Magnolol against enterovirus 71 by targeting Nrf2-SLC7A11-GSH pathway.

Enterovirus 71 (EV71), a prominent pathogen associated with hand, foot, and mouth disease (HFMD), has been reported worldwide. To date, the advancement of effective drugs targeting EV71 remains in the preliminary experimental stage. In this study, magnolol demonstrated a significant dose-dependent inhibition of EV71 replication in vitro. It upregulated the overall expression level of nuclear factor erythroid 2 - related factor 2 (Nrf2) and facilitated its nucleus translocation, resulting in the increased expression of various ferroptosis inhibitory genes. This process led to a reduction in reactive oxygen species (ROS) accumulation induced by viral infection. Additionally, magnolol exhibited a broad-spectrum antiviral effect against enteroviruses. Notably, treatment with magnolol substantially enhanced the survival rate of EV71-infected mice, attenuated viral load in heart, liver, brain, and limb tissues, and mitigated tissue inflammation. Taken together, magnolol emerges as a promising candidate for the development of anti-EV71 drugs.

Fluoxetine Successfully Treats Intracranial Enterovirus E18 Infection in a Patient with CD79a Deficiency Arising from Segmental Uniparental Disomy of Chromosome 19.

The pre BCR complex plays a crucial role in B cell production, and its successful expression marks the B cell differentiation from the pro-B to pre-B. The CD79a and CD79b mutations, encoding Igα and Igß respectively, have been identified as the cause of autosomal recessive agammaglobulinemia (ARA). Here, we present a case of a patient with a homozygous CD79a mutation, exhibiting recurrent respiratory infections, diarrhea, growth and development delay, unique facial abnormalities and microcephaly, as well as neurological symptoms including tethered spinal cord, sacral canal cyst, and chronic enteroviral E18 meningitis. Complete blockade of the early B cell development in the bone marrow of the patient results in the absence of peripheral circulating mature B cells. Whole exome sequencing revealed a Loss of Heterozygosity (LOH) of approximately 19.20Mb containing CD79a on chromosome 19 in the patient. This is the first case of a homozygous CD79a mutation caused by segmental uniparental diploid (UPD). Another key outcome of this study is the effective management of long-term chronic enteroviral meningitis using a combination of intravenous immunoglobulin (IVIG) and fluoxetine. This approach offers compelling evidence of fluoxetine's utility in treating enteroviral meningitis, particularly in immunocompromised patients.

Doxycycline inhibits neurotropic enterovirus proliferation in vitro.

Human enteroviruses (EVs) represent a global public health concern due to their association with a range of serious pediatric illnesses. Despite the high morbidity and mortality exerted by EVs, no broad-spectrum antivirals are currently available. Herein, we presented evidence that doxycycline can inhibit in vitro replication of various neurotropic EVs, including enterovirus A71 (EV-A71), enterovirus D68 (EV-D68), and coxsackievirus (CV)-A6, in a dose-dependent manner. Further investigations indicated that the drug primarily acted at the post-entry stage of virus infection in vitro, with inhibitory effects reaching up to 89 % for EV-A71 when administered two hours post-infection. These findings provide valuable insights for the development of antiviral drugs against EV infections.

Inhibition of ESCRT-independent extracellular vesicles biogenesis suppresses enterovirus 71 replication and pathogenesis in mice.

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), neurological complications, and even fatalities in infants. Clinically, the increase of extracellular vesicles (EVs) in EV71 patients' serum was highly associated with the severity of HFMD. EV71 boosts EVs biogenesis in an endosomal sorting complex required for transport (ESCRT)-dependent manner to facilitate viral replication. Yet, the impact of EVs-derived from ESCRT-independent pathway on EV71 replication and pathogenesis is highly concerned. Here, we assessed the effects of EV71-induced EVs from ESCRT-independent pathway on viral replication and pathogenesis by GW4869, a neutral sphingomyelinase inhibitor. Detailly, in EV71-infected mice, blockade of the biogenesis of tissue-derived EVs in the presence of GW4869 restored body weight loss, attenuated clinical scores, and improved survival rates. Furthermore, GW4869 dampens EVs biogenesis to reduce viral load and pathogenesis in multiple tissues of EV71-infected mice. Consistently, GW4869 treatment in a human intestinal epithelial HT29 cells decreased the biogenesis of EVs, in which the progeny EV71 particle was cloaked, leading to the reduction of viral infection and replication. Collectively, GW4869 inhibits EV71-induced EVs in an ESCRT-independent pathway and ultimately suppresses EV71 replication and pathogenesis. Our study provides a novel strategy for the development of therapeutic agents in the treatment for EV71-associated HFMD.

Disseminated Enterovirus Infection in a Patient Affected by Follicular Lymphoma Treated with Obinutuzumab: A Case Report and a Narrative Review of the Literature.

Background and Objectives: the principal purpose of this literature review is to cluster adults with hematological malignancies after treatment or on maintenance with obinutuzumab who experienced disseminated EV infection to understand clinical characteristics and outcome of this rare condition in these patients. We report the first clinical case of a male affected by follicular lymphoma treated with immune-chemotherapy including obinutuzumab who was affected by disseminated EV infection with cardiovascular involvement. Materials and Methods: this narrative review summarizes all the research about disseminated EV infection in immunosuppressed adult patients treated with obinutuzumab from January 2000 to January 2024 using the Scale for the Assessment of Narrative Review Articles (SANRA) flow-chart. We performed a descriptive statistic using the standard statistical measures for quantitative data. Results: we included six studies, five case reports, and one case report with literature analysis. We collected a total of seven patients, all female, with disseminated EV infection. The most common signs and clinical presentations of EV infection were fever and encephalitis symptoms (N = 6, 85.7%), followed by hepatitis/acute liver failure (N = 5, 71.4%). Conclusions: onco-hematological patients who receive immune-chemotherapy with a combination of treatments which depress adaptative immunity, which includes the antiCD20 obinutuzumab, could be at higher risk of disseminated EV infection, including CNS and cardiac involvement.

Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

Miltefosine reduces coxsackievirus B3 lethality of mice with enhanced STAT3 activation.

Coxsackievirus B3 (CVB3), one serotype of enteroviruses, can induce fatal myocarditis and hepatitis in neonates, but both treatment and vaccine are unavailable. Few reports tested antivirals to reduce CVB3. Several antivirals were developed against other enterovirus serotypes, but these antivirals failed in clinical trials due to side effects and drug resistance. Repurposing of clinical drugs targeting cellular factors, which enhance viral replication, may be another option. Parasite and cancer studies showed that the cellular protein kinase B (Akt) decreases interferon (IFN), apoptosis, and interleukin (IL)-6-induced STAT3 responses, which suppress CVB3 replication. Furthermore, miltefosine, the Akt inhibitor used in the clinic for parasite infections, enhances IL-6, IFN, and apoptosis responses in treated patients, suggesting that miltefosine could be the potential antiviral for CVB3. This study was therefore designated to test the antiviral effects of miltefosine against CVB3 in vitro and especially, in mice, as few studies test miltefosine in vitro, but not in vivo. In vitro results showed that miltefosine inhibited viral replication with enhanced activation of the cellular transcription factor, STAT3, which is reported to reduce CVB3 both in vitro and in mice. Notably, STAT3 knockdown abolished the anti-CVB3 activity of miltefosine in vitro. Mouse studies demonstrated that miltefosine pretreatment reduced CVB3 lethality of mice with decreased virus loads, organ damage, and apoptosis, but enhanced STAT3 activation. Miltefosine could be prophylaxis for CVB3 by targeting Akt to enhance STAT3 activation in the mechanism, which is independent of IFN responses and hardly reported in pathogen infections.

The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

Advances in anti-EV-A71 drug development research.

