase, Protein Transport, 030104 developmental biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Nucleoproteins, chemistry, Ribonucleoproteins, Virus Diseases, Phosphoprotein, Respiratory Syncytial Virus, Human, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Recombinant DNA, biology.protein, RNP complex
Abstract: International audience; Peptide-based inhibitors hold promising potential in the development of antiviral therapy. Here, we investigated the antiviral potential of fragmented viral proteins derived from ribonucleoprotein (RNP) components of the human respiratory syncytial virus (HRSV). Based on a mimicking approach that targets the functional domains of viral proteins, we designed various fragments of nucleoprotein (N), matrix protein M2-1 and phosphoprotein (P) and tested the antiviral activity in an RSV mini-genome system. We found that the fragment comprising residues 130–180 and 212–241 in the C-terminal region of P (81 amino acid length), denoted as P Fr, significantly inhibited the polymerase activity through competitive binding to the full-length P. Further deletion analysis of P Fr suggested that three functional domains in P Fr (oligomerization, L-binding and nucleocapsid binding) are required for maximum inhibitory activity. More importantly, a purified recombinant P Fr displayed significant antiviral activity at low nanomolar range in RSV-infected HEp-2 cells. These results highlight P as an important target for the development of antiviral compounds against RSV and other paramyxoviruses.
Published: 2019
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40. Respiratory syncytial virus tropism for olfactory sensory neurons in mice

Author: Maxence Frétaud, Laura Sedano, Jean-François Eléouët, Delphyne Descamps, Christelle Langevin, Bertrand Bryche, Marie Galloux, Nicolas Meunier, Audrey Saint-Albin Deliot, Ronan Le Goffic, Marie-Anne Rameix-Welti, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Neurobiologie de l'Olfaction et de la Prise Alimentaire (NOPA), Institut National de la Recherche Agronomique (INRA), Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme d'Infectiologie Expérimentale des Rongeurs et Poissons (IERP (UE 0907)), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Versailles Saint-Quentin-en-Yvelines - UFR Sciences de la santé Simone Veil (UVSQ Santé), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Microbiologie et Hygiène [AP-HP Hôpital Ambroise-Paré], Hôpital Ambroise Paré [AP-HP], and Region Ile-de-France
Subjects: 0301 basic medicine, Nasal cavity, Olfactory system, Central Nervous System, Central nervous system, Sensory system, Olfaction, Biology, Virus Replication, Biochemistry, Tropism, odorant binding protein, Olfactory Receptor Neurons, 03 medical and health sciences, Cellular and Molecular Neuroscience, Olfactory mucosa, Mice, 0302 clinical medicine, Imaging, Three-Dimensional, Olfactory Mucosa, Central Nervous System Diseases, medicine, Animals, Steno's gland, Mice, Inbred BALB C, neurotropism, nasal cavity, Olfactory Bulb, 3. Good health, Olfactory bulb, Respiratory Syncytial Viruses, [SDV.GEN.GA]Life Sciences [q-bio]/Genetics/Animal genetics, Nasal Mucosa, 030104 developmental biology, medicine.anatomical_structure, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, RNA, Viral, Female, olfactory epithelium, Neuroscience, Olfactory epithelium, Head, 030217 neurology & neurosurgery, olfaction
Abstract: The olfactory mucosa, where the first step of odor detection occurs, is a privileged pathway for environmental toxicants and pathogens toward the central nervous system. Indeed, some pathogens can infect olfactory sensory neurons including their axons projecting to the olfactory bulb allowing them to bypass the blood-brain barrier and reach the central nervous system (CNS) through the so-called olfactory pathway. The respiratory syncytial virus (RSV) is a major respiratory tract pathogen but there is growing evidence that RSV may lead to CNS impairments. However, the mechanisms involved in RSV entering into the CNS have been poorly described. In this study, we wanted to explore the capacity of RSV to reach the CNS via the olfactory pathway and to better characterize RSV cellular tropism in the nasal cavity. We first explored the distribution of RSV infectious sites in the nasal cavity by in vivo bioluminescence imaging and a tissue clearing protocol combined with deep-tissue imaging and 3D image analyses. This whole tissue characterization was confirmed with immunohistochemistry and molecular biology approaches. Together, our results provide a novel 3D atlas of mouse nasal cavity anatomy and show that RSV can infect olfactory sensory neurons giving access to the central nervous system by entering the olfactory bulb. Cover Image for this issue: doi: 10.1111/jnc.14765.
