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Background: Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines., Methods: In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its in vivo efficacy in an EV-A71-infected murine model., Findings: We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D in vitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3D pol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice., Interpretation: Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application., Funding: MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC)., Competing Interests: Declaration of interests HF received grants from Synthetic Biology Translational Research Program, School of Medicine, National University of Singapore., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus M pro s exhibit overlapping substrate specificity in all binding pockets, whereas the 3C pro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 M pro can be successfully applied to investigate the activity of the M pro from other coronaviruses as well as the 3C pro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds., (© 2024 The Protein Society.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Enterovirus genomic replication initiates at a predicted RNA cloverleaf (5'CL) at the 5' end of the RNA genome. The 5'CL contains one stem (SA) and three stem-loops (SLB, SLC, SLD). Here, we present an analysis of 5'CL conservation and divergence for 209 human health-related serotypes from the enterovirus genus, including enterovirus and rhinovirus species. Phylogenetic analysis indicates six distinct 5'CL serotypes that only partially correlate with the species definition. Additional findings include that 5'CL sequence conservation is higher between the EV species than between the RV species, the 5'CL of EVA and EVB are nearly identical, and RVC has the lowest 5'CL conservation. Regions of high conservation throughout all species include SA and the loop and nearby bases of SLB, which is consistent with known protein interactions at these sites. In addition to the known protein binding site for the Poly-C binding protein in the loop of SLB, other conserved consecutive cytosines in the stems of SLB and SLC provide additional potential interaction sites that have not yet been explored. Other sites of conservation, including the predicted bulge of SLD and other conserved stem, loop, and junction regions, are more difficult to explain and suggest additional interactions or structural requirements that are not yet fully understood. This more intricate understanding of sequence and structure conservation and variability in the 5'CL may assist in the development of broad-spectrum antivirals against a wide range of enteroviruses , while better defining the range of virus isotypes expected to be affected by a particular antiviral.

The ongoing challenge of viral transmission, exemplified by the Covid pandemic and recurrent viral outbreaks, necessitates the exploration of sustainable antiviral solutions. This study investigates the underexplored antiviral potential of wooden surfaces. We evaluated the antiviral efficacy of various wood types, including coniferous and deciduous trees, against enveloped coronaviruses and nonenveloped enteroviruses like coxsackie virus A9. Our findings revealed excellent antiviral activity manifesting already within 10 to 15 min in Scots pine and Norway spruce, particularly against enveloped viruses. In contrast, other hardwoods displayed varied efficacy, with oak showing effectiveness against the enterovirus. This antiviral activity was consistently observed across a spectrum of humidity levels (20 to 90 RH%), while the antiviral efficacy manifested itself more rapidly at 37 °C vs 21 °C. Key to our findings is the chemical composition of these woods. Resin acids and terpenes were prevalent in pine and spruce, correlating with their antiviral performance, while oak's high phenolic content mirrored its efficacy against enterovirus. The pine surface absorbed a higher fraction of the coronavirus in contrast to oak, whereas enteroviruses were not absorbed on those surfaces. Thermal treatment of wood or mixing wood with plastic, such as in wood-plastic composites, strongly compromised the antiviral functionality of wood materials. This study highlights the role of bioactive chemicals in the antiviral action of wood and opens new avenues for employing wood surfaces as a natural and sustainable barrier against viral transmissions.

Hand, foot, and mouth disease (HFMD) is a common pediatric illness mainly caused by enteroviruses, which are important human pathogens. Currently, there are no available antiviral agents for the therapy of enterovirus infection. In this study, an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed. Using this screening system, we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors. Fangchinoline (FAN), a bis-benzylisoquinoline alkaloid, exhibits potential inhibitory effects against various enteroviruses that cause HFMD, such as EV-A71, CV-A10, CV-B3 and CV-A16. Further investigations revealed that FAN targets the early stage of the enterovirus life cycle. Through the selection of FAN-resistant EV-A71 viruses, we demonstrated that the VP1 protein could be a potential target of FAN, as two mutations in VP1 (E145G and V258I) resulted in viral resistance to FAN. Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses., Competing Interests: Conflict of interest Prof. Bo Zhang is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Importance: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants., Competing Interests: The authors declare no conflict of interest.

