4-Trifluoromethyl bithiazoles as broad-spectrum antimicrobial agents for virus-related bacterial infections or co-infections.

Respiratory tract infections involving a variety of microorganisms such as viruses, bacteria, and fungi are a prominent cause of morbidity and mortality globally, exacerbating various pre-existing respiratory and non-respiratory conditions. Moreover, the ability of bacteria and viruses to coexist might impact the development and severity of lung infections, promoting bacterial colonization and subsequent disease exacerbation. Secondary bacterial infections following viral infections represent a complex challenge to be overcome from a therapeutic point of view. We report herein our efforts in the development of new bithiazole derivatives showing broad-spectrum antimicrobial activity against both viruses and bacteria. A series of 4-trifluoromethyl bithiazole analogues was synthesized and screened against selected viruses (hRVA16, EVD68, and ZIKV) and a panel of Gram-positive and Gram-negative bacteria. Among them, two promising broad-spectrum antimicrobial compounds (8a and 8j) have been identified: both compounds showed low micromolar activity against all tested viruses, 8a showed synergistic activity against E. coli and A. baumannii in the presence of a subinhibitory concentration of colistin, while 8j showed a broader spectrum of activity against Gram-positive and Gram-negative bacteria. Activity against antibiotic-resistant clinical isolates is also reported. Given the ever-increasing need to adequately address viral and bacterial infections or co-infections, this study paves the way for the development of new agents with broad antimicrobial properties and synergistic activity with common antivirals and antibacterials.

Non-Steroidal Estrogens Inhibit Influenza Virus by Interacting with Hemagglutinin and Preventing Viral Fusion.

Influenza virus is one of the main causes of respiratory infections worldwide. Despite the availability of seasonal vaccines and antivirals, influenza virus infections cause an important health and economic burden. Therefore, the need to identify alternative antiviral strategies persists. In this study, we identified non-steroidal estrogens as potent inhibitors of influenza virus due to their interaction with the hemagglutinin protein, preventing viral entry. This activity is maintained in vitro, ex vivo, and in vivo. Therefore, we found a new domain to target on the hemagglutinin and a class of compounds that could be further optimized for influenza treatment.

Small Molecules Targeting Viral RNA.

The majority of antivirals available target viral proteins; however, RNA is emerging as a new and promising antiviral target due to the presence of highly structured RNA in viral genomes fundamental for their replication cycle. Here, we discuss methods for the identification of RNA-targeting compounds, starting from the determination of RNA structures either from purified RNA or in living cells, followed by in silico screening on RNA and phenotypic assays to evaluate viral inhibition. Moreover, we review the small molecules known to target the programmed ribosomal frameshifting element of SARS-CoV-2, the internal ribosomal entry site of different viruses, and RNA elements of HIV.

Variant enterovirus A71 found in immune-suppressed patient binds to heparan sulfate and exhibits neurotropism in B-cell-depleted mice.

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease outbreaks with neurological complications and deaths. We previously isolated an EV-A71 variant in the stool, cerebrospinal fluid, and blood of an immunocompromised patient who had a leucine-to-arginine substitution on the VP1 capsid protein, resulting in increased heparin sulfate binding. We show here that this mutation increases the virus's pathogenicity in orally infected mice with depleted B cells, which mimics the patient's immune status, and increases susceptibility to neutralizing antibodies. However, a double mutant with even greater heparin sulfate affinity is not pathogenic, suggesting that increased heparin sulfate affinity may trap virions in peripheral tissues and reduce neurovirulence. This research sheds light on the increased pathogenicity of variant with heparin sulfate (HS)-binding ability in individuals with decreased B cell immunity.

Geneticin shows selective antiviral activity against SARS-CoV-2 by interfering with programmed -1 ribosomal frameshifting.

SARS-CoV-2 is currently causing an unprecedented pandemic. While vaccines are massively deployed, we still lack effective large-scale antiviral therapies. In the quest for antivirals targeting conserved structures, we focused on molecules able to bind viral RNA secondary structures. Aminoglycosides are a class of antibiotics known to interact with the ribosomal RNA of both prokaryotes and eukaryotes and have previously been shown to exert antiviral activities by interacting with viral RNA. Here we show that the aminoglycoside geneticin is endowed with antiviral activity against all tested variants of SARS-CoV-2, in different cell lines and in a respiratory tissue model at non-toxic concentrations. The mechanism of action is an early inhibition of RNA replication and protein expression related to a decrease in the efficiency of the -1 programmed ribosomal frameshift (PRF) signal of SARS-CoV-2. Using in silico modeling, we have identified a potential binding site of geneticin in the pseudoknot of frameshift RNA motif. Moreover, we have selected, through virtual screening, additional RNA binding compounds, interacting with the same site with increased potency.

An anti-influenza combined therapy assessed by single cell RNA-sequencing.

Influenza makes millions of people ill every year, placing a large burden on the healthcare system and the economy. To develop a treatment against influenza, we combined virucidal sialylated cyclodextrins with interferon lambda and demonstrated, in human airway epithelia, that the two compounds inhibit the replication of a clinical H1N1 strain more efficiently when administered together rather than alone. We investigated the mechanism of action of the combined treatment by single cell RNA-sequencing analysis and found that both the single and combined treatments impair viral replication to different extents across distinct epithelial cell types. We showed that each cell type comprises multiple sub-types, whose proportions are altered by H1N1 infection, and assessed the ability of the treatments to restore them. To the best of our knowledge this is the first study investigating the effectiveness of an antiviral therapy against influenza virus by single cell transcriptomic studies.

Lignin: A Sustainable Antiviral Coating Material.

Transmission of viruses through contact with contaminated surfaces is an important pathway for the spread of infections. Antiviral surface coatings are useful to minimize such risks. Current state-of-the-art approaches toward antiviral surface coatings either involve metal-based materials or complex synthetic polymers. These approaches, however, even if successful, will have to face great challenges when it comes to large-scale applications and their environmental sustainability. Here, an antiviral surface coating was prepared by spin-coating lignin, a natural biomass residue of the paper production industry. We show effective inactivation of herpes simplex virus type 2 (>99% after 30 min) on a surface coating that is low-cost and environmentally sustainable. The antiviral mechanism of the lignin surface was investigated and is attributed to reactive oxygen species generated upon oxidation of lignin phenols. This mechanism does not consume the surface coating (as opposed to the release of a specific antiviral agent) and does not require regeneration. The coating is stable in ambient conditions, as demonstrated in a 6 month aging study that did not reveal any decrease in antiviral activity. This research suggests that natural compounds may be used for the development of affordable and sustainable antiviral coatings.

Geneticin shows selective antiviral activity against SARS-CoV-2 by interfering with programmed -1 ribosomal frameshifting.

SARS-CoV-2 is currently causing an unprecedented pandemic. While vaccines are massively deployed, we still lack effective large-scale antiviral therapies. In the quest for antivirals targeting conserved structures, we focused on molecules able to bind viral RNA secondary structures. Aminoglycosides are a class of antibiotics known to interact with the ribosomal RNA of both prokaryotes and eukaryotes and have previously been shown to exert antiviral activities by interacting with viral RNA. Here we show that the aminoglycoside geneticin is endowed with antiviral activity against all tested variants of SARS-CoV-2, in different cell lines and in a respiratory tissue model at non-toxic concentrations. The mechanism of action is an early inhibition of RNA replication and protein expression related to a decrease in the efficiency of the -1 programmed ribosomal frameshift (PRF) signal of SARS-CoV-2. Using in silico modelling, we have identified a potential binding site of geneticin in the pseudoknot of frameshift RNA motif. Moreover, we have selected, through virtual screening, additional RNA binding compounds, interacting with the same site with increased potency.

Polyanionic Amphiphilic Dendritic Polyglycerols as Broad-Spectrum Viral Inhibitors with a Virucidal Mechanism.

Heparin has been known to be a broad-spectrum inhibitor of viral infection for almost 70 years, and it has been used as a medication for almost 90 years due to its anticoagulant effect. This nontoxic biocompatible polymer efficiently binds to many types of viruses and prevents their attachment to cell membranes. However, the anticoagulant properties are limiting their use as an antiviral drug. Many heparin-like compounds have been developed throughout the years; however, the reversible nature of the virus inhibition mechanism has prevented their translation to the clinics. In vivo, such a mechanism requires the unrealistic maintenance of the concentration above the binding constant. Recently, we have shown that the addition of long hydrophobic linkers to heparin-like compounds renders the interaction irreversible while maintaining the low-toxicity and broad-spectrum activity. To date, such hydrophobic linkers have been used to create heparin-like gold nanoparticles and ß-cyclodextrins. The former achieves a nanomolar inhibition concentration on a non-biodegradable scaffold. The latter, on a fully biodegradable scaffold, shows only a micromolar inhibition concentration. Here, we report that the addition of hydrophobic linkers to a new type of multifunctional scaffold (dendritic polyglycerol, dPG) creates biocompatible compounds endowed with nanomolar activity. Furthermore, we present an in-depth analysis of the molecular design rules needed to achieve irreversible virus inhibition. The most active compound (dPG-5) showed nanomolar activity against herpes simplex virus 2 (HSV-2) and respiratory syncytial virus (RSV), giving a proof-of-principle for broad-spectrum while keeping low-toxicity. In addition, we demonstrate that the virucidal activity leads to the release of viral DNA upon the interaction between the virus and our polyanionic dendritic polymers. We believe that this paper will be a stepping stone toward the design of a new class of irreversible nontoxic broad-spectrum antivirals.

Alpha and Omicron SARS-CoV-2 Adaptation in an Upper Respiratory Tract Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing an unprecedented pandemic. Although vaccines and antivirals are limiting the spread, SARS-CoV-2 is still under selective pressure in human and animal populations, as demonstrated by the emergence of variants of concern. To better understand the driving forces leading to new subtypes of SARS-CoV-2, we infected an ex vivo cell model of the human upper respiratory tract with Alpha and Omicron BA.1 variants for one month. Although viral RNA was detected during the entire course of the infection, infectious virus production decreased over time. Sequencing analysis did not show any adaptation in the spike protein, suggesting a key role for the adaptive immune response or adaptation to other anatomical sites for the evolution of SARS-CoV-2.

Clinical severe acute respiratory syndrome coronavirus 2 isolation and antiviral testing.

Severe acute respiratory syndrome coronavirus 2 is an RNA virus currently causing a pandemic. Due to errors during replication, mutations can occur and result in cell adaptation by the virus or in the rise of new variants. This can change the attachment receptors' usage, result in different morphology of plaques, and can affect as well antiviral development. Indeed, a molecule can be active on laboratory strains but not necessarily on circulating strains or be effective only against some viral variants. Experiments with clinical samples with limited cell adaptation should be performed to confirm the efficiency of drugs of interest. In this protocol, we present a method to culture severe acute respiratory syndrome coronavirus 2 from nasopharyngeal swabs, obtain a high viral titer while limiting cell adaptation, and assess antiviral efficiency.

Viruses Like Sugars: How to Assess Glycan Involvement in Viral Attachment.

The first step of viral infection requires interaction with the host cell. Before finding the specific receptor that triggers entry, the majority of viruses interact with the glycocalyx. Identifying the carbohydrates that are specifically recognized by different viruses is important both for assessing the cellular tropism and for identifying new antiviral targets. Advances in the tools available for studying glycan-protein interactions have made it possible to identify them more rapidly; however, it is important to recognize the limitations of these methods in order to draw relevant conclusions. Here, we review different techniques: genetic screening, glycan arrays, enzymatic and pharmacological approaches, and surface plasmon resonance. We then detail the glycan interactions of enterovirus D68 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlighting the aspects that need further clarification.

Methylene Blue has a potent antiviral activity against SARS-CoV-2 and H1N1 influenza virus in the absence of UV-activation in vitro.

Methylene blue is an FDA (Food and Drug Administration) and EMA (European Medicines Agency) approved drug with an excellent safety profile. It displays broad-spectrum virucidal activity in the presence of UV light and has been shown to be effective in inactivating various viruses in blood products prior to transfusions. In addition, its use has been validated for methemoglobinemia and malaria treatment. In this study, we first evaluated the virucidal activity of methylene blue against influenza virus H1N1 upon different incubation times and in the presence or absence of light activation, and then against SARS-CoV-2. We further assessed the therapeutic activity of methylene blue by administering it to cells previously infected with SARS-CoV-2. Finally, we examined the effect of co-administration of the drug together with immune serum. Our findings reveal that methylene blue displays virucidal preventive or therapeutic activity against influenza virus H1N1 and SARS-CoV-2 at low micromolar concentrations and in the absence of UV-activation. We also confirm that MB antiviral activity is based on several mechanisms of action as the extent of genomic RNA degradation is higher in presence of light and after long exposure. Our work supports the interest of testing methylene blue in clinical studies to confirm a preventive and/or therapeutic efficacy against both influenza virus H1N1 and SARS-CoV-2 infections.

A high-throughput cell- and virus-free assay shows reduced neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma.

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies in the serum of an individual indicates previous infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral spike protein are more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, inflexible, and potentially biohazardous. Here, we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 spike protein binding to the angiotensin-converting enzyme 2 (ACE2) receptor. This high-throughput method matches the performance of the gold standard live virus infection assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific immunoglobulin G titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 spike protein variants of concern. We used our assay to profile serum samples from 59 patients hospitalized with coronavirus disease 2019 (COVID-19). We found that although most sera had high activity against the 2019-nCoV parental spike protein and, to a lesser extent, the α (B.1.1.7) variant, only 58% of serum samples could efficiently neutralize a spike protein derivative containing mutations present in the ß (B.1.351) variant. Thus, we have developed an assay that can evaluate effective neutralizing antibody responses to SARS-CoV-2 spike protein variants of concern after natural infection and that can be applied to characterize vaccine-induced antibody responses or to assess the potency of monoclonal antibodies.

Non-Toxic Virucidal Macromolecules Show High Efficacy Against Influenza Virus Ex Vivo and In Vivo.

Influenza is one of the most widespread viral infections worldwide and represents a major public health problem. The risk that one of the next pandemics is caused by an influenza strain is high. It is important to develop broad-spectrum influenza antivirals to be ready for any possible vaccine shortcomings. Anti-influenza drugs are available but they are far from ideal. Arguably, an ideal antiviral should target conserved viral domains and be virucidal, that is, irreversibly inhibit viral infectivity. Here, a new class of broad-spectrum anti-influenza macromolecules is described that meets these criteria and display exceedingly low toxicity. These compounds are based on a cyclodextrin core modified on its primary face with long hydrophobic linkers terminated either in 6'sialyl-N-acetyllactosamine (6'SLN) or in 3'SLN. SLN enables nanomolar inhibition of the viruses while the hydrophobic linkers confer irreversibility to the inhibition. The combination of these two properties allows for efficacy in vitro against several human or avian influenza strains, as well as against a 2009 pandemic influenza strain ex vivo. Importantly, it is shown that, in mice, one of the compounds provides therapeutic efficacy when administered 24 h post-infection allowing 90% survival as opposed to no survival for the placebo and oseltamivir.

The tyrosine kinase inhibitor nilotinib inhibits SARS-CoV-2 in vitro.

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019, no vaccine has been approved to counter this infection and the available treatments are mainly directed against the immune pathology caused by the infection. The coronavirus disease 2019 (COVID-19) is currently causing a worldwide pandemic, pointing the urgent need for effective treatment. In such emergency, drug repurposing presents the best option for a rapid antiviral response. We assess here the in vitro activity of nilotinib, imatinib and dasatinib, three Abl tyrosine kinase inhibitors, against SARS-CoV-2. Although the last two compounds do not show antiviral efficacy, we observe inhibition with nilotinib in Vero-E6 cells and Calu-3 cells with EC50s of 1.44 µM and 3.06 µM, respectively. These values are close to the mean peak concentration of nilotinib observed at steady state in serum, making this compound a potential candidate for treatment of COVID-19 in vivo.

Do we need a contact tracing app?

The goal of this paper is to shed some light on the usefulness of a contact tracing smartphone app for the containment of the COVID-19 pandemic. We review the basics of contact tracing during the spread of a virus, we contextualize the numbers to the case of COVID-19 and we analyze the state of the art for proximity detection using Bluetooth Low Energy. Our contribution is to assess if there is scientific evidence of the benefit of a contact tracing app in slowing down the spread of the virus using present technologies. Our conclusion is that such evidence is lacking, and we should re-think the introduction of such a privacy-invasive measure.

SARS-CoV-2 Inhibition by Sulfonated Compounds.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) depends on angiotensin converting enzyme 2 (ACE2) for cellular entry, but it might also rely on attachment receptors such as heparan sulfates. Several groups have recently demonstrated an affinity of the SARS-CoV2 spike protein for heparan sulfates and a reduced binding to cells in the presence of heparin or heparinase treatment. Here, we investigated the inhibitory activity of several sulfated and sulfonated molecules, which prevent interaction with heparan sulfates, against vesicular stomatitis virus (VSV)-pseudotyped-SARS-CoV-2 and the authentic SARS-CoV-2. Sulfonated cyclodextrins and nanoparticles that have recently shown broad-spectrum non-toxic virucidal activity against many heparan sulfates binding viruses showed inhibitory activity in the micromolar and nanomolar ranges, respectively. In stark contrast with the mechanisms that these compounds present for these other viruses, the inhibition against SARS-CoV-2 was found to be simply reversible.

Sulfonated Nanomaterials with Broad-Spectrum Antiviral Activity Extending beyond Heparan Sulfate-Dependent Viruses.

Viral infections are among the main causes of death worldwide, and we lack antivirals for the majority of viruses. Heparin-like sulfated or sulfonated compounds have been known for decades for their ability to prevent infection by heparan sulfate proteoglycan (HSPG)-dependent viruses but only in a reversible way. We have previously shown that gold nanoparticles and ß-cyclodextrins coated with mercapto-undecane sulfonic acid (MUS) inhibit HSPG-dependent viruses irreversibly while retaining the low-toxicity profile of most heparin-like compounds. In this work, we show that, in stark contrast to heparin, these compounds also inhibit different strains of influenza virus and vesicular stomatitis virus (VSV), which do not bind HSPG. The antiviral action is virucidal and irreversible for influenza A virus (H1N1), while for VSV, there is a reversible inhibition of viral attachment to the cell. These results further broaden the spectrum of activity of MUS-coated gold nanoparticles and ß-cyclodextrins.