A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses.

Enteroviruses have been a historical concern since the identification of polioviruses in humans. Wild polioviruses have almost been eliminated, while multiple species of non-polio enteroviruses and their variants co-circulate annually. To date, at least 116 types have been found in humans and are grouped into the species Enterovirus A-D and Rhinovirus A-C. However, there are few available antiviral drugs, especially with a universal pharmaceutical effect. Here, we demonstrate that peptide P25 from EV-D68 has broad antiviral activity against EV A-D enteroviruses in vitro. P25, derived from the HI loop and ß-I sheet of VP1, operates through a conserved hydrophilic motif -R---K-K--K- and the hydrophobic F near the N-terminus. It could prevent viral infection of EV-A71 by competing for the heparan sulfate (HS) receptor, binding and stabilizing virions by suppressing the release of the viral genome. P25 also inhibited the generation of infectious viral particles by reducing viral protein synthesis. The molecular docking revealed that P25 might bind to the pocket opening area, a potential target for broad-spectrum antivirals. Our findings implicate the multiple antiviral effects of peptide P25, including blocking viral binding to the HS receptor, impeding viral genome release, and reducing progeny particles, which could be a novel universal anti-enterovirus drug candidate.

Elucidating the Substrate Envelope of Enterovirus 68-3C Protease: Structural Basis of Specificity and Potential Resistance.

Enterovirus-D68 (EV68) has emerged as a global health concern over the last decade with severe symptomatic infections resulting in long-lasting neurological deficits and death. Unfortunately, there are currently no FDA-approved antiviral drugs for EV68 or any other non-polio enterovirus. One particularly attractive class of potential drugs are small molecules inhibitors, which can target the conserved active site of EV68-3C protease. For other viral proteases, we have demonstrated that the emergence of drug resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of substrate specificity. However, the structural characterization of EV68-3C protease bound to its substrates has been lacking. Here, we have determined the substrate specificity of EV68-3C protease through molecular modeling, molecular dynamics (MD) simulations, and co-crystal structures. Molecular models enabled us to successfully characterize the conserved hydrogen-bond networks between EV68-3C protease and the peptides corresponding to the viral cleavage sites. In addition, co-crystal structures we determined have revealed substrate-induced conformational changes of the protease which involved new interactions, primarily surrounding the S1 pocket. We calculated the substrate envelope, the three-dimensional consensus volume occupied by the substrates within the active site. With the elucidation of the EV68-3C protease substrate envelope, we evaluated how 3C protease inhibitors, AG7088 and SG-85, fit within the active site to predict potential resistance mutations.

Pharmacological activation of TLR7 exerts inhibition on the replication of EV-D68 in respiratory cells.

The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.

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.

Structure of Human Enterovirus 70 and Its Inhibition by Capsid-Binding Compounds.

Enterovirus 70 (EV70) is a human pathogen belonging to the family Picornaviridae. EV70 is transmitted by eye secretions and causes acute hemorrhagic conjunctivitis, a serious eye disease. Despite the severity of the disease caused by EV70, its structure is unknown. Here, we present the structures of the EV70 virion, altered particle, and empty capsid determined by cryo-electron microscopy. The capsid of EV70 is composed of the subunits VP1, VP2, VP3, and VP4. The partially collapsed hydrophobic pocket located in VP1 of the EV70 virion is not occupied by a pocket factor, which is commonly present in other enteroviruses. Nevertheless, we show that the pocket can be targeted by the antiviral compounds WIN51711 and pleconaril, which block virus infection. The inhibitors prevent genome release by stabilizing EV70 particles. Knowledge of the structures of complexes of EV70 with inhibitors will enable the development of capsid-binding therapeutics against this virus. IMPORTANCE Globally distributed enterovirus 70 (EV70) causes local outbreaks of acute hemorrhagic conjunctivitis. The discharge from infected eyes enables the high-efficiency transmission of EV70 in overcrowded areas with low hygienic standards. Currently, only symptomatic treatments are available. We determined the structures of EV70 in its native form, the genome release intermediate, and the empty capsid resulting from genome release. Furthermore, we elucidated the structures of EV70 in complex with two inhibitors that block virus infection, and we describe the mechanism of their binding to the virus capsid. These results enable the development of therapeutics against EV70.

Discovery of Potent and Broad-Spectrum Pyrazolopyridine-Containing Antivirals against Enteroviruses D68, A71, and Coxsackievirus B3 by Targeting the Viral 2C Protein.

The enterovirus genus of the picornavirus family contains many important human pathogens. EV-D68 primarily infects children, and the disease manifestations range from respiratory illnesses to neurological complications such as acute flaccid myelitis (AFM). EV-A71 is a major pathogen for the hand, foot, and mouth disease (HFMD) in children and can also lead to AFM and death in severe cases. CVB3 infection can cause cardiac arrhythmias, acute heart failure, as well as type 1 diabetes. There is currently no FDA-approved antiviral for any of these enteroviruses. In this study, we report our discovery and development of pyrazolopyridine-containing small molecules with potent and broad-spectrum antiviral activity against multiple strains of EV-D68, EV-A71, and CVB3. Serial viral passage experiments, coupled with reverse genetics and thermal shift binding assays, suggested that these molecules target the viral protein 2C. Overall, the pyrazolopyridine inhibitors represent a promising class of candidates for the urgently needed nonpolio enterovirus antivirals.

Functional and structural characterization of a two-MAb cocktail for delayed treatment of enterovirus D68 infections.

Enterovirus D68 (EV-D68) is an emerging pathogen associated with respiratory diseases and/or acute flaccid myelitis. Here, two MAbs, 2H12 and 8F12, raised against EV-D68 virus-like particle (VLP), show distinct preference in binding VLP and virion and in neutralizing different EV-D68 strains. A combination of 2H12 and 8F12 exhibits balanced and potent neutralization effects and confers broader protection in mice than single MAbs when given at onset of symptoms. Cryo-EM structures of EV-D68 virion complexed with 2H12 or 8F12 show that both antibodies bind to the canyon region of the virion, creating steric hindrance for sialic acid receptor binding. Additionally, 2H12 binding can impair virion integrity and trigger premature viral uncoating. We also capture an uncoating intermediate induced by 2H12 binding, not previously described for picornaviruses. Our study elucidates the structural basis and neutralizing mechanisms of the 2H12 and 8F12 MAbs and supports further development of the 2H12/8F12 cocktail as a broad-spectrum therapeutic agent against EV-D68 infections in humans.

Chemical Evolution of Antivirals Against Enterovirus D68 through Protein-Templated Knoevenagel Reactions.

The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C-C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development.

Caspase-3 inhibitor inhibits enterovirus D68 production.

Enterovirus D68 (EVD68) is an emerging pathogen that recently caused a large worldwide outbreak of severe respiratory disease in children. However, the relationship between EVD68 and host cells remains unclear. Caspases are involved in cell death, immune response, and even viral production. We found that caspase-3 was activated during EVD68 replication to induce apoptosis. Caspase-3 inhibitor (Z-DEVD-FMK) inhibited viral production, protected host cells from the cytopathic effects of EVD68 infection, and prevented EVD68 from regulating the host cell cycle at G0/G1. Meanwhile, caspase-3 activator (PAC-1) increased EVD68 production. EVD68 infection therefore activates caspase-3 for virus production. This knowledge provides a potential direction for the prevention and treatment of disease related to EVD68.

Synthesis and antiviral effect of novel fluoxetine analogues as enterovirus 2C inhibitors.

Enteroviruses (EV) are a group of positive-strand RNA (+RNA) viruses that include many important human pathogens (e.g. poliovirus, coxsackievirus, echovirus, numbered enteroviruses and rhinoviruses). Fluoxetine was identified in drug repurposing screens as potent inhibitor of enterovirus B and enterovirus D replication. In this paper we are reporting the synthesis and the antiviral effect of a series of fluoxetine analogues. The results obtained offer a preliminary insight into the structure-activity relationship of its chemical scaffold and confirm the importance of the chiral configuration. We identified a racemic fluoxetine analogue, 2b, which showed a similar antiviral activity compared to (S)-fluoxetine. Investigating the stereochemistry of 2b revealed that the S-enantiomer exerts potent antiviral activity and increased the antiviral spectrum compared to the racemic mixture of 2b. In line with the observed antiviral effect, the S-enantiomer displayed a dose-dependent shift in the melting temperature in thermal shift assays, indicative for direct binding to the recombinant 2C protein.

Methyl-ß-cyclodextrin inhibits EV-D68 virus entry by perturbing the accumulation of virus particles and ICAM-5 in lipid rafts.