A novel series of peptidomimetic aldehydes was designed and synthesized to target 3C protease (3C pro ) of enterovirus 71 (EV71). Most of the compounds exhibited high antiviral activity, and among them, compound 18p demonstrated potent enzyme inhibitory activity and broad-spectrum antiviral activity on a panel of enteroviruses and rhinoviruses. The crystal structure of EV71 3C pro in complex with 18p determined at a resolution of 1.2 Å revealed that 18p covalently linked to the catalytic Cys147 with an aldehyde group. In addition, these compounds also exhibited good inhibitory activity against the 3CL pro and the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially compound 18p (IC 50 = 0.034 μM, EC 50 = 0.29 μM). According to our previous work, these compounds have no reasons for concern regarding acute toxicity. Compared with AG7088 , compound 18p also exhibited good pharmacokinetic properties and more potent anticoronavirus activity, making it an excellent lead for further development.

Enteroviruses are a group of clinically relevant RNA viruses that causes human diseases [...].

The structural diversity and unique physicochemical properties of sulphated polysaccharides of red algae carrageenans (CRGs), to a great extent, determine the wide range of their antiviral properties. This work aimed to compare the antiviral activities of different structural types of CRGs: against herpes simplex virus type 1 (HSV-1) and enterovirus (ECHO-1). We found that CRGs significantly increased the resistance of Vero cells to virus infection (preventive effect), directly affected virus particles (virucidal effect), inhibited the attachment and penetration of virus to cells, and were more effective against HSV-1. CRG1 showed the highest virucidal effect on HSV-1 particles with a selective index (SI) of 100. CRG2 exhibited the highest antiviral activity by inhibiting HSV-1 and ECHO-1 plaque formation, with a SI of 110 and 59, respectively, when it was added before virus infection. CRG2 also significantly reduced the attachment of HSV-1 and ECHO-1 to cells compared to other CRGs. It was shown by molecular docking that tetrasaccharides-CRGs are able to bind with the HSV-1 surface glycoprotein, gD, to prevent virus-cell interactions. The revealed differences in the effect of CRGs on different stages of the lifecycle of the viruses are apparently related to the structural features of the investigated compounds.

Enterovirus 71 (EV71) is an etiological agent of hand foot and mouth disease and can also cause neurological complications in young children. However, there are no approved drugs as of yet to treat EV71 infections. In this study, we conducted antiviral drug screening by using a Food and Drug Administration (FDA)-approved drug library. We identified five drugs that showed dose-dependent inhibition of viral replication. Sertraline was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index among the five hits. The antiviral activity of sertraline was noted for other EV serotypes. The drug's antiviral effect is not likely associated with its approved indications as an antidepressant and its mode-of-action as a selective serotonin reuptake inhibitor. The time-of-addition assay revealed that sertraline inhibited an EV71 infection at the entry stage. We also showed that sertraline partitioned into acidic compartments, such as endolysosomes, to neutralize the low pH levels. In agreement with the findings, the antiviral effect of sertraline could be greatly relieved by exposing virus-infected cells to extracellular low-pH culture media. Ultimately, we have identified a use for an FDA-approved antidepressant in broad-spectrum EV inhibition by blocking viral entry through the alkalization of the endolysosomal route.

A series of phosphoamidate derivatives of nucleoside 2'-acetylene-7-deaza-adenosine (NITD008) were synthesized and evaluated for their in vitro antiviral activities against the enteroviruses EV71 and EV-D68. The phosphoamidate (15f) containing a hexyl ester of l-alanine exhibited the most promising activity against EV71 (IC 50  = 0.13 ± 0.08 μM) and was 4-times more potent than NITD008. Meanwhile, the derivative containing a cyclohexyl ester of l-alanine (15l) exhibited the most potent activity with high selectivity index against both EV71 (IC 50  = 0.19 ± 0.27 μM, SI = 117.00) and EV-D68 (IC 50  = 0.17 ± 0.16 μM, SI = 130.76), which were both higher than that of NITD008. The results indicated that the phosphoamidate 15l was the most promising candidate for further development as antiviral agents for the treatment of both EV71 and EV-D68 infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-β in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Chen et al.)

Background: As frequent viral outbreaks continue to pose threat to public health, the unavailability of antiviral drugs and challenges associated with vaccine development underscore the need for antiviral drugs discovery in emergent moments (endemic or pandemic). Plants in response to microbial and pest attacks are able to produce defence molecules such as antimicrobial peptides as components of their innate immunity, which can be explored for viral therapeutics., Methods: In this study, partially purified peptide-rich fraction (P-PPf) were obtained from aqueous extracts of seven plants by reverse-phase solid-phase extraction and cysteine-rich peptides detected by a modified TLC method. The peptide-enriched fractions and the aqueous (crude polar) were screened for antiviral effect against three non-polio enterovirus species C members using cytopathic effect reduction assay., Results: In this study, peptide fraction obtained from Euphorbia hirta leaf showed most potent antiviral effect against Coxsackievirus A13, Coxsackievirus A20, and Enterovirus C99 (EV-C99) with IC 50  < 2.0 µg/mL and selective index ≥ 81. EV-C99 was susceptible to all partially purified peptide fractions except Allamanda blanchetii leaf., Conclusion: These findings establish the antiviral potentials of plants antimicrobial peptides and provides evidence for the anti-infective use of E. hirta in ethnomedicine. This study provides basis for further scientific investigation geared towards the isolation, characterization and mechanistic pharmacological study of the detected cysteine-rich peptides., (© 2021. The Author(s).)

As one of the principal etiological agents of hand, foot, and mouth disease (HFMD), enterovirus 71 (EV71) is associated with severe neurological complications or fatal diseases, while without effective medications thus far. Here we applied dually activated Michael acceptor to develop a series of reversible covalent compounds for EV71 3C protease (3C pro ), a promising antiviral drug target that plays an essential role during viral replication by cleaving the precursor polyprotein, inhibiting host protein synthesis, and evading innate immunity. Among them, cyanoacrylate and Boc-protected cyanoarylamide derivatives (SLQ-4 and SLQ-5) showed effective antiviral activity against EV71. The two inhibitors exhibited broad antiviral effects, acting on RD, 293T, and Vero cell lines, as well as on EV71 A, B, C, CVA16, and CVB3 viral strains. We further determined the binding pockets between the two inhibitors and 3C pro based on docking studies. These results, together with our previous studies, provide evidence to elucidate the mechanism of action of these two reversible covalent inhibitors and contribute to the development of clinically effective medicines to treat EV71 infections., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Between April and June 2016, an outbreak of rhombencephalitis (RE) caused by enterovirus (EV) A71 was detected in Catalonia, Spain-the first documented in Western Europe. The clinical characteristics and outcome of patients with this condition differed from those reported in outbreaks occurring in Southeast Asia., Methods: Observational, multicenter study analyzing characteristics, treatment and outcome of patients with EV-A71 rhombencephalitis diagnosed in 6 publicly funded hospitals within the Catalonian Health Institute. A review of clinical characteristics, diagnosis, treatment and outcome of these patients was conducted., Results: Sixty-four patients met the clinical and virologic criteria for rhombencephalitis caused by EV-A71. All patients had symptoms suggesting viral disease, mainly fever, lethargy, ataxia and tremor, with 30% of hand-foot-mouth disease. Intravenous immunoglobulin therapy was given to 44/64 (69%) patients and methylprednisolone to 27/64 (42%). Six patients (9%) required pediatric intensive care unit admission. Three patients had acute flaccid paralysis of 1 limb, and another had autonomic nervous system (ANS) dysfunction with cardiorespiratory arrest. Outcome in all patients (except the patient with hypoxic-ischemic encephalopathy) was good, with complete resolution of the symptoms., Conclusions: During the 2016 outbreak, rhombencephalitis without ANS symptoms was the predominant form of presentation and most patients showed no hand-foot-mouth disease. These findings contrast with those of other patient series reporting associated ANS dysfunction (10%-15%) and hand-foot-mouth disease (60%-80%). Complete recovery occurred in almost all cases. In light of the favorable outcome in untreated mild cases, therapies for this condition should be reserved for patients with moderate-severe infection. The main relevance of this study is to provide useful information for setting priorities, management approaches and adequate use of resources in future EV-A71 associated rhombencephalitis outbreaks., Competing Interests: The authors have no conflicts of interest or funding to disclose., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

The anti-enteroviral activity of three stilbenoids isolated from the leaves of Macaranga barteri was investigated using the cytopathic effect reduction assay. The stilbenes were inactive against echovirus E13 but showed activity against echoviruses E7 and E19. In particular, vedelianin ( 2 ), schweinfurthin G ( 3 ) and mappain ( 1 ) elicited antiviral activity on E19 with IC 50 values of 0.0036 nM, 0.018 nM and 0.24 µM, respectively. Vedelianin ( 2 ) showed the best selectivity profile amongst the isolated compounds with selectivity index values of 31 and 216 against E7 and E19, respectively. It is possible these compounds may be responsible for the traditional use of Macaranga barteri in the treatment of viral infections.

Introduction : Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes. Areas covered : This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral proteases have been evaluated. Expert opinion : The main protease of coronaviruses and enteroviruses share similarities in their structure and function. These proteases process their viral polyproteins and thus drugs that bind to the active site have potential to target both virus groups. It is important to develop drugs that target more evolutionarily conserved processes and proteins. Moreover, it is a wise strategy to concentrate on processes that are similar between several virus families.

Seventeen new prenylated C 6 -C 3 derivatives, namely, illifargeins A-M (1-13), including three pairs of enantiomers (1, 5, and 12) and one norillifargeal A (14), together with eight known analogues (15-22), were isolated from the stems and leaves of Illicium fargesii. The structures of the new compounds were elucidated using spectroscopic data (UV, IR, 1D and 2D NMR, and HRESIMS). Their absolute configurations were determined by using experimental and calculated ECD data analysis, as well as a modified Mosher's method. Compounds 1a, 1b, 2, 3, 5a, 7, 10, 11, 15, 16, 19, and 20 showed potential activity against Coxsackie virus B3, with IC 50 values ranging from 6.23 to 33.33 µM. Compounds 9 and 15 exhibited potential activity against influenza virus A, with IC 50 values of 11.11 and 19.24 µM, respectively. Compounds 2, 3, and 18 exhibited potential anti-oxidant activity, with IC 50 values ranging from 1.43 to 6.71 µM., (Copyright © 2021. Published by Elsevier Inc.)

Lactoferrin is part of the innate immune system, with antiviral activity against numerous DNA and RNA viruses. Rhinoviruses, the leading cause of the common cold, are associated with exacerbation of respiratory illnesses such as asthma. Here, we explored the effect of bovine lactoferrin (BLf) on RV-B14 infectivity. Using different assays, we show that the effect of BLf is strongest during adhesion of the virus to the cell and entry. Tracking the internalisation of BLf and virus revealed a degree of colocalisation, although their interaction was only confirmed in vitro using empty viral particles, indicating a possible additional influence of BLf on other infection steps.

The frequency of bacterial factors causing central nervous system infections has decreased as a result of the development of our national immunization program. In this study, it is aimed to obtain the data of our local surveillance by defining the viral etiology in cases diagnosed with meningoencephalitis for 1 year. Previously healhty 186 children, who applied with findings suggesting viral meningoencephalitis to 8 different tertiary health centers between August 2018 and August 2019, in Istanbul, were included. The cerebrospinal fluid (CSF) sample was evaluated by polymerase chain reaction. The M:F ratio was 1.24 in the patient group, whose age ranged from 1 to 216 months (mean 40.2 ± 48.7). Viral factor was detected in 26.8%. Enterovirus was the most common agent (24%) and followed by Adenovirus (22%) and HHV type 6 (22%). In the rest of the samples revealed HHV type 7 (10%), EBV (6%), CMV (6%), HSV type 1 (6%), Parvovirus (4%) and VZV (2%). The most common symptoms were fever (79%) and convulsions (45.7%). Antibiotherapy and antiviral therapy was started 48.6% and 4% respectively. Mortality and sequela rate resulted 0.53% and 3.7%, respectively. This highlights the importance of monitoring trends in encephalitis in Turkey with aview to improving pathogen diagnosis for encephalitis and rapidly identifying novel emerging encephalitis-causing pathogens that demand public health action especially in national immunisation programme.

Based on the fact that a search for influenza antivirals among nucleoside analogues has drawn very little attention of chemists, the present study reports the synthesis of a series of 1,2,3-triazolyl nucleoside analogues in which a pyrimidine fragment is attached to the ribofuranosyl-1,2,3-triazol-4-yl moiety by a polymethylene linker of variable length. Target compounds were prepared by the Cu alkyne-azide cycloaddition (CuAAC) reaction. Derivatives of uracil, 6-methyluracil, 3,6-dimethyluracil, thymine and quinazolin-2,4-dione with ω-alkyne substituent at the N1 (or N5) atom and azido 2,3,5-tri-O-acetyl-D-β-ribofuranoside were used as components of the CuAAC reaction. All compounds synthesized were evaluated for antiviral activity against influenza virus A/PR/8/34/(H1N1) and coxsackievirus B3. The best values of IC 50 (inhibiting concentration) and SI (selectivity index) were demonstrated by the lead compound 4i in which the 1,2,3-triazolylribofuranosyl fragment is attached to the N1 atom of the quinazoline-2,4-dione moiety via a butylene linker (IC 50  = 30 μM, SI = 24) and compound 8n in which the 1,2,3-triazolylribofuranosyl fragment is attached directly to the N5 atom of the 6-methyluracil moiety (IC 50  = 15 μM, SI = 5). According to theoretical calculations, the antiviral activity of the 1,2,3-triazolyl nucleoside analogues 4i and 8n against H1N1 (A/PR/8/34) influenza virus can be explained by their influence on the functioning of the polymerase acidic protein (PA) of RNA-dependent RNA polymerase (RdRP).

Viruses from the genus Enterovirus (EV) of the Picornaviridae family are known to cause diseases such as hand foot and mouth disease (HFMD), respiratory diseases, encephalitis and myocarditis. The capsid of EV is an attractive target for the development of direct-acting small molecules that can interfere with viral entry. Some of the capsid binders have been evaluated in clinical trials but the majority have failed due to insufficient efficacy or unacceptable off-target effects. Furthermore, most of the capsid binders exhibited a low barrier to resistance. Alternatively, host-targeting inhibitors such as peptides derived from the capsid of EV that can recognize cellular receptors have been identified. However, the majority of these peptides displayed low anti-EV potency (µM range) as compared to the potency of small molecule compounds (nM range). Nonetheless, the development of anti-EV peptides is warranted as they may complement the small-molecules in a drug combination strategy to treat EVs. Lastly, structure-based approach to design antiviral peptides should be utilized to unearth potent anti-EV peptides.

Enteroviruses 71 (EV71) is a single-stranded, neurotrophic RNA virus responsible for the numerous outbreaks of hand, foot, and mouth disease (HFMD) in the Asia-Pacific regions. HFMD primarily affects children to cause range of infection, from mild symptoms to acute flaccid paralysis, and hemorrhage. Despite increased incidence of EV71 epidemics globally and research against EV71 becoming prioritized, no antiviral agent against EV71 has yet been licensed and approved worldwide. In this chapter, detailed EV71 antiviral screening techniques are described, including plaque assay which determines viral titers through the use of a semisolid overlay, carboxymethyl cellulose to allow even viral spread and infection across the host cellular monolayers as well as a crystal violet, a distinct counterstain to visualize circular regions of infectious zones-plaques. qRT-PCR is used to quantify the viral genomic RNA in the infected samples and MTS cell viability assay to quantify the cell viability after infection or toxicity of the compound on the cells. Furthermore, various antiviral inhibition assays including prophylactic, post infection, and virucidal assays are demonstrated for estimation of the antiviral activity of potential antiviral drugs against EV71. These methods can be effectively utilized in virology laboratories for effective high-throughput screening of antiviral molecules against EV71 that can assist in the future development of antiviral drugs.

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine-which also targets 2C-but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed., Competing Interests: The authors have declared that no competing interests exist.

This study screened mastic gum (Pistacia lentiscus L.) for antiviral activity against herpes simplex virus type 2 (HSV-2), coxsackievirus type B3, and adenovirus type 5. The organs of this plant (leaves, stem, and seed) were macerated sequentially using solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and methanol). Only the methanol extract of stem exhibited significant activity against HSV-2. This extract showed anti-HSV-2 activity with a selectivity index of 51 (50% cytotoxic concentration = 186 µg/mL; 50% inhibitory concentration = 3.63 µg/mL), and demonstrated direct inhibition against this virus with a virucidal selectivity index of 620 (50% virucidal concentration = 0.30 µg/mL). A bio-guided assay involving thin-layer chromatography led to the isolation of two active compounds, which have been identified as dammaradienone and dammaradienol using high-performance liquid chromatography-diode array detection coupled with electrospray ionization mass spectrometry. P. lentiscus has been widely studied for other biological activities. However, to our knowledge, this is the first report of P. lentiscus L. exhibiting antiviral activity.

Non-polio enteroviruses such as enterovirus A71 (EV-A71), EV-D68, and coxsackievirus B3 (CVB3) are significant human pathogens with disease manifestations ranging from mild flu-like symptoms to more severe encephalitis, myocarditis, acute flaccid paralysis/myelitis, and even death. There is currