Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 µg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 µM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 µM. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

Inhalation of 2, 4-di-tert-butylphenol-Loaded micelles suppresses respiratory syncytial virus infection in mice.

Human respiratory syncytial virus (RSV) is a common cause of respiratory infections in infants, young children, and elderly people. However, there are no effective treatments or vaccines available in most countries. In this study, we explored the anti-RSV potential of 2, 4-Di-tert-butylphenol (2, 4-DTBP), a compound derived from Houttuynia cordata Thunb. To overcome the poor solubility of 2, 4-DTBP, we encapsulated it in polymeric micelles and delivered it by inhalation. We found that 2, 4-DTBP-loaded micelles inhibited RSV infection in vitro and improved survival, lung pathology, and viral clearance in RSV-infected mice. Our results suggested that 2, 4-DTBP-loaded micelle is a promising novel therapeutic agent for RSV infection.

In-vivo and human evidence for potential efficacy of therapeutic polyclonal RSV neutralizing antibodies for palivizumab-resistant RSV infections.

BACKGROUND: Monoclonal antibody (palivizumab), intravenous immune globulin (IGIV), or respiratory syncytial virus (RSV)-polyclonal-hyperimmune-globulin (RSV-IG as Respigam®, RI-001, RI-002) are used with ribavirin in RSV-infected immunocompromised patients, with debated efficacy. Palivizumab-resistance (PR) can arise during treatment of persistent infections in this population. RSV-IG may confer benefit in PR-RSV infection. METHODS: RSV-IG [RI-001] was provided for an immunocompromised infant with RSV-pneumonitis refractory to ribavirin and palivizumab. RSV-neutralizing antibody, respiratory RSV load (qPCR), and F-gene-sequence-detection of PR was determined. Prophylactic RSV-IG [RI-002] or palivizumab was administered in a cotton-rat model infected with wild-type and PR-RSV. Lung RSV load and neutralizing antibody were measured. RESULTS: As protective RI-001-neutralizing antibody titers waned in the infant, a subpopulation of PR-escape mutants were detected with a fatal RSV-burden in the lungs. In PR-RSV-infected cotton rats, prophylactic RI-002 reduced RSV-load in the lungs (2.45 vs 0.28 log10 PFU/g lung-tissue reduction, respectively, p < 0.05) and provided protective RSV-neutralizing antibody. CONCLUSIONS: RSV-IG and ribavirin use in immunocompromised patients requires further study.

[EDP-938, a new antiviral with inhibitory activity against the nucleoprotein of the respiratory syncytial virus]. / EDP-938, un nuevo antiviral con actividad inhibidora frente a la nucleoproteína del virus respiratorio sincitial.

The absence of an effective vaccine against respiratory syncytial virus (RSV) has led to the development of various drugs with the ability to inhibit or block its replicative activity. The first generation, called fusion inhibitors, bind to the protein on the viral surface and prevent the virus from binding and entering the cell. However, its low efficacy has determined the start of studies with second-generation compounds capable of binding or blocking the nucleoprotein (N); most of these compounds are analogs of 1,4-benzodiazepines. EDP-938 has shown high efficacy against RSV. The first trials in humans have shown that this antiviral is rapidly absorbed after oral administration and has a half-life of between 11-18 hours Administration for seven days of multiple oral doses of up to 600 mg/day or 300 mg/day/twice a day, there were hardly any significant adverse effects and the viral load in the lower respiratory tract decreased significantly.

EDP-938, a Respiratory Syncytial Virus Inhibitor, in a Human Virus Challenge.

BACKGROUND: Respiratory syncytial virus (RSV) infection causes substantial morbidity and mortality among infants, older adults, and immunocompromised adults. EDP-938, a nonfusion replication inhibitor of RSV, acts by modulating the viral nucleoprotein. METHODS: In a two-part, phase 2a, randomized, double-blind, placebo-controlled challenge trial, we assigned participants who had been inoculated with RSV-A Memphis 37b to receive EDP-938 or placebo. Different doses of EDP-938 were assessed. Nasal-wash samples were obtained from day 2 until day 12 for assessments. Clinical symptoms were assessed by the participants, and pharmacokinetic profiles were obtained. The primary end point was the area under the curve (AUC) for the RSV viral load, as measured by reverse-transcriptase-quantitative polymerase-chain-reaction assay. The key secondary end point was the AUC for the total symptom score. RESULTS: In part 1 of the trial, 115 participants were assigned to receive EDP-938 (600 mg once daily [600-mg once-daily group] or 300 mg twice daily after a 500-mg loading dose [300-mg twice-daily group]) or placebo. In part 2, a total of 63 participants were assigned to receive EDP-938 (300 mg once daily after a 600-mg loading dose [300-mg once-daily group] or 200 mg twice daily after a 400-mg loading dose [200-mg twice-daily group]) or placebo. In part 1, the AUC for the mean viral load (hours × log10 copies per milliliter) was 204.0 in the 600-mg once-daily group, 217.7 in the 300-mg twice-daily group, and 790.2 in the placebo group. The AUC for the mean total symptom score (hours × score, with higher values indicating greater severity) was 124.5 in the 600-mg once-daily group, 181.8 in the 300-mg twice-daily group, and 478.8 in the placebo group. The results in part 2 followed a pattern similar to that in part 1: the AUC for the mean viral load was 173.9 in the 300-mg once-daily group, 196.2 in the 200-mg twice-daily group, and 879.0 in the placebo group, and the AUC for the mean total symptom score was 99.3, 89.6, and 432.2, respectively. In both parts, mucus production was more than 70% lower in each EDP-938 group than in the placebo group. The four EDP-938 regimens had a safety profile similar to that of placebo. Across all dosing regimens, the EDP-938 median time to maximum concentration ranged from 4 to 5 hours, and the geometric mean half-life ranged from 13.7 to 14.5 hours. CONCLUSIONS: All EDP-938 regimens were superior to placebo with regard to lowering of the viral load, total symptom scores, and mucus weight without apparent safety concerns. (ClinicalTrials.gov number, NCT03691623.).

Chitinase 3-like 1 is involved in the induction of IL-8 expression by double-stranded RNA in airway epithelial cells.

Viral respiratory infection causes inflammatory lung disease. Chitinase 3-like 1 (CHI3L1) contributes to airway inflammation, but its role in human airway epithelial cells following viral infection is unclear. Thus, we investigated whether CHI3L1 regulates inflammatory responses caused by viral infections in airway epithelial cells. Human bronchial epithelial cells, BEAS-2B, were stimulated with a synthetic analog of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly(I:C)). To confirm the specific role of CHI3L1, CHI3L1 was knocked down in BEAS-2B cells using shRNA lentivirus. The expression of CHI3L1 and proinflammatory cytokines such as IL-8 and phosphorylation of mitogen-activated protein kinase (MAPK) pathways were analyzed. In addition to poly(I:C), BEAS-2B cells were infected with the human respiratory syncytial virus (RSV) A2 strain, and CHI3L1 and IL-8 expression was analyzed. Stimulating the cells with poly(I:C) increased CHI3L1 and IL-8 expression, whereas IL-8 expression was abrogated in CHI3L1 knockdown BEAS-2B cells. Poly(I:C) stimulation of BEAS-2B cells resulted in phosphorylation of MAPK pathways, and inhibition of MAPK pathways significantly abolished IL-8 secretion. Phosphorylation of MAPK pathways was diminished in CHI3L1 knockdown BEAS-2B cells. Infection with RSV increased CHI3L1 and IL-8 expression. IL-8 expression induced by RSV infection was abrogated in CHI3L1 knockdown cells. In conclusion, CHI3L1 may be involved in IL-8 secretion by regulating MAPK pathways during respiratory viral infections in airway epithelial cells.

Respiratory syncytial virus infection induces the release of transglutaminase 2 from human airway epithelial cells.

