Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

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.

Repurposing screen identifies novel candidates for broad-spectrum coronavirus antivirals and druggable host targets.

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiological processes in humans, providing unique opportunities for the discovery of host-targeting antivirals. We screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) repurposing library with approximately 12,000 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus and mapping to 58 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor, the dopamine receptor, and cyclin-dependent kinases. Gene knock-out of the drugs' host targets including cathepsin B and L (CTSB/L; VBY-825), the aryl hydrocarbon receptor (AHR; Phortress), the farnesyl-diphosphate farnesyltransferase 1 (FDFT1; P-3622), and the kelch-like ECH-associated protein 1 (KEAP1; Omaveloxolone), significantly modulated HCoV-229E infection, providing evidence that these compounds inhibited the virus through acting on their respective host targets. Counter-screening of all 134 primary compound candidates with SARS-CoV-2 and validation in primary cells identified Phortress, an AHR activating ligand, P-3622-targeting FDFT1, and Omaveloxolone, which activates the NFE2-like bZIP transcription factor 2 (NFE2L2) by liberating it from its endogenous inhibitor KEAP1, as antiviral candidates for both an Alpha- and a Betacoronavirus. This study provides an overview of HCoV-229E repurposing candidates and reveals novel potentially druggable viral host dependency factors hijacked by diverse coronaviruses.

Persicamidines-Unprecedented Sesquarterpenoids with Potent Antiviral Bioactivity against Coronaviruses.

A new family of highly unusual sesquarterpenoids (persicamidines A-E) exhibiting significant antiviral activity was isolated from a newly discovered actinobacterial strain, Kibdelosporangium persicum sp. nov., collected from a hot desert in Iran. Extensive NMR analysis unraveled a hexacyclic terpenoid molecule with a modified sugar moiety on one side and a highly unusual isourea moiety fused to the terpenoid structure. The structures of the five analogues differed only in the aminoalkyl side chain attached to the isourea moiety. Persicamidines A-E showed potent activity against hCoV-229E and SARS-CoV-2 viruses in the nanomolar range together with very good selectivity indices, making persicamidines promising as starting points for drug development.

Respiratory Syncytial Virus Two-Step Infection Screen Reveals Inhibitors of Early and Late Life Cycle Stages.

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.

Thiamyxins: Structure and Biosynthesis of Myxobacterial RNA-Virus Inhibitors.

During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with potent antiviral activity. We isolated four congeners of this unprecedented natural product family with the non-cyclized thiamyxin D fused to a glycerol unit at the C-terminus. Alongside their structure elucidation, we present a concise biosynthesis model based on biosynthetic gene cluster analysis and isotopically labelled precursor feeding. We report incorporation of a 2-(hydroxymethyl)-4-methylpent-3-enoic acid moiety by a GCN5-related N-acetyltransferase-like decarboxylase domain featuring polyketide synthase. The thiamyxins show potent inhibition of RNA viruses in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate the potential for further development of the thiamyxins.

Target capture sequencing reveals a monoclonal outbreak of respiratory syncytial virus B infections among adult hematologic patients.

BACKGROUND: Respiratory syncytial virus (RSV) causes community-acquired respiratory tract infections during winter. However, outbreaks in hospitals also occur repeatedly. In particular, patients with hematologic malignancies are at an increased risk for a severe and potentially fatal course of RSV infection. Here we present the investigation of an RSV outbreak in a hematology ward for adults following the ORION statement. METHODS: An epidemiologic and molecular outbreak analysis was performed. We developed and employed a minimal oligonucleotide probe set in target capture probe sequencing that allows cost-effective RSV-A or -B capturing to reconstruct RSV genomes from clinical samples. RESULTS: Four adult patients were involved in the outbreak caused by RSV-B in March 2019. The enforcement of the pre-existing infection control measures by effective training of hospital staff contributed to a successful containment. PCR-based RSV screening on the ward enabled early detection of new cases and rapid isolation measures. The molecular analysis demonstrated that the outbreak sequences were highly related and distinct to other RSV-B strains circulating at the same time. CONCLUSIONS: A multimodal infection control concept is essential for the timely detection and control of RSV outbreaks in patients with hematological disease. Among other measures, preventive screening for respiratory viruses is recommended. Furthermore, the integration of conventional and molecular epidemiology, such as whole-genome sequencing and variant calling, significantly contributes to the understanding of transmission pathways. Based on this, appropriate conclusions can be drawn for targeted prevention measures that have prepared us for the COVID-19 pandemic beyond the RSV approach described here.

The Human Liver-Expressed Lectin CD302 Restricts Hepatitis C Virus Infection.

C-type lectin domain-containing proteins (CTLDcps) shape host responses to pathogens and infectious disease outcomes. Previously, we identified the murine CTLDcp Cd302 as restriction factor, limiting hepatitis C virus (HCV) infection of murine hepatocytes. In this study, we investigated in detail the human orthologue's ability to restrict HCV infection in human liver cells. CD302 overexpression in Huh-7.5 cells potently inhibited infection of diverse HCV chimeras representing seven genotypes. Transcriptional profiling revealed abundant CD302 mRNA expression in human hepatocytes, the natural cellular target of HCV. Knockdown of endogenously expressed CD302 modestly enhanced HCV infection of Huh-7.5 cells and primary human hepatocytes. Functional analysis of naturally occurring CD302 transcript variants and engineered CD302 mutants showed that the C-type lectin-like domain (CTLD) is essential for HCV restriction, whereas the cytoplasmic domain (CPD) is dispensable. Coding single nucleotide polymorphisms occurring in human populations and mapping to different domains of CD302 did not influence the capacity of CD302 to restrict HCV. Assessment of the anti-HCV phenotype at different life cycle stages indicated that CD302 preferentially targets the viral entry step. In contrast to the murine orthologue, overexpression of human CD302 did not modulate downstream expression of nuclear receptor-controlled genes. Ectopic CD302 expression restricted infection of liver tropic hepatitis E virus (HEV), while it did not affect infection rates of two respiratory viruses, including respiratory syncytial virus (RSV) and the alpha coronavirus HVCoV-229E. Together, these findings suggest that CD302 contributes to liver cell-intrinsic defense against HCV and might mediate broader antiviral defenses against additional hepatotropic viruses. IMPORTANCE The liver represents an immunoprivileged organ characterized by enhanced resistance to immune responses. However, the importance of liver cell-endogenous, noncytolytic innate immune responses in pathogen control is not well defined. Although the role of myeloid cell-expressed CTLDcps in host responses to viruses has been characterized in detail, we have little information about their potential functions in the liver and their relevance for immune responses in this organ. Human hepatocytes endogenously express the CTLDcp CD302. Here, we provide evidence that CD302 limits HCV infection of human liver cells, likely by inhibiting a viral cell entry step. We confirm that the dominant liver-expressed transcript variant, as well as naturally occurring coding variants of CD302, maintain the capacity to restrict HCV. We further show that the CTLD of the protein is critical for the anti-HCV activity and that overexpressed CD302 limits HEV infection. Thus, CD302 likely contributes to human liver-intrinsic antiviral defenses.

IRIS: Infection with RespIratory Syncytial Virus in infants-a prospective observational cohort study.

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection in infants. Globally, RSV is responsible for approximately 3.2 million hospital admissions and about 60,000 in-hospital deaths per year. METHODS: Infection with RespIratory Syncytial Virus (IRIS) is an observational, multi-centre study enrolling infants with severe RSV infection and healthy controls. Inclusion criteria are age between 0 and 36 months and hospitalisation due to RSV infection at three German sites. Exclusion criteria are premature birth, congenital or acquired bronchopulmonary or cardiac diseases, and immunodeficiency. Healthy control probands are enrolled via recruitment of patients undergoing routine surgical procedures. Blood and respiratory specimens are collected upon admission, and RSV and other pathogens are analysed by multiplex polymerase chain reaction. Different biomaterials, including plasma, nasal lining fluid, blood cells, DNA, and RNA specimens, are sampled in a dedicated biobank. Detailed information on demographic characteristics and medical history is recorded, and comprehensive clinical data, including vital signs, medication, and interventions. DISCUSSION: The IRIS study aims to discover host and viral factors controlling RSV disease courses in infants. The approach including multi-omics characterisation in clinically well-characterized children with RSV bronchiolitis seeks to improve our understanding of the immune response against this virus. It may disclose novel diagnostic and treatment approaches for respiratory infections in infants. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04925310. Registered 01 October 2021-Retrospectively registered. https://clinicaltrials.gov/ct2/show/NCT04925310?cond=NCT04925310&draw=2&rank=1.

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

Sandacrabins - Structurally Unique Antiviral RNA Polymerase Inhibitors from a Rare Myxobacterium.

Structure elucidation and total synthesis of five unprecedented terpenoid-alkaloids, the sandacrabins, are reported, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS-CoV-2 RNA-dependent RNA polymerase complex, highlighting them as structurally distinct non-nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition opens the possibility for their medicinal chemistry optimization towards selective inhibitors.

A condensate-hardening drug blocks RSV replication in vivo.

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.

Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants.

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.

Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

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.

Labyrinthopeptins Exert Broad-Spectrum Antiviral Activity through Lipid-Binding-Mediated Virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.

Physicochemical Properties Govern the Activity of Potent Antiviral Flavones.

Ladanein (i.e., 5,6,7-trihydroxylated flavone) was demonstrated to act as a powerful virucidal agent toward a broad range of enveloped virus particles. Fe(III) coordination and pH are indeed among the key parameters that might favor both bioactivation of the flavone and consequent host cell entry inhibition. In this present work, the impact of fluorinated groups on the physicochemical and antiviral properties of the flavone was investigated, thus allowing a deeper understanding of the antiviral mode of action. The improved synthesis of ladanein allowed accessing a broad range of analogues, some of them being significantly more active than the former ladanein lead compound. We first determined the acido-basic properties of this homogenous series of compounds and then investigated their electrochemical behavior. Fe(III) coordination properties (stability, spectral behavior, and kinetics) of ladanein and its analogues were then examined (quasiphysiological conditions) and provided key information of their stability and reactivity. Using the determined physicochemical parameters, the critical impact of the iron complexation and medium acidity was confirmed on hepatitis C virus (HCV) particles (pre)treated with ladanein. Finally, a preliminary structure-HCV entry inhibition relationship study evidenced the superior antiviral activity of the ladanein analogues bearing an electron-withdrawing group in para position (FCF 3 > FOCF 3 > FFCF 3 > FF > FOMe) on the B cycle in comparison with the parent ladanein itself.

Improved protocol for simultaneous analysis of leukocyte subsets and epithelial cells from murine and human lung.

PURPOSE: To study and isolate lung cells by flow cytometry, enzymatic digestion and generation of single cell suspensions is required. This significantly influences expression of cellular epitopes and protocols need to be adapted for the best isolation and subsequent analysis of specific cellular subsets. MATERIALS AND METHODS: We optimized protocols for the simultaneous isolation and characterization of specific human and murine lung cell types. For alveolar epithelial cells (AEC), a primarily dispase based digestion method and for leukocytes, a primarily collagenase based technique was adapted. Protocols were applied in parallel in either single experimental mice or human lung specimens. RESULTS: Optimized dispase/DNase digestion yielded a high percentage of Epcam+CD45-CD31- AEC as assessed by flow cytometry. Epcam+CD45-CD3-CD11b-CD11c-CD16/32-CD19-CD31-F4/80- AEC were readily sortable with high purity and typical morphology and function upon in vitro stimulation with lipopolysaccharide or respiratory-syncytial-virus (RSV) infection. To analyze lung leukocytes, specimens were digested with an adapted collagenase/DNase protocol yielding high percentages of viable leukocytes with typical morphology, function, and preserved subset specific leukocyte markers. Both protocols could be applied simultaneously in a single experimental mouse post mortem. Application of both digestion methods in primary human lung specimens yielded similar results with high proportions of Epcam+CD45- human AEC after dispase/DNase digestion and preservation of human T cell epitopes after collagenase/DNase digestion. CONCLUSION: The here described protocols were optimized for the simple and efficient isolation of murine and human lung cells. In contrast to previously described techniques, they permit simultaneous in-depth characterization of pulmonary epithelial cells and leukocyte subsets such as T helper, cytotoxic T, and B cells from one sample. As such, they may help to comprehensively and sustainably characterize murine and human lung specimens and facilitate studies on the role of lung immune cells in different respiratory pathologies.

Molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease.

Background: Respiratory syncytial virus (RSV) is responsible for upper and lower respiratory tract infection in adults and children. Especially immunocompromised patients are at high risk for a severe course of infection, and mortality is increased. Moreover RSV can spread in healthcare settings and can cause outbreaks. Herein we demonstrate the successful control and characteristics of a RSV outbreak that included 8 patients in our Department of Pediatric Hematology and Oncology. Methods: We performed an epidemiologic investigation and a molecular analysis of the outbreak strains. Moreover we present the outbreak control bundle and our concept for RSV screening in the winter season. Results: RSV A and B strains caused the outbreak. RSV B strains affected 3 patients, 2 of whom were co-infected with RSV A. Exactly this RSV A strain was detected in another 5 patients. Our multimodal infection control bundle including prophylactic RSV screening was able to rapidly stop the outbreak. Conclusion: An infection control bundle in RSV outbreaks should address all potential transmission pathways. In pediatric settings the restriction of social activities might have a temporal negative impact on quality of life but helps to limit transmission opportunities. Molecular analysis allows better understanding of RSV outbreaks and, if done in a timely manner, might be helpful for guidance of infection control measures.

Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1.

UNLABELLED: To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. CONCLUSION: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as a cost-effective component of HCV combination therapies.

Efficient virus assembly, but not infectivity, determines the magnitude of hepatitis C virus-induced interferon alpha responses of plasmacytoid dendritic cells.

UNLABELLED: Worldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV. IMPORTANCE: Although pegylated IFN-α plus ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here, we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood-derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the nonstructural genes of Japanese fulminant hepatitis C virus (JFH1) induced massive type I interferon responses, whereas the original genotype 2a JFH1 strain did not. Our detailed analyses revealed that, not the virus infectivity, but rather, the efficiency of virus assembly and core protein envelopment critically determined the magnitude of interferon responses. To our knowledge, this is the first example of hepatitis C virus-associated genetic variations that determine the magnitude of innate host responses.