RESUMEN
Tick-borne encephalitis (TBE) virus is the most prevalent tick-transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)-based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. The RVP-based SNT for TBEV exhibited a highly significant correlation with the traditional virus-based SNT (R2 = 0.8637, p < 0.0001). The RVP-based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%-97.4%) and specificity of 100% (95% CI: 81.6%-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV-positive by ELISA but negative by RVP-based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP-based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.
Asunto(s)
Anticuerpos Antivirales , Reacciones Cruzadas , Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Ensayo de Inmunoadsorción Enzimática , Pruebas de Neutralización , Sensibilidad y Especificidad , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Humanos , Anticuerpos Antivirales/sangre , Pruebas de Neutralización/métodos , Encefalitis Transmitida por Garrapatas/diagnóstico , Encefalitis Transmitida por Garrapatas/virología , Ensayo de Inmunoadsorción Enzimática/métodos , Virión/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , AnimalesRESUMEN
We currently lack antivirals for most human viruses. In a quest for new molecules, focusing on viral RNA, instead of viral proteins, can represent a promising strategy. In this study, new inhibitors were identified starting from a published crystal structure of the tertiary SARS-CoV-2 RNA involved in the -1 programmed ribosomal frameshift. The pseudoknot structure was refined, and a virtual screening was performed using the repository of binders to the nucleic acid library, taking into consideration RNA flexibility. Hit compounds were validated against the wild-type virus and with a dual-luciferase assay measuring the frameshift efficiency. Several active molecules were identified. Our study reveals new inhibitors of SARS-CoV-2 but also highlights the feasibility of targeting RNA starting from virtual screening, a strategy that could be broadly applied to drug development.
Asunto(s)
Antivirales , Evaluación Preclínica de Medicamentos , ARN Viral , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , ARN Viral/metabolismo , ARN Viral/antagonistas & inhibidores , ARN Viral/genética , Evaluación Preclínica de Medicamentos/métodos , Humanos , Conformación de Ácido Nucleico , Simulación del Acoplamiento Molecular , Interfaz Usuario-Computador , Modelos MolecularesRESUMEN
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.
Asunto(s)
COVID-19 , ARN Viral , Humanos , ARN Viral/genética , SARS-CoV-2/genética , Antivirales/farmacología , Antivirales/uso terapéutico , BioensayoRESUMEN
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.
Asunto(s)
Estrógenos no Esteroides , Gripe Humana , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Humanos , Hemaglutininas , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/prevención & control , Orthomyxoviridae/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Antivirales/farmacologíaRESUMEN
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.
Asunto(s)
Nanopartículas del Metal , Virus , Anticoagulantes/farmacología , Antivirales/química , Antivirales/farmacología , Glicerol , Oro , Heparina/farmacología , Polímeros/farmacologíaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Nanopartículas del Metal , Virus , Antivirales/farmacología , Oro , Heparitina Sulfato/farmacologíaRESUMEN
Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient's respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus's dissemination.
Asunto(s)
Proteínas de la Cápside/genética , Enterovirus Humano A/fisiología , Enfermedad de Boca, Mano y Pie/virología , Heparitina Sulfato/metabolismo , Tropismo Viral/genética , Animales , Enterovirus Humano A/patogenicidad , Enfermedad de Boca, Mano y Pie/genética , Enfermedad de Boca, Mano y Pie/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Ratones , Mutación , Receptores Depuradores/metabolismo , Replicación Viral/genéticaRESUMEN
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 (â¼190 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.
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Antivirales , Materiales Biomiméticos , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 2/metabolismo , Nanopartículas , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/metabolismo , Animales , Antivirales/química , Antivirales/farmacología , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/farmacología , Herpes Simple/metabolismo , Herpes Simple/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/patologíaRESUMEN
BACKGROUND: The leading cause of acute illnesses, respiratory viruses, typically cause self-limited diseases, although severe complications can occur in fragile patients. Rhinoviruses (RVs), respiratory enteroviruses (EVs), influenza virus, respiratory syncytial viruses (RSVs), and coronaviruses are highly prevalent respiratory pathogens, but because of the lack of reliable animal models, their differential pathogenesis remains poorly characterized. OBJECTIVE: We sought to compare infections by respiratory viruses isolated from clinical specimens using reconstituted human airway epithelia. METHODS: Tissues were infected with RV-A55, RV-A49, RV-B48, RV-C8, and RV-C15; respiratory EV-D68; influenza virus H3N2; RSV-B; and human coronavirus (HCoV)-OC43. Replication kinetics, cell tropism, effect on tissue integrity, and cytokine secretion were compared. Viral adaptation and tissue response were assessed through RNA sequencing. RESULTS: RVs, RSV-B, and HCoV-OC43 infected ciliated cells and caused no major cell death, whereas H3N2 and EV-D68 induced ciliated cell loss and tissue integrity disruption. H3N2 was also detected in rare goblet and basal cells. All viruses, except RV-B48 and HCoV-OC43, altered cilia beating and mucociliary clearance. H3N2 was the strongest cytokine inducer, and HCoV-OC43 was the weakest. Persistent infection was observed in all cases. RNA sequencing highlighted perturbation of tissue metabolism and induction of a transient but important immune response at 4 days after infection. No majority mutations emerged in the viral population. CONCLUSION: Our results highlight the differential in vitro pathogenesis of respiratory viruses during the acute infection phase and their ability to persist under immune tolerance. These data help to appreciate the range of disease severity observed in vivo and the occurrence of chronic respiratory tract infections in immunocompromised hosts.
Asunto(s)
Infecciones por Virus ARN/fisiopatología , Infecciones por Virus ARN/virología , Mucosa Respiratoria/virología , Humanos , Virus ARNRESUMEN
BACKGROUND: Human rotavirus (HRoV) is the leading cause of severe gastroenteritis in infants and children under the age of five years. No specific antiviral drug is available for HRoV infections and the treatment of viral diarrhea is mainly based on rehydration and zinc treatment. In this study, we explored medicinal plants endemic to Turkey flora as a source of anti-HRoV compunds. METHODS: We performed an antiviral screening on Ballota macrodonta, Salvia cryptantha and Rindera lanata extracts by focus reduction assay. The extract with the highest selectivity index (SI) was selected; its antiviral activity was further confirmed against other HRoV strains and by virus yield reduction assay. The step of viral replicative cycle putatively inhibited was investigated by in vitro assays. RESULTS: The methanolic extract of R. lanata (Boraginaceae) showed the most favourable selectivity index. This extract exhibited a dose-dependent inhibitory activity against three different HRoV strains (EC50 values ranging from 5.8 µg/ml to 25.5 µg/ml), but was inactive or barely active against other RNA viruses, namely human rhinovirus and respiratory syncytial virus. The R. lanata extract targets the early steps of HRoV infection, likely by hampering virus penetration into the cells. CONCLUSION: These results make the R. lanata methanolic extract a promising starting material for a bioguided-fractionation aimed at identifying anti-HRoV compounds. Further work is required to isolate the active principle and assess its clinical potential.
Asunto(s)
Antivirales/análisis , Boraginaceae/química , Infecciones por Rotavirus/tratamiento farmacológico , Rotavirus/efectos de los fármacos , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Chlorocebus aethiops , Pruebas de Sensibilidad Microbiana , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéuticoRESUMEN
Ficus religiosa extracts have been used in traditional Indian medicine to treat sexually transmitted infections such as gonorrhea and genital ulcers. The aim of this study was to investigate the antiviral activity of F. religiosa extracts against herpes simplex virus type 2 (HSV-2), the main causative agent of genital ulcers and sores. Water and chloroform bark extracts were the most active against HSV-2, and also against an acyclovir-resistant strain. We demonstrate that the water extract has a direct virus-inactivating activity. By contrast, the chloroform extract inhibits viral attachment and entry and limits the production of viral progeny.
Asunto(s)
Antivirales/farmacología , Ficus/química , Herpesvirus Humano 2/efectos de los fármacos , Corteza de la Planta/química , Extractos Vegetales/farmacología , Antivirales/aislamiento & purificación , Herpesvirus Humano 2/fisiología , Humanos , Extractos Vegetales/aislamiento & purificación , Acoplamiento Viral/efectos de los fármacos , Inactivación de Virus , Internalización del Virus/efectos de los fármacosRESUMEN
The agmatine-containing poly(amidoamine) polymer AGMA1 was recently shown to inhibit the infectivity of several viruses, including human papillomavirus 16 (HPV-16), that exploit cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The aim of this work was to assess the antiviral activity of AGMA1 and its spectrum of activity against a panel of low-risk and high-risk HPVs and to elucidate its mechanism of action. AGMA1 was found to be a potent inhibitor of mucosal HPV types (i.e., types 16, 31, 45, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 0.34 µg/ml and 0.73 µg/ml, and no evidence of cytotoxicity was observed. AGMA1 interacted with immobilized heparin and with cellular heparan sulfates, exerting its antiviral action by preventing virus attachment to the cell surface. The findings from this study indicate that AGMA1 is a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.
Asunto(s)
Agmatina/análogos & derivados , Antivirales/metabolismo , Antivirales/farmacología , Heparitina Sulfato/metabolismo , Papillomaviridae/efectos de los fármacos , Poliaminas/metabolismo , Poliaminas/farmacología , Agmatina/metabolismo , Agmatina/farmacología , Línea Celular , Humanos , Acoplamiento Viral/efectos de los fármacosRESUMEN
Poly(amidoamine)s (PAAs) are multifunctional tert-amine polymers endowed with high structural versatility. Here we report on the screening of a minilibrary of PAAs against a panel of viruses. The PAA AGMA1 showed antiviral activity against herpes simplex virus, human cytomegalovirus, human papillomavirus 16, and respiratory syncytial virus but not against human rotavirus and vesicular stomatitis virus. The results suggest the contribution of both a polycationic nature and side guanidine groups in imparting antiviral activity.
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Agmatina/química , Antivirales/química , Antivirales/farmacología , Poliaminas/química , Poliaminas/farmacología , Rotavirus/efectos de los fármacos , Citomegalovirus/efectos de los fármacos , Humanos , Simplexvirus/efectos de los fármacos , Vesiculovirus/efectos de los fármacosRESUMEN
Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The interaction between RSV and HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was used to generate a collection of sulfated K5 derivatives with a backbone structure that mimics the heparin/heparan sulfate biosynthetic precursor. The screening of a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation revealed the highly sulfated K5 derivatives K5-N,OS(H) and K5-OS(H) to be inhibitors of RSV. Their 50% inhibitory concentrations were between 1.07 nM and 3.81 nM in two different cell lines, and no evidence of cytotoxicity was observed. Inhibition of RSV infection was maintained in binding and attachment assays but not in preattachment assays. Moreover, antiviral activity was also evident when the K5 derivatives were added postinfection, both in cell-to-cell spread and viral yield reduction assays. Finally, both K5-N,OS(H) and K5-OS(H) prevented RSV infection in human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. Together, these features put K5-N,OS(H) and K5-OS(H) forward as attractive candidates for further development as RSV inhibitors.
Asunto(s)
Antivirales/farmacología , Cápsulas Bacterianas/química , Polisacáridos Bacterianos/farmacología , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Acoplamiento Viral/efectos de los fármacos , Antivirales/aislamiento & purificación , Bronquios/efectos de los fármacos , Bronquios/virología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/virología , Escherichia coli/química , Células Gigantes/efectos de los fármacos , Células Gigantes/ultraestructura , Heparina/farmacología , Humanos , Polisacáridos Bacterianos/aislamiento & purificación , Virus Sincitial Respiratorio Humano/fisiología , Técnicas de Cultivo de Tejidos , Tráquea/efectos de los fármacos , Tráquea/virología , Carga Viral/efectos de los fármacos , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacosRESUMEN
AIMS: Several studies have recently reported the detection of oncogenic human papillomaviruses (HPV) in human milk of a minority of lactating mothers. These findings raised safety concerns in the context of human donor milk banking given the potential risk of HPV transmission to recipient infants. The aim of this study was to investigate whether the Holder pasteurization, a procedure currently in use in human donor milk banks for milk pasteurization, completely inactivates high-risk and low-risk HPV. METHODS: HPV pseudoviruses (PsV) were generated, spiked into cell culture medium or donor human milk and subjected to thermal inactivation. HPV PsV infectivity and morphological integrity was analyzed by cell-based assay and by electron microscopy, respectively. RESULTS: The Holder pasteurization completely inactivated the infectivity of high-risk (types 16 and 18) and low-risk (type 6) HPV both in cell culture medium and in human milk causing PsV particle disassembly. CONCLUSIONS: The results presented here indicate that the Holder pasteurization is an efficient procedure to inactivate high-risk and low-risk HPV thus preventing the potential risk of their transmission through human donor milk.
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Alphapapillomavirus , Bancos de Leche Humana , Leche Humana/virología , Pasteurización/métodos , Alphapapillomavirus/aislamiento & purificación , Alphapapillomavirus/fisiología , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía Electrónica , Riesgo , Inactivación de VirusRESUMEN
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.
RESUMEN
Most viruses start their invasion by binding to glycoproteins' moieties on the cell surface (heparan sulfate proteoglycans [HSPG] or sialic acid [SA]). Antivirals mimicking these moieties multivalently are known as broad-spectrum multivalent entry inhibitors (MEI). Due to their reversible mechanism, efficacy is lost when concentrations fall below an inhibitory threshold. To overcome this limitation, we modify MEIs with hydrophobic arms rendering the inhibitory mechanism irreversible, i.e., preventing the efficacy loss upon dilution. However, all our HSPG-mimicking MEIs only showed reversible inhibition against HSPG-binding SARS-CoV-2. Here, we present a systematic investigation of a series of small molecules, all containing a core and multiple hydrophobic arms terminated with HSPG-mimicking moieties. We identify the ones that have irreversible inhibition against all viruses including SARS-CoV-2 and discuss their design principles. We show efficacy in vivo against SARS-CoV-2 in a Syrian hamster model through both intranasal instillation and aerosol inhalation in a therapeutic setting (12 h postinfection). We also show the utility of the presented design rules in producing SA-mimicking MEIs with irreversible inhibition against SA-binding influenza viruses.
RESUMEN
BACKGROUND: Nauclea latifolia Smith, a shrub belonging to the family Rubiaceae is a very popular medicinal plant in Cameroon and neighboring countries where it is used to treat jaundice, yellow fever, rheumatism, abdominal pains, hepatitis, diarrhea, dysentery, hypertension, as well as diabetes. The ethno-medicinal use against yellow fever, jaundice and diarrhea prompted us to investigate on the antiviral activity of the root bark of N. latifolia. In this study, HSV-2 was chosen as a viral model because of its strong impact on HIV transmission and acquisition. METHODS: The crude extract under study was prepared by maceration of air-dried and powdered roots barks of N. latifolia in CH2Cl2/MeOH (50:50) mixture for 48 hours, then it was subjected to filtration and evaporation under vacuum. A phytochemical analysis of the crude extract was performed by High Performance Liquid Chromatography coupled with a photodiode array and mass spectrometry (HPLC-PDA-ESI-qMS). The anti-HSV-2 activity was assayed in vitro by plaque reduction and virus yield assays and the major mechanism of action was investigated by virucidal and time of addition assays. Data values were compared using the Extra sum of squares F test of program GraphPad PRISM 4. RESULTS: The main components detected in the extract belong to the class of indole alkaloids characteristic of Nauclea genus. Strictosamide, vincosamide and pumiloside were tentatively identified together with quinovic acid glycoside. N. latifolia crude extract inhibited both acyclovir sensitive and acyclovir resistant HSV-2 strains, with IC50 values of 5.38 µg/ml for the former and 7.17 µg/ml for the latter. The extract was found to be most active when added post-infection, with IC50 of 3.63 µg/ml. CONCLUSION: The results of this work partly justify the empirical use of N. latifolia in traditional medicine for the treatment of viral diseases. This extract could be a promising rough material for the development of a new and more effective modern anti-HSV-2 medication also active against acyclovir-resistant HSV-2 strains.
Asunto(s)
Antivirales/farmacología , Herpesvirus Humano 2/efectos de los fármacos , Extractos Vegetales/farmacología , Raíces de Plantas/química , Rubiaceae/química , Antivirales/química , Camerún , Medicina Tradicional , Extractos Vegetales/químicaRESUMEN
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.