Enterovirus D68 (EV-D68) is a member of the Picornavirus family and a causative agent of respiratory diseases in children. The incidence of EV-D68 infection has increased worldwide in recent years. Thus far, there are no approved antiviral agents or vaccines for EV-D68. Here, we show that methyl-ß-cyclodextrin (MßCD), a common drug that disrupts lipid rafts, specifically inhibits EV-D68 infection without producing significant cytotoxicity at virucidal concentrations. The addition of exogenous cholesterol attenuated the anti-EV-D68 activity of MßCD. MßCD treatment had a weak influence on the attachment of viral particles to the cell membrane but significantly inhibited EV-D68 entry into host cells. We demonstrated that EV-D68 facilitated the translocation of the viral receptor ICAM-5 to membrane rafts in infected cells. The colocalization of viral particles with ICAM-5 in lipid rafts was thoroughly abolished in cells after treatment with MßCD. Finally, we showed that MßCD inhibited the replication of isolated circulating EV-D68 strains. In summary, our results demonstrate that MßCD suppresses EV-D68 replication by perturbing the accumulation of virus particles and ICAM-5 in lipid rafts. This mechanism represents a promising strategy for drug development.

A Novel Capsid Binding Inhibitor Displays Potent Antiviral Activity against Enterovirus D68.

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68; we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and release of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, Western blot, RT-qPCR, viral heat inactivation, serial viral passage, and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, a recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild-type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.

Fluoxetine Inhibits Enterovirus Replication by Targeting the Viral 2C Protein in a Stereospecific Manner.

Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as a replication inhibitor of enterovirus B and D species. Fluoxetine likely targets the nonstructural viral protein 2C, but detailed mode-of-action studies are missing because structural information on 2C of fluoxetine-sensitive enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is stereospecific concomitant with binding to recombinant 2C. (S)-Fluoxetine inhibits with a 5-fold lower 50% effective concentration (EC50) than racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered coxsackievirus B3 (CVB3) resistant to fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.

Discovery of Quinoline Analogues as Potent Antivirals against Enterovirus D68 (EV-D68).

Enterovirus D68 (EV-D68) is an atypical nonpolio enterovirus that mainly infects the respiratory system of humans, leading to moderate-to-severe respiratory diseases. In rare cases, EV-D68 can spread to the central nervous system and cause paralysis in infected patients, especially young children and immunocompromised individuals. There is currently no approved vaccine or antiviral available for the prevention and treatment of EV-D68. In this study, we aimed to improve the antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through structure-activity relationship studies. In total, 60 compounds were synthesized and tested against EV-D68 using the viral cytopathic effect assay. Three compounds 10a, 12a, and 12c were identified to have significantly improved potency (EC50 < 1 µM) and a high selectivity index (>180) compared with dibucaine against five different strains of EV-D68 viruses. These compounds also showed potent antiviral activity in neuronal cells, such as A172 and SH-SY5Y cells, suggesting they might be further developed for the treatment of both respiratory infection as well as neuronal infection.

Validating Enterovirus D68-2Apro as an Antiviral Drug Target and the Discovery of Telaprevir as a Potent D68-2Apro Inhibitor.

Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2Apro) and characterization of its enzymatic activity. Furthermore, we discovered that telaprevir, an FDA-approved drug used for the treatment of hepatitis C virus (HCV) infections, is a potent antiviral against EV-D68 by targeting the 2Apro enzyme. Using a fluorescence resonance energy transfer-based substrate cleavage assay, we showed that the purified EV-D68 2Apro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2Apro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar-to-low-micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2Apro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2Apro and the identification of telaprevir as a potent EV-D68 2Apro inhibitor. These findings implicate EV-D68 2Apro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection.IMPORTANCE A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2Apro, and identify telaprevir-an FDA-approved drug used to treat hepatitis C virus (HCV) infections-as a potent antiviral with a novel mechanism of action toward 2Apro 2Apro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. The binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2Apro inhibition. This is the first inhibitor to selectively target the 2Apro from EV-D68 and can be used as a starting point for the development of therapeutics with selective activity against EV-D68.

Molecular basis for the acid-initiated uncoating of human enterovirus D68.

Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid-labile. The molecular mechanism by which the acid-sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryoelectron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate [the A (altered) particle] of EV-D68 at 2.2- and 2.7-Å resolution, respectively. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N termini from the capsid interior, and formation of pores around the icosahedral twofold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68, cryo-EM analyses of a mixed population of particles at neutral pH and following acid treatment demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded 1 ("E1") particle, shows a majority of internal regions (e.g., the VP1 N termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A particles. Together, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses.

Susceptibilities of enterovirus D68, enterovirus 71, and rhinovirus 87 strains to various antiviral compounds.

Compounds were evaluated for antiviral activity in rhabdomyosarcoma (RD) cells against a recent 2014 clinical isolate of enterovirus D68 (EV-D68), a 1962 strain of EV-68D, rhinovirus 87 (RV-87, serologically the same as EV-D68), and enterovirus 71 (EV-71). Test substances included known-active antipicornavirus agents (enviroxime, guanidine HCl, pirodavir, pleconaril, and rupintrivir), nucleobase/nucleoside analogs (3-deazaguanine and ribavirin), and three novel epidithiodiketopiperazines (KCN-2,2'-epi-19, KCN-19, and KCN-21). Of these, rupintrivir was the most potent, with 50% inhibition of viral cytopathic effect (EC50) and 90% inhibition (EC90) of virus yield at 0.0022-0.0053 µM against EV-D68. Enviroxime, pleconaril and the KCN compounds showed efficacy at 0.01-0.3 µM; 3-deazaguanine and pirodavir inhibited EV-D68 at 7-13 µM, and guanidine HCl and ribavirin were inhibitory at 80-135 µM. Pirodavir was active against EV-71 (EC50 of 0.78 µM) but not against RV-87 or EV-D68, and all other compounds were less effective against EV-71 than against RV-87 and EV-D68. The most promising compound inhibiting both virus infections at low concentrations was rupintrivir. Antiviral activity was confirmed for the ten compounds in virus yield reduction (VYR) assays in RD cells, and for enviroxime, guanidine HCl, and pirodavir by cytopathic effect (CPE) assays in A549, HeLa-Ohio-1, and RD cells. These studies may serve as a basis for further pre-clinical discovery of anti-enterovirus inhibitors. Furthermore, the antiviral profiles and growth characteristics observed herein support the assertion that EV-D68 should be classified together with RV-87.

Potent inhibition of enterovirus D68 and human rhinoviruses by dipeptidyl aldehydes and α-ketoamides.

Enterovirus D68 (EV-D68) is an emerging pathogen responsible for mild to severe respiratory infections that occur mostly in infants, children and teenagers. EV-D68, one of more than 100 non-polio enteroviruses, is acid-labile and biologically similar to human rhinoviruses (HRV) (originally classified as HRV87). However, there is no approved preventive or therapeutic measure against EV-D68, HRV, or other enteroviruses. In this study, we evaluated the antiviral activity of series of dipeptidyl compounds against EV-D68 and HRV strains, and demonstrated that several peptidyl aldehyde and α-ketoamide peptidyl compounds are potent inhibitors of EV-D68 and HRV strains with high in-vitro therapeutic indices (>1000). One of the α-ketoamide compounds is shown to have favorable pharmacokinetics profiles, including a favorable oral bioavailability in rats. Recent successful development of α-ketoamide protease inhibitors against hepatitis C virus suggests these compounds may have a high potential for further optimization and development against emerging EV-D68, as well as HRV.

Antiviral Activity of Broad-Spectrum and Enterovirus-Specific Inhibitors against Clinical Isolates of Enterovirus D68.

We investigated the susceptibility of 10 enterovirus D68 (EV-D68) isolates (belonging to clusters A, B, and C) to (entero)virus inhibitors with different mechanisms of action. The 3C-protease inhibitors proved to be more efficient than enviroxime and pleconaril, which in turn were more effective than vapendavir and pirodavir. Favipiravir proved to be a weak inhibitor. Resistance to pleconaril maps to V69A in the VP1 protein, and resistance to rupintrivir maps to V104I in the 3C protease. A structural explanation of why both substitutions may cause resistance is provided.

In Vitro Efficacy of Antiviral Compounds against Enterovirus D68.

In 2014, the United States experienced a large outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68). We used a homogeneous, cell-based assay to assess the antiviral activity of compounds developed for EV/rhinovirus infection or other indications. Three of 15 compounds were highly active against all four strains tested (the prototype and three 2014 strains), with 50% effective concentrations of 0.0012 to 0.027 µM. Additional studies are needed to assess their in vivo efficacy against EV-D68.