Published: 2019
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41. High-throughput screening of active compounds against human respiratory syncytial virus

Author: Yue-Ying Jiao, Marie-Anne Rameix-Welti, Xiang-Lei Peng, Yuan-Hui Fu, Nan Jiang, Zhu-Xin Xu, Shan Cen, Jean-François Eléouët, Ye Wang, Jin-Sheng He, Yan-Peng Zheng, Beijing Jiaotong University (BJTU), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Hôpital Ambroise Paré [AP-HP], Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Chinese Academy of Medical Sciences, National Major Scientific and Technological Special Project for 'AIDS and Viral Hepatitis and Other Major Infectious Diseases Prevention and Control' during the Twelfth Five-year Plan Period [2013ZX10004601], Natural Science Foundation of ChinaNational Natural Science Foundation of China [81771777], National Mega -Project for Infectious Disease [2018ZX10301408], and CAMS Innovation Fund for Medical Sciences [2018-I2M-3-004]
Subjects: [SDV]Life Sciences [q-bio], viruses, High-throughput screening, Green Fluorescent Proteins, Drug Evaluation, Preclinical, Biology, rsv inhibitor, high-throughput screening, Antiviral Agents, Virus, law.invention, Green fluorescent protein, 03 medical and health sciences, law, In vivo, Transcription (biology), Genes, Reporter, Virology, Lower respiratory tract infection, medicine, Humans, 6-mercaptopurine, IC50, 030304 developmental biology, 0303 health sciences, azathioprine, Staining and Labeling, 030302 biochemistry & molecular biology, virus diseases, respiratory system, medicine.disease, Recombinant Proteins, 3. Good health, High-Throughput Screening Assays, Respiratory Syncytial Virus, Human, Recombinant DNA, human respiratory syncytial virus, HeLa Cells
Abstract: International audience; Human respiratory syncytial virus (RSV) is one of the predominant pathogens causing lower respiratory tract infection in infants and young children worldwide, whereas there is so far no vaccine or drug against RSV infection for clinical use. In this work, we developed and validated a fluorescence-based high-throughput screening (HTS) assay to identify compounds active against RSV, using RSV-mGFP, a recombinant RSV encoding enhanced green fluorescent protein (EGFP). Thereafter, among 54,800 compounds used for our screen, we obtained 62 compounds active against RSV. Among these hits, azathioprine (AZA) and 6-mercaptopurine (6-MP) were identified as RSV inhibitors with half maximal inhibitory concentration (IC50) values of 6.69 +/- 1.41 and 3.13 +/- 0.98 mu M respectively. Further experiments revealed that they functioned by targeting virus transcription or/and genome replication. In conclusion, the established HTS assay is suitable to screen anti-RSV compounds, and the screened two hits of AZA and 6-MP, as potential anti-RSV agents targeting RSV genome replication/transcription, are worthy of further investigation on their anti-RSV activity in vivo.
Published: 2019
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42. Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen

Author: Barney S. Graham, Jean-François Eléouët, Patricia Corthésy, Bruno E. Correia, Pablo Gainza, Che Yang, Fabian Sesterhenn, Marie Galloux, Marie-Anne Rameix-Welti, Sabrina Vollers, Stéphane Rosset, Delphyne Descamps, Jaume Bonet, Sabine Riffault, Man Chen, Simon Friedensohn, Sai T. Reddy, Lucia Csepregi, Institute of Bioengineering [Lausanne, Suisse], Ecole Polytechnique Fédérale de Lausanne (EPFL), Swiss Institute of Bioinformatics (SIB), Swiss Institute of Bioinformatics, Molecular Immunology and Virology (VIM-UR892), Institut National de la Recherche Agronomique (INRA), Department of Biosystems Science and Engineering [ETH Zürich] (D-BSSE), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich), Viral Pathogenesis Laboratory [Bethesda, MD, États-Unis], Vaccine Research Center [Bethesda, MD, États-Unis], National Institute of Allergy and Infectious Diseases [Bethesda] (NIAID-NIH), National Institutes of Health [Bethesda] (NIH)-National Institutes of Health [Bethesda] (NIH)-National Institute of Allergy and Infectious Diseases [Bethesda] (NIAID-NIH), National Institutes of Health [Bethesda] (NIH)-National Institutes of Health [Bethesda] (NIH), Infection et inflammation chronique (2I), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Laboratoire de Microbiologie [Boulogne-Billancourt], Hôpital Ambroise Paré, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), FS was supported by the Swiss initiative for systems biology (SystemsX.ch). BEC was supported by the European Research Council (Starting grant - 716058) and the Swiss National Science Foundation (Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, 310030_163139)., Bodescot, Myriam, Swiss Institute of Bioinformatics [Lausanne] (SIB), Université de Lausanne = University of Lausanne (UNIL), Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Ambroise Paré [AP-HP], and Université de Lausanne (UNIL)
Subjects: 0301 basic medicine, RNA viruses, rsv antibody, Immunogen, Physiology, Gene Expression, Antibody Response, hiv, dependent enhancement, Antibodies, Viral, Pathology and Laboratory Medicine, Biochemistry, Epitope, Epitopes, Mice, Immunologic Adjuvants, 0302 clinical medicine, Immunogenicity, Vaccine, Immune Physiology, Medicine and Health Sciences, Public and Occupational Health, Biology (General), Cloning, Molecular, Enzyme-Linked Immunoassays, Neutralizing antibody, Immune Response, 0303 health sciences, hemagglutinin-stem, Mice, Inbred BALB C, Immune System Proteins, biology, General Neuroscience, Immunogenicity, respiratory syncytial virus, fusion-glycoprotein vaccine, structural basis, dengue virus, infection, cells, Vaccination and Immunization, 3. Good health, Respiratory Syncytial Viruses, Vaccination, Medical Microbiology, Viral Pathogens, Viruses, [SDV.IMM]Life Sciences [q-bio]/Immunology, Computer-Aided Design, Female, Antibody, Pathogens, General Agricultural and Biological Sciences, medicine.drug, Research Article, Palivizumab, Subdominant, [SDV.IMM] Life Sciences [q-bio]/Immunology, QH301-705.5, Recombinant Fusion Proteins, Genetic Vectors, Immunology, Receptors, Antigen, B-Cell, Immunodominance, Antibodies, Monoclonal, Humanized, Research and Analysis Methods, Microbiology, General Biochemistry, Genetics and Molecular Biology, Antibodies, 03 medical and health sciences, Antigen, Vaccine Development, medicine, Escherichia coli, Respiratory Syncytial Virus Vaccines, Animals, Antigens, Immunoassays, Microbial Pathogens, 030304 developmental biology, General Immunology and Microbiology, Organisms, Biology and Life Sciences, Proteins, Virology, Antibodies, Neutralizing, 030104 developmental biology, Structural Homology, Protein, Paramyxoviruses, biology.protein, Immunologic Techniques, Nanoparticles, Immunization, Respiratory Syncytial Virus, Preventive Medicine, Viral Fusion Proteins, 030217 neurology & neurosurgery
Abstract: Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies., A computationally designed epitope-focused immunogen presenting a single neutralization epitope from Respiratory Syncytial Virus elicits superior epitope-specific responses compared to the viral fusion protein. Furthermore, epitope-focused immunogens can reshape established antibody hierarchies., Author summary Vaccines are one of the most valuable instruments to prevent and control infectious diseases. Their primary correlate of protection is the level of induction of neutralizing antibodies that target critical antigenic sites and thereby block infection. Natural infections with pathogens such as the respiratory syncytial virus (RSV) or influenza induce a broad repertoire of antibodies that target multiple epitopes. Among those, functional antibodies with key specificities are often subdominant (present in low titers). Thus, a central goal for vaccine development is to focus antibody responses on such neutralization epitopes. Here, we show that a computationally designed, epitope-focused immunogen mimicking an important RSV neutralization epitope (site II) can focus antibodies onto this well-defined epitope. In a scenario of preexisting immunity, in which site II–specific antibodies were subdominant, the epitope-focused immunogen selectively boosted site II–specific antibodies, resulting in an increased viral neutralization through this epitope. We propose that rationally designed immunogens spotlighting defined epitopes have a unique potential to focus antibody responses on functionally conserved sites in cases of preexisting immunity. Our results have broad implications for vaccine design as a strategy to steer preexisting antibody responses away from immunodominant, variable epitopes and toward subdominant epitopes that confer broad and potent neutralization.
Published: 2019
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43. Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism

Author: Barbara Sanavio, Yanxiao Han, Marie Anne Rameix Welti, Chiara Martinelli, Manuela Donalisio, Patrizia Andreozzi, Marta Vallino, Ahmet Bekdemir, Samuel T. Jones, Laurent Kaiser, Caroline Tapparel, Marie Galloux, Emma Rose Janecek, Francesco Stellacci, Petr Král, Jan Weber, Jean-François Eléouët, Ronan Le Goffic, Silke Krol, Paulo Jacob Silva, David Lembo, Marco D'Alicarnasso, Jan Hodek, Valeria Cagno, Lela Vuković, Soumyo Sen, Marie Mueller, Università degli studi di Torino (UNITO), Institute of Materials, Ruhr-Universität Bochum [Bochum], Université de Genève (UNIGE), IFOM - FIRC, Centro de Investigación Cooperativa en Biomateriales (CIC biomaGUNE), Fondazione CEN, Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Université Paris Saclay (COmUE), University of Manchester [Manchester], Istituto per la Protezione Sostenibile delle Piante (CNR), Czech Academy of Sciences [Prague] (CAS), University of Illinois, University of Illinois System, Fondazione IRCCS Istituto Neurologico 'Carlo Besta', Laboratoire de Microbiologie, Hôpital Raymond Poincareé, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Hôpitaux Universitaires de Genève (HUG), University of Texas, IRCCS Ist Tumori Giovanni Paolo II, Bari, Italy, Partenaires INRAE, Fdn IRCCS Ist Neurol Carlo Besta, IFOM IEO Campus, Milan, Italy, Ecole Polytechnique Fédérale de Lausanne (EPFL), Swiss National Science Foundation [NRP 64], NCCR on bio-inspired materials, University of Turin, Ministry of Education, Youth and Sports of the Czech Republic [LK11207], Leenaards Foundation, NSF [DMR-1506886], UTEP, Ile de France region (SESAME), European Project: 646364,H2020,H2020-NMP-PILOTS-2014,NANOFACTURING(2015), Università degli studi di Torino = University of Turin (UNITO), Université de Genève = University of Geneva (UNIGE), CNR Istituto per la Protezione Sostenibile delle Piante [Torino, Italia] (IPSP), and National Research Council of Italy | Consiglio Nazionale delle Ricerche (CNR)
Subjects: Drug, media_common.quotation_subject, [SDV]Life Sciences [q-bio], Herpesvirus 2, Human, viruses, 02 engineering and technology, Respiratory Syncytial Virus Infections, 010402 general chemistry, medicine.disease_cause, 01 natural sciences, Antiviral Agents, Virus, Dengue fever, antiviral drugs, Mice, human papilloma virus, In vivo, Biomimetic Materials, medicine, Animals, Humans, General Materials Science, broad-spectrum, Cytotoxicity, media_common, ddc:616, Mice, Inbred BALB C, Chemistry, Mechanical Engineering, Herpes Simplex, General Chemistry, self-assembly, herpes simplex virus, 021001 nanoscience & nanotechnology, Condensed Matter Physics, medicine.disease, Virology, antiviral, drug development, In vitro, 3. Good health, 0104 chemical sciences, Respiratory Syncytial Viruses, Herpes simplex virus, Mechanics of Materials, Nanoparticles, 0210 nano-technology, Ex vivo, Heparan Sulfate Proteoglycans
Abstract: International audience; Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (similar to 190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.
Published: 2018
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44. Labyrinthopeptin A1 and A2 efficiently inhibit cell entry of hRSV isolates

Author: Gesine Hansen, Jean-François Eléouët, Volker Thiel, Hans Prochnow, Sebastian Blockus, Thomas Pietschmann, Svenja M. Wiechert, Mark Brönstrup, Ronald Dijkman, Bettina Wiegmann, Martin Wetzke, Paul Duprex, Marie-Anne Rameix-Welti, and Sibylle Haid
Subjects: 0301 basic medicine, Cell entry, Palivizumab, business.industry, Cell, Treatment options, Lantibiotics, Virology, Virus, 03 medical and health sciences, 030104 developmental biology, medicine.anatomical_structure, Ic50 values, Medicine, business, Cytotoxicity, medicine.drug
Abstract: Introduction: Human respiratory syncytial virus (hRSV) is the most common cause of lower respiratory tract disease in infants. Due to the lack of treatment options there is a need for new antiviral drugs. Objective: We aimed to identify small molecules with antiviral activity against hRSV and to investigate the mode-of-action of selected compounds. Methods: A cell-based high-throughput screening system was used to screen a compound library. Time-of-drug-addition, lentiviral hRSV pseudotype assays, infection competition and virus dilution assays were conducted to explore the mode of action of lead compounds. Results: The two lantibiotics Labyrinthopeptin A1 and A2 emerged as potent inhibitors of clinical hRSV strains. IC50 values were in the low micromolar range without cytotoxicity up to a 100-fold higher dosage. The antiviral effect could be shown in carcinoma derived lung cells as wells as in physiologically relevant air-liquid interphase (ALI) cultures involving primary human lung epithelial cells. Application during virus inoculation and therapeutic administration of compounds after hRSV infection reduced virus load in the ALI cultures. Moreover, laboratory and clinical strains of hRSV were inhibited. In addition, lentiviral hRSV pseudotypes with or without resistance mutations against palivizumab or fusion inhibitors in clinical development were susceptible to labyrinthopeptin A1 and A2. Time-of-drug-addition as well as the pseudotype assays indicated that both compounds inhibit cell entry of hRSV. Conclusion: Labyrinthopeptin A1 and A2 exert broad antiviral activity against hRSV subtype A and B strains. Thus, these molecules are candidates for further development as antivirals for treatment of hRSV infections.
Published: 2017
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45. Respiratory Syncytial Virus Infects Regulatory B Cells in Human Neonates via Chemokine Receptor CX3CR1 and Promotes Lung Disease Severity

Author: Pierre Tissieres, Benoit Beitz, Jean-François Eléouët, Maryline M Ripaux-Lefevre, Delphyne Descamps, Fabrice Porcheray, Marie-Anne Rameix-Welti, Sébastien Lemoine, Nicoletta Casartelli, Jenna Fix, Richard Lo-Man, Valérie Lorin, Sabine Riffault, Ahsen Morva, Hugo Mouquet, Pierre-Louis Hervé, Françoise Mascart, Liliane Schandené, Jordi Miatello, Dania Zhivaki, Pierre-Olivier Vidalain, Edith Dériaud, Brigitte Lemercier, Xiaoming X Zhang, Olivier Schwartz, Annick Lim, Histopathologie humaine et Modèles animaux, Institut Pasteur [Paris], Université Paris Diderot - Paris 7 (UPD7), Régulation Immunitaire et Vaccinologie, Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Département d'Immunologie - Department of Immunology, Génomique virale et vaccination, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Immunobiology Clinic, Hôpital Erasme [Bruxelles] (ULB), Faculté de Médecine [Bruxelles] (ULB), Université libre de Bruxelles (ULB)-Université libre de Bruxelles (ULB)-Faculté de Médecine [Bruxelles] (ULB), Université libre de Bruxelles (ULB)-Université libre de Bruxelles (ULB), Virus et Immunité - Virus and immunity, Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Laboratoire de Microbiologie, Hôpital Raymond Poincareé, Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Microbiology Technology Institute, BIOASTER Microbiology Technology Institute [Lyon], School of Medicine, University of Patras [Greece], UMR9196, Physiologie et Pathologie Moléculaires des Rétrovirus Endogènes et Infectieux, Centre National de la Recherche Scientifique (CNRS), APHP, Pediatric ICU and Neonatal Medicine, Hôpitaux Universitaires Paris Sud, Réponse humorale aux pathogènes, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Vaccinology and Mucosal Immunity, Université libre de Bruxelles (ULB), Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP), This work was supported by an ANR grant (ANR 13-BSV3-0016) and by the Fondation pour la Recherche Médicale (grant no. DEQ20120323719). This study also received funding from the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant no. ANR-10-LABX-62-IBEID). X.Z. and S.L. were supported by ANR and by the European Commission FP7 ADITEC program (HEALTH-F4-2011-280873). X.Z. was also partially supported by the Major Basic Research Project of Shanghai Science and Technology Commission (no. 13JC1405600), National Natural Science Foundation of China (31270961, 31470879), Interdisciplinary Innovation Team and External Cooperation Program (no. GJHZ201312), and Chinese Academy of Sciences. D.Z. was supported by DIM Malinf of Region IdF. Work in the O.S. lab is also supported by ANRS, Sidaction and Fondation Areva. We acknowledge the Center for Human Immunology at Institut Pasteur for support in conducting these studies., We are very grateful to T. Domet (Therapie Cellulaire, Hopital Saint-Louis) for the cord blood sample collection and M. Lucas-Hourani for some viral preparation. We thank F. Huetz for helpful discussion about B lymphocytes., ANR-13-BSV3-0016,SyncBreg,Signature moléculaire et fonction des lymphocytes B régulateurs dans l'infection VRS(2013), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 280873,EC:FP7:HEALTH,FP7-HEALTH-2011-single-stage,ADITEC(2011), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Virus et Immunité - Virus and immunity (CNRS-UMR3569), University of Patras, Physiologie et physiopathologie des rétrovirus endogènes et infectieux (RETRO-ENDO), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Hôpitaux Universitaires Paris Sud [AP-HP] (HUPS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Hôpital Erasme, Virus et Immunité, Infection et inflammation chronique (2I), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Unité de recherche Virologie et Immunologie Moléculaires (VIM), BIOASTER, Université Libre de Bruxelles [Bruxelles] (ULB), ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), and University of Patras [Patras]
Subjects: CD4-Positive T-Lymphocytes, 0301 basic medicine, Enzyme-Linked Immunospot Assay, Regulatory B cells, respiratory syncytial virus, [SDV]Life Sciences [q-bio], Immunology, B-cell receptor, CX3C Chemokine Receptor 1, Enzyme-Linked Immunosorbent Assay, Respiratory Syncytial Virus Infections, Biology, Lymphocyte Activation, Article, 03 medical and health sciences, Chemokine receptor, 0302 clinical medicine, newborn, CX3CR1, medicine, Bronchiolitis, Viral, Humans, Immunology and Allergy, B cell, Oligonucleotide Array Sequence Analysis, B-Lymphocytes, Regulatory, Respiratory tract infections, Gene Expression Profiling, Infant, Newborn, medicine.disease, Virology, Respiratory Syncytial Viruses, 3. Good health, Breg cell, Interleukin 10, 030104 developmental biology, Infectious Diseases, medicine.anatomical_structure, Bronchiolitis, 030220 oncology & carcinogenesis, [SDV.IMM]Life Sciences [q-bio]/Immunology, Receptors, Chemokine, bronchiolitis, Transcriptome
Abstract: International audience; Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.
Published: 2017
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46. A short double-stapled peptide inhibits respiratory syncytial virus entry and spreading

Author: Geneviève Mottet-Osman, Origène Nyanguile, Elodie Baechler, Miriam Arrell, Abdelhak Boukadiri, Ronan Le Goffic, Marie Galloux, Julien Héritier, Thibaut Larcher, Marie-Anne Rameix-Welti, Jean-Manuel Segura, Vanessa Gaillard, Dominique Garcin, Jean-François Eléouët, Haute École Spécialisée de Suisse occidentale Valais (HES-SO Valais-Wallis), Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Department of Microbiology and Molecular Medicine, University of Geneva [Switzerland], Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Laboratoire de Microbiologie, Hôpital Raymond Poincareé, Université de Versailles Saint-Quentin-en-Yvelines - UFR Sciences de la santé Simone Veil (UVSQ Santé), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Génétique Animale et Biologie Intégrative (GABI), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Commission for Technology and Innovation CTI [12.868.1], Swiss Confederation, Unité de recherche Virologie et Immunologie Moléculaires (VIM), Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, and UFR des Sciences de la Santé Simone Veil
Subjects: 0301 basic medicine, Protein Conformation, alpha-Helical, viruses, respiratory syncytial virus, [SDV]Life Sciences [q-bio], Peptide, Plasma protein binding, Respiratory syncytial virus, stapled peptides, Virus Replication, respiratory syncytial viruses, Mice, Pharmacology (medical), Peptide sequence, chemistry.chemical_classification, ddc:616, Mice, Inbred BALB C, Protein Stability, virus respiratoire syncytial, 3. Good health, Amino acid, Stapled peptides, Infectious Diseases, Female, Protein Binding, Respiratory Syncytial Virus Infections, Biology, Antiviral Agents, Virus, 03 medical and health sciences, Viral entry, Animals, Humans, Protein Interaction Domains and Motifs, Amino Acid Sequence, Administration, Intranasal, Pharmacology, Binding Sites, Sequence Homology, Amino Acid, Virus Internalization, Virology, Heptad repeat, 030104 developmental biology, chemistry, Viral replication, Amino Acid Substitution, Respiratory Syncytial Virus, Human, Proteolysis, Peptides, Sequence Alignment, Viral Fusion Proteins, HeLa Cells
Abstract: Synthetic peptides derived from the heptad repeat (HR) of fusion (F) proteins can be used as dominant negative inhibitors to inhibit the fusion mechanism of class I viral F proteins. Here, we have performed a stapled-peptide scan across the HR2 domain of the respiratory syncytial virus (RSV) F protein with the aim to identify a minimal domain capable of disrupting the formation of the postfusion six-helix bundle required for viral cell entry. Constraining the peptides with a single staple was not sufficient to inhibit RSV infection. However, the insertion of double staples led to the identification of novel short stapled peptides that display nanomolar potency in HEp-2 cells and are exceptionally robust to proteolytic degradation. By replacing each amino acid of the peptides by an alanine, we found that the substitution of residues 506 to 509, located in a patch of polar contacts between HR2 and HR1, severely affected inhibition. Finally, we show that intranasal delivery of the most potent peptide to BALB/c mice significantly decreased RSV infection in upper and lower respiratory tracts. The discovery of this minimal HR2 sequence as a means for inhibition of RSV infection provides the basis for further medicinal chemistry efforts toward developing RSV fusion antivirals.
Published: 2017
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47. Host Restriction of Avian Influenza Viruses at the Level of the Ribonucleoproteins

Author: Andru Tomoiu, Nadia Naffakh, Marie-Anne Rameix-Welti, and Sylvie van der Werf
Subjects: Models, Molecular, Genes, Viral, Orthomyxoviridae, Reassortment, RNA-dependent RNA polymerase, Context (language use), medicine.disease_cause, Microbiology, Virus, Evolution, Molecular, Viral Proteins, medicine, Animals, Humans, Phylogeny, Ribonucleoprotein, Genetics, biology, biology.organism_classification, Virology, Influenza A virus subtype H5N1, Nucleoprotein, Ribonucleoproteins, Influenza A virus, Host-Pathogen Interactions, Mutation
Abstract: Although transmission of avian influenza viruses to mammals, particularly humans, has been repeatedly documented, adaptation and sustained transmission in the new host is a rare event that in the case of humans may result in pandemics. Host restriction involves multiple genetic determinants. Among the known determinants of host range, key determinants have been identified on the genes coding for the nucleoprotein and polymerase proteins that, together with the viral RNA segments, form the ribonucleoproteins (RNPs). The RNP genes form host-specific lineages and harbor host-associated genetic signatures. The functional significance of these determinants has been studied by reassortment and reverse genetics experiments, underlining the influence of the global genetic context. In some instances the molecular mechanisms have been approached, pointing to the importance of the polymerase activity and interaction with cellular host factors. Better knowledge of determinants of host restriction will allow monitoring of the pandemic potential of avian influenza viruses.
Published: 2008
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48. Sensitivity of H5N1 influenza viruses to oseltamivir: an update

Author: Sylvie van der Werf, Nadia Naffakh, and Marie-Anne Rameix-Welti
Subjects: Drug, Oseltamivir, biology, business.industry, Clinical effectiveness, viruses, media_common.quotation_subject, virus diseases, Drug resistance, medicine.disease_cause, Virology, Influenza A virus subtype H5N1, Treatment efficacy, chemistry.chemical_compound, chemistry, Pandemic, Immunology, biology.protein, Medicine, business, Neuraminidase, media_common
Abstract: Continued worldwide circulation of highly pathogenic influenza A (H5N1) viruses has raised concerns that they might adapt to humans and cause a pandemic. Preparedness plans rely on influenza vaccines and on the prophylactic and therapeutic use of neuraminidase inhibitors, especially the orally available drug oseltamivir. Although the efficacy of oseltamivir is well established for uncomplicated seasonal influenza, questions remain about its clinical effectiveness for human H5N1 infections. In this review, available data on the sensitivity of H5N1 viruses to oseltamivir in vitro, in animal models and in human patients are discussed. The factors that might limit the efficacy of oseltamivir treatment for human H5N1 infections are also discussed, in addition to options for improving treatment efficacy and limiting the development of drug resistance.
Published: 2008
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49. Identification and characterization of the binding site of the respiratory syncytial virus phosphoprotein to RNA-free nucleoprotein

Author: Jean-François Eléouët, Magali Aumont-Nicaise, Charles-Adrien Richard, Julien Sourimant, Gaëlle Gabiane, Marie-Anne Rameix-Welti, Marie Galloux, Mohammed Moudjou, Patrick England, Jenna Fix, Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Infection et inflammation (2I), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Institut Pasteur [Paris], Université Paris-Sud - Paris 11 (UP11), Laboratoire de Microbiologie, Hôpital Raymond Poincareé, French Agence Nationale de la Recherche, specific program ANR Blanc 'Bronchioliteaser' [ANR-11-BSV8-0024], ProdInra, Migration, BLANC - Etude structurale et fonctionnelle du complexe polymérase du virus respiratoire syncytial et recherche d'inhibiteurs - - BRONCHIOLITEASER2011 - ANR-11-BSV8-0024 - BLANC - VALID, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Biophysique des macromolécules et de leurs interactions (Plate-forme), and ANR-11-BSV8-0024,BRONCHIOLITEASER,Etude structurale et fonctionnelle du complexe polymérase du virus respiratoire syncytial et recherche d'inhibiteurs(2011)
Subjects: Paramyxoviridae, viruses, [SDV]Life Sciences [q-bio], Immunology, DNA Mutational Analysis, Centrifugation, Microbiology, Cell Line, chemistry.chemical_compound, Virology, RNA polymerase, Cricetinae, Protein Interaction Mapping, Animals, Binding site, Polymerase, Viral Structural Proteins, Binding Sites, biology, RNA, Rhabdoviridae, Nucleocapsid Proteins, Surface Plasmon Resonance, biology.organism_classification, Molecular biology, Nucleoprotein, Genome Replication and Regulation of Viral Gene Expression, [SDV] Life Sciences [q-bio], chemistry, Insect Science, Phosphoprotein, Respiratory Syncytial Virus, Human, biology.protein, Mutagenesis, Site-Directed, Protein Binding
Abstract: The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae , it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N 0 -P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N 0 monomer. We used this N mutant, designated N mono , as a substitute for N 0 in order to characterize the P regions involved in the N 0 -P complex formation. Using a series of P fragments, we determined by glutathione S -transferase (GST) pulldown assays that the N and C termini of P are able to interact with N mono . We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N mono in vitro . Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N 0 -P interaction could constitute a potential antiviral strategy. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N 0 ) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.
Published: 2015
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50. Interactome Analysis of the Human Respiratory Syncytial Virus RNA Polymerase Complex Identifies Protein Chaperones as Important Cofactors That Promote L-Protein Stability and RNA Synthesis

Author: Jean-François Eléouët, Olivier Touzelet, John N. Barr, Sarah L. Noton, Andressa Peres de Oliveira, Armando Morais Ventura, Nikki Smith, Weining Wu, Jenna M. Dawson, Andrew J. Easton, Paul Digard, Fernando Moreira Simabuco, David J. Hughes, Jenna Fix, Julian A. Hiscox, Stuart D. Armstrong, Diane C. Munday, Marie-Anne Rameix-Welti, Rachel Fearns, Marie Galloux, Waleed Aljabr, Institute of Infection and Global Health, University of Liverpool, Roslin Institute, University of Edinburgh, Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Boston University [Boston] (BU), School of Life Sciences, University of Warwick [Coventry], Universidade de São Paulo (USP), Universidade Estadual de Campinas (UNICAMP), School of molecular and cellular biology, University of Leeds, NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Medical Research Council (MRC) [MR/K000276/1], MRC studentship, NIHR, NIH [R01AI074903], and Ministry of Education, Kingdom of Saudi Arabia
Subjects: Viral protein, Immunology, RNA-dependent RNA polymerase, Biology, Virus Replication, medicine.disease_cause, Microbiology, Viral Proteins, 03 medical and health sciences, VP40, Viral entry, Virology, Protein Interaction Mapping, Viral structural protein, medicine, Humans, HSP70 Heat-Shock Proteins, HSP90 Heat-Shock Proteins, Protein Interaction Maps, Polymerase, 030304 developmental biology, 0303 health sciences, Protein Stability, 030302 biochemistry & molecular biology, DNA-Directed RNA Polymerases, Virus-Cell Interactions, 3. Good health, Viral replication, Respiratory Syncytial Virus, Human, Insect Science, Chaperone (protein), VIRUS DE RNA, Host-Pathogen Interactions, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, biology.protein, Molecular Chaperones, Protein Binding
Abstract: The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.
Published: 2015
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