BACKGROUND: Enterovirus A71 (EV-A71) is capable of causing hand, foot and mouth disease (HFMD), which may lead to neurological sequelae and even death. As EV-A71 is resistant to environmental changes and mutates easily, there is still a lack of effective treatments or globally available vaccines. AIM OF REVIEW: For more than 50 years since the HFMD epidemic, related drug research has been conducted. Progress in this area can promote the further application of existing potential drugs and develop more efficient and safe antiviral drugs, and provide useful reference for protecting the younger generation and maintaining public health security. KEY SCIENTIFIC CONCEPTS OF REVIEW: At present, researchers have identified hundreds of EV-A71 inhibitors based on screening repurposed drugs, targeted structural design, and rational modification of previously effective drugs as the main development strategies. This review systematically introduces the current potential drugs to inhibit EV-A71 infection, including viral inhibitors targeting key sites such as the viral capsid, RNA-dependent RNA polymerase (RdRp), 2C protein, internal ribosome entry site (IRES), 3C proteinase (3Cpro), and 2A proteinase (2Apro), starting from each stage of the viral life cycle. Meanwhile, the progress of host-targeting antiviral drugs and their development are summarized in terms of regulating host immunity, inhibiting autophagy or apoptosis, and regulating the cellular redox environment. In addition, the current clinical methods for the prevention and treatment of HFMD are summarized and discussed with the aim of providing support and recommendations for the treatment of enterovirus infections including EV-A71.

Licochalcone A regulates viral IRES activity to inhibit enterovirus replication.

Enterovirus D68 (EV-D68), belonging to the genus Enterovirus of the Picornavirus family, is an emerging pathogen that can cause neurological and respiratory diseases in children. However, there is little understanding of the pathogenesis of EV-D68, and no effective vaccine or drug for the prevention or treatment of the diseases caused by this virus is available. Autophagy is a cellular process that targets cytoplasmic proteins or organelles to the lysosomes for degradation. Enteroviruses strategically harness the host autophagy pathway to facilitate the completion of their life cycle. Therefore, we selected an autophagy compound library to screen for autophagy-related compounds that may affect viral growth. By using the neutralization screening assay, we identified a compound, 'licochalcone A' that significantly inhibited EV-D68 replication. To investigate the mechanism by which licochalcone A inhibits EV-D68 replication and to identify the viral life cycle stage it inhibits, the time-of-addition, viral attachment, viral entry, and dual-luciferase reporter assays were performed. The results of the time-of-addition assay showed that licochalcone A, a characteristic chalcone found in liquorice roots and widely used in traditional Chinese medicine, inhibits EV-D68 replication during the early stages of the viral life cycle, while those of the dual-luciferase reporter assay showed that licochalcone A does not regulate viral attachment and entry, but inhibits EV-D68 IRES-dependent translation. Licochalcone A also inhibited enterovirus A71 and coxsackievirus B3 but did not significantly inhibit dengue virus 2 or human coronavirus 229E replication. Licochalcone A regulates IRES translation to inhibit EV-D68 viral replication.

Recent Advances in Enterovirus A71 Infection and Antiviral Agents.

Enterovirus A71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease (HFMD) that majorly affects children. Most of the time, HFMD is a mild disease but can progress to severe complications, such as meningitis, brain stem encephalitis, acute flaccid paralysis, and even death. HFMD caused by EV-A71 has emerged as an acutely infectious disease of highly pathogenic potential in the Asia-Pacific region. In this review, we introduced the properties and life cycle of EV-A71, and the pathogenesis and the pathophysiology of EV-A71 infection, including tissue tropism and host range of virus infection, the diseases caused by the virus, as well as the genes and host cell immune mechanisms of major diseases caused by enterovirus 71 (EV-A71) infection, such as encephalitis and neurologic pulmonary edema. At the same time, clinicopathologic characteristics of EV-A71 infection were introduced. There is currently no specific medication for EV-A71 infection, highlighting the urgency and significance of developing suitable anti-EV-A71 agents. This overview also summarizes the targets of existing anti-EV-A71 agents, including virus entry, translation, polyprotein processing, replication, assembly and release; interferons; interleukins; the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase B signaling pathways; the oxidative stress pathway; the ubiquitin-proteasome system; and so on. Furthermore, it overviews the effects of natural products, monoclonal antibodies, and RNA interference against EV-A71. It also discusses issues limiting the research of antiviral drugs. This review is a systematic and comprehensive summary of the mechanism and pathological characteristics of EV-A71 infection, the latest progress of existing anti-EV-A71 agents. It would provide better understanding and guidance for the research and application of EV-A71 infection and antiviral inhibitors.

Antiviral activity of SP81 peptide against Enterovirus A71 (EV-A71).

The hand, food, and mouth disease (HFMD) is primarily caused by Enterovirus A71 (EV-A71). EV-A71 outbreaks in the Asia Pacific have been associated with severe neurological disease and high fatalities. Currently, there are no FDA-approved antivirals for the treatment of EV-A71 infections. In this study, the SP81 peptide, derived from the VP1 capsid protein of EV-A71 was shown to be a promising antiviral candidate for the treatment of EV-A71 infections. SP81 peptide was non-toxic to RD cells up to 45 µM, with a half-maximal cytotoxic concentration (CC50) of 90.32 µM. SP81 peptide exerted antiviral effects during the pre- and post-infection stages with 50% inhibitory concentrations (IC50) of 4.529 µM and 1.192 µM, respectively. Direct virus inactivation of EV-A71 by the SP81 peptide was also observed with an IC50 of 8.076 µM. Additionally, the SP81 peptide exhibited direct virus inactivation of EV-A71 at 95% upon the addition of the SP81 peptide within 5 min. This study showed that the SP81 peptide exhibited significant inhibition of EV-A71 and could serve as a promising antiviral agent for further clinical development against EV-A71 infections.

Effects of glycine 64 substitutions in RNA-dependent RNA polymerase on ribavirin sensitivity and pathogenicity of coxsackievirus A6.

Hand, foot, and mouth disease (HFMD) caused by a group of enteroviruses is a global public health problem. In recent years, coxsackievirus A6 (CVA6) has emerged as an important HFMD agent. Previous studies have shown that mutations of glycine 64 in RNA-dependent RNA polymerase (3D polymerase), which is central to viral replication, cause phenotypic changes such as ribavirin resistance, increased replication fidelity, and virulence attenuation in poliovirus and enterovirus A71. In this study, we constructed CVA6 mutants with G64R, G64S, and G64T substitutions by site-directed mutagenesis in full-length cDNA of an infectious CVA6 strain cloned in pcDNA3.1. Viral RNA was obtained by in vitro transcription, and the rescued virus strains were propagated in RD cells. Sequencing after six passages revealed that G64S and G64T mutations were stably inherited, whereas G64R was genetically unstable and reversed to the wild type. Comparison of the biological characteristics of the wild-type and mutant CVA6 strains in an in vivo model (one-day-old ICR mice) revealed that the pathogenicity of CVA6-G64S and CVA6-G64T was significantly reduced compared to wild-type CVA6. In vitro experiments indicated the mutant CVA6-G64S and CVA6-G64T strains had increased resistance to 0.8 mM ribavirin and a decreased replication rate in the presence of 0.8 mM guanidine hydrochloride. Our results show that mutation of residue 64 reduces CVA6 susceptibility to ribavirin and increases CVA6 susceptibility to guanidine hydrochloride, together with increased replication fidelity and attenuated viral pathogenicity, thus laying a foundation for the development of safe and effective live attenuated CVA6 vaccine.

2C protein of Enterovirus: key protein of viral replication and antiviral target.

Enteroviruses (EVs) include many human pathogens of increasing public health concern. These EVs are often associated with mild clinical manifestations, but they can lead to serious complications such as encephalitis, meningitis, pneumonia, myocarditis or poliomyelitis. Despite significant advances, there is no approved antiviral therapy for the treatment of enterovirus infections. Due to the high genotypic diversity of EVs, molecules targeting highly conserved viral proteins may be considered for developing a pan-EV treatment. In this regard, the ATPase/Helicase 2C, which is a highly conserved non-structural protein among EVs, has essential functions for viral replication and is therefore an attractive antiviral target. Recent functional and structural studies on the 2C protein led to the identification of molecules showing ex vivo anti-EV activity and associated with resistance mutations on the coding sequence of the 2C protein. This review presents the current state of knowledge about the 2C protein from an antiviral target perspective and the mode of action of specific inhibitors for this therapeutic target.

Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIß.

Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect was related to a cellular phosphatidylinositol 4-kinase type IIIß (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevented infection efficiently in acute cell models, eradicated infection in a chronic cell model, and lowered virus amounts in pancreas and heart in an acute mouse model. Altogether, instead of acting through the RAF/MEK/ERK pathway, vemurafenib affects the cellular PI4KB and, hence, enterovirus replication, opening new possibilities to evaluate further the potential of vemurafenib as a repurposed drug in clinical care. IMPORTANCE Despite the prevalence and medical threat of enteroviruses, presently, there are no antivirals against them. Here, we show that vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIß (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses.

Neutralizing activity of intravenous immune globulin products against enterovirus D68 strains isolated in Japan.

BACKGROUND: Enterovirus D68 (EV-D68), belonging to Enterovirus D, is a unique human enterovirus mainly associated with common respiratory diseases. However, EV-D68 can cause severe respiratory diseases, and EV-D68 endemic is epidemiologically linked to current global epidemic of acute flaccid myelitis. METHODS: In this study, we measured neutralizing antibody titers against six clinical EV-D68 isolates in nine intravenous immune globulin (IVIG) products commercially available in Japan to assess their potential as therapeutic options for severe EV-D68 infection. RESULTS: Seven IVIG products manufactured from Japanese donors contained high neutralizing antibody titers (IC50 = 0.22-85.01 µg/mL) against all six EV-D68 strains. Apparent differences in neutralizing titers among the six EV-D68 strains were observed for all IVIG products derived from Japanese and non-Japanese blood donors. CONCLUSIONS: High levels of EV-D68-neutralizing antibodies in IVIG products manufactured from Japanese donors suggest that anti-EV-D68 antibodies are maintained in the Japanese donor population similarly as found in foreign blood donors. Apparent differences in neutralizing antibody titers against the six EV-D68 strains suggest distinct antigenicity among the strains used in this study regardless of the genetic similarity of EV-D68.

Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5.

Enteroviruses (EV) cause a number of life-threatening infectious diseases. EV-D68 is known to cause respiratory illness in children that can lead to acute flaccid myelitis. Coxsackievirus B5 (CVB5) is commonly associated with hand-foot-mouth disease. There is no antiviral treatment available for either. We have developed an isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of EV-D68 (IC50 58 nM) as well as other enteroviruses including the pleconaril-resistant Coxsackievirus B3-Woodruff (IC50 6-20 nM) and CVB5 (EC50 1 nM). Cryo-electron microscopy structures of EV-D68 in complex with 11526092 and pleconaril demonstrate destabilization of the EV-D68 MO strain VP1 loop, and a strain-dependent effect. A mouse respiratory model of EV-D68 infection, showed 3-log decreased viremia, favorable cytokine response, as well as statistically significant 1-log reduction in lung titer reduction at day 5 after treatment with 11526092. An acute flaccid myelitis neurological infection model did not show efficacy. 11526092 was tested in a mouse model of CVB5 infection and showed a 4-log TCID50 reduction in the pancreas. In summary, 11526092 represents a potent in vitro inhibitor of EV with in vivo efficacy in EV-D68 and CVB5 animal models suggesting it is worthy of further evaluation as a potential broad-spectrum antiviral therapeutic against EV.