Respiratory syncytial virus (RSV) is an important human pathogen that causes severe lower respiratory tract infections in young children, the elderly, and the immunocompromised, yet no effective treatments or vaccines are available. The precise mechanism underlying RSV-induced acute airway disease and associated sequelae are not fully understood; however, early lung inflammatory and immune events are thought to play a major role in the outcome of the disease. Moreover, oxidative stress responses in the airways play a key role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium (Ca2+) levels, which in turn activate Ca2+-dependent enzymes, including transglutaminase 2 (TG2). Transglutaminase 2 is a multifunctional cross-linking enzyme implicated in various physiological and pathological conditions; however, its involvement in respiratory virus-induced airway inflammation is largely unknown. In this study, we demonstrated that RSV-induced oxidative stress promotes enhanced activation and release of TG2 from human lung epithelial cells as a result of its translocation from the cytoplasm and subsequent release into the extracellular space, which was mediated by Toll-like receptor (TLR)-4 and NF-κB pathways. Antioxidant treatment significantly inhibited RSV-induced TG2 extracellular release and activation via blocking viral replication. Also, treatment of RSV-infected lung epithelial cells with TG2 inhibitor significantly reduced RSV-induced matrix metalloprotease activities. These results suggested that RSV-induced oxidative stress activates innate immune receptors in the airways, such as TLRs, that can activate TG2 via the NF-κB pathway to promote cross-linking of extracellular matrix proteins, resulting in enhanced inflammation.

4'-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.

Interactions between the Nucleoprotein and the Phosphoprotein of Pneumoviruses: Structural Insight for Rational Design of Antivirals.

Pneumoviruses include pathogenic human and animal viruses, the most known and studied being the human respiratory syncytial virus (hRSV) and the metapneumovirus (hMPV), which are the major cause of severe acute respiratory tract illness in young children worldwide, and main pathogens infecting elderly and immune-compromised people. The transcription and replication of these viruses take place in specific cytoplasmic inclusions called inclusion bodies (IBs). These activities depend on viral polymerase L, associated with its cofactor phosphoprotein P, for the recognition of the viral RNA genome encapsidated by the nucleoprotein N, forming the nucleocapsid (NC). The polymerase activities rely on diverse transient protein-protein interactions orchestrated by P playing the hub role. Among these interactions, P interacts with the NC to recruit L to the genome. The P protein also plays the role of chaperone to maintain the neosynthesized N monomeric and RNA-free (called N0) before specific encapsidation of the viral genome and antigenome. This review aims at giving an overview of recent structural information obtained for hRSV and hMPV P, N, and more specifically for P-NC and N0-P complexes that pave the way for the rational design of new antivirals against those viruses.

Monoterpene-Containing Substituted Coumarins as Inhibitors of Respiratory Syncytial Virus (RSV) Replication.

Respiratory syncytial virus (RSV) is a critical cause of infant mortality. However, there are no vaccines and adequate drugs for its treatment. We showed, for the first time, that O-linked coumarin-monoterpene conjugates are effective RSV inhibitors. The most potent compounds are active against both RSV serotypes, A and B. According to the results of the time-of-addition experiment, the conjugates act at the early stages of virus cycle. Based on molecular modelling data, RSV F protein may be considered as a possible target.

mTOR kinase is a therapeutic target for respiratory syncytial virus and coronaviruses.

Therapeutic interventions targeting viral infections remain a significant challenge for both the medical and scientific communities. While specific antiviral agents have shown success as therapeutics, viral resistance inevitably develops, making many of these approaches ineffective. This inescapable obstacle warrants alternative approaches, such as the targeting of host cellular factors. Respiratory syncytial virus (RSV), the major respiratory pathogen of infants and children worldwide, causes respiratory tract infection ranging from mild upper respiratory tract symptoms to severe life-threatening lower respiratory tract disease. Despite the fact that the molecular biology of the virus, which was originally discovered in 1956, is well described, there is no vaccine or effective antiviral treatment against RSV infection. Here, we demonstrate that targeting host factors, specifically, mTOR signaling, reduces RSV protein production and generation of infectious progeny virus. Further, we show that this approach can be generalizable as inhibition of mTOR kinases reduces coronavirus gene expression, mRNA transcription and protein production. Overall, defining virus replication-dependent host functions may be an effective means to combat viral infections, particularly in the absence of antiviral drugs.

Antibiotic use for respiratory syncytial virus in the Middle East: A surveillance study in hospitalized Jordanian children.

INTRODUCTION: In developing countries where point-of-care testing is limited, providers rely on clinical judgement to discriminate between viral and bacterial respiratory infections. We performed a cross-sectional cohort study of hospitalized Jordanian children to evaluate antibiotic use for respiratory syncytial virus (RSV) infections. MATERIALS AND METHODS: Admitting diagnoses from a prior viral surveillance cohort of hospitalized Jordanian children were dichotomized into suspected viral-like, non-pulmonary bacterial-like, and pulmonary bacterial-like infection. Stratifying by sex, we performed a polytomous logistic regression adjusting for age, underlying medical condition, maternal education, and region of residence to estimate prevalence odds ratios (PORs) for antibiotic use during hospitalization. Sensitivity and specificity of admission diagnoses and research laboratory results were compared. RESULTS: Children with a suspected viral-like admission diagnosis, compared to those with suspected non-pulmonary bacterial-like, were 88% and 86% less likely to be administered an empiric/first-line antibiotic (male, aPOR: 0.12; female, aPOR: 0.14; p-value = <0.001). There were slight differences by sex with males having a lower prevalence than females in being administered an expanded coverage antibiotic; but they had a higher prevalence of macrolide administration than males with non-pulmonary bacterial-like infection. Overall, children with RSV had a 34% probability (sensitivity) of being assigned to a suspected viral-like diagnosis; whereas RSV-negative children had a 76% probability (specificity) of being assigned to a suspected pulmonary bacterial-like diagnosis. CONCLUSIONS: Hospitalized children with a suspected viral-like admission diagnosis were less likely to receive an empiric/first-line and expanded coverage antibiotic compared to suspected non-pulmonary and pulmonary infections; however, when evaluating the accuracy of admission diagnosis to RSV-laboratory results there were considerable misclassifications. These results highlight the need for developing antibiotic interventions for Jordan and the rest of the Middle East.

Synthesis of New Modified with Rhodamine B Peptides for Antiviral Protection of Textile Materials.

Here we report on the synthesis and characterization of three new N-modified analogues of hemorphin-4 with rhodamine B. Modified with chloroacetyl, chloride cotton fabric has been dyed and color coordinates of the obtained textile materials were determined. Antiviral and virucidal activities of both the peptide-rhodamine B compounds and the dyed textile material were studied. Basic physicochemical properties (acid-base behavior, solvent influence, kinetics) related to the elucidation of structural activity of the new modified peptides based on their steric open/closed ring effect were studied. The obtained results lead to the conclusion that in protic solvent with change in pH of the environment, direct control over the dyeing of textiles can be achieved. Both the new hybrid peptide compounds and the modification of functionalized textile materials with these bioactive hemorphins showed virucidal activity against the human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5 (HAdV-5) for different time intervals (30 and 60 min) and the most active compound was Rh-3.

Respiratory syncytial virus: from pathogenesis to potential therapeutic strategies.

Respiratory syncytial virus (RSV) is one of the most important viral pathogens causing respiratory tract infection in infants, the elderly and people with poor immune function, which causes a huge disease burden worldwide every year. It has been more than 60 years since RSV was discovered, and the palivizumab monoclonal antibody, the only approved specific treatment, is limited to use for passive immunoprophylaxis in high-risk infants; no other intervention has been approved to date. However, in the past decade, substantial progress has been made in characterizing the structure and function of RSV components, their interactions with host surface molecules, and the host innate and adaptive immune response to infection. In addition, basic and important findings have also piqued widespread interest among researchers and pharmaceutical companies searching for effective interventions for RSV infection. A large number of promising monoclonal antibodies and inhibitors have been screened, and new vaccine candidates have been designed for clinical evaluation. In this review, we first briefly introduce the structural composition, host cell surface receptors and life cycle of RSV virions. Then, we discuss the latest findings related to the pathogenesis of RSV. We also focus on the latest clinical progress in the prevention and treatment of RSV infection through the development of monoclonal antibodies, vaccines and small-molecule inhibitors. Finally, we look forward to the prospects and challenges of future RSV research and clinical intervention.

Antiviral iridoid glycosides from Clerodendrum myricoides.

The methanol root extract of Clerodendrum myricoides (Hochst.) Vatke afforded two new (1, 2) and two known (3, 4) iridoid glycosides. The structures of the isolated compounds were established based on NMR, IR, UV and MS data analyses. The crude extract and the isolated constituents were assayed for antiviral activity against the human respiratory syncytial virus (RSV) in human laryngeal epidermoid carcinoma (HEp-2) cells. The crude extract inhibited RSV infectivity at EC50 = 0.21 µg/ml, while it showed cytotoxicity against HEp-2 cells with CC50 = 9 µg/ml. Compound 2 showed 43.2% virus inhibition at 100 µM, while compounds 1 as well as 3 and 4 had only weak antiviral and cytotoxic activities.

Statin-mediated disruption of Rho GTPase prenylation and activity inhibits respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory tract infections in children. To uncover new antiviral therapies, we developed a live cell-based high content screening approach for rapid identification of RSV inhibitors and characterized five drug classes which inhibit the virus. Among the molecular targets for each hit, there was a strong functional enrichment in lipid metabolic pathways. Modulation of lipid metabolites by statins, a key hit from our screen, decreases the production of infectious virus through a combination of cholesterol and isoprenoid-mediated effects. Notably, RSV infection globally upregulates host protein prenylation, including the prenylation of Rho GTPases. Treatment by statins or perillyl alcohol, a geranylgeranyltransferase inhibitor, reduces infection in vitro. Of the Rho GTPases assayed in our study, a loss in Rac1 activity strongly inhibits the virus through a decrease in F protein surface expression. Our findings provide new insight into the importance of host lipid metabolism to RSV infection and highlight geranylgeranyltransferases as an antiviral target for therapeutic development.

Reversible disruption of XPO1-mediated nuclear export inhibits respiratory syncytial virus (RSV) replication.

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract disease in infants, young children, the elderly and immunocompromised individuals. Therapy for RSV infections is limited to high risk infants and there are no safe and efficacious vaccines. Matrix (M) protein is a major RSV structural protein with a key role in virus assembly. Interestingly, M is localised to the nucleus early in infection and its export into the cytoplasm by the nuclear exporter, exportin-1 (XPO1) is essential for RSV assembly. We have shown previously that chemical inhibition of XPO1 function results in reduced RSV replication. In this study, we have investigated the anti-RSV efficacy of Selective Inhibitor of Nuclear Export (SINE) compounds, KPT-335 and KPT-185. Our data shows that therapeutic administration of the SINE compounds results in reduced RSV titre in human respiratory epithelial cell culture. Within 24 h of treatment, RSV replication and XPO1 expression was reduced, M protein was partially retained in the nucleus, and cell cycle progression was delayed. Notably, the effect of SINE compounds was reversible within 24 h after their removal. Our data show that reversible inhibition of XPO1 can disrupt RSV replication by affecting downstream pathways regulated by the nuclear exporter.

Chloroquine and pyrimethamine inhibit the replication of human respiratory syncytial virus A.

Human respiratory syncytial virus (hRSV) is a major cause of respiratory illness in young children and can cause severe infections in the elderly or in immunocompromised adults. To date, there is no vaccine to prevent hRSV infections, and disease management is limited to preventive care by palivizumab in infants and supportive care for adults. Intervention with small-molecule antivirals specific for hRSV represents a good alternative, but no such compounds are currently approved. The investigation of existing drugs for new therapeutic purposes (drug repositioning) can be a faster approach to address this issue. In this study, we show that chloroquine and pyrimethamine inhibit the replication of human respiratory syncytial virus A (long strain) and synergistically increase the anti-replicative effect of ribavirin in cellulo. Moreover, chloroquine, but not pyrimethamine, inhibits hRSV replication in the mouse model. Our results show that chloroquine can potentially be an interesting compound for treatment of hRSV infection in monotherapy or in combination with other antivirals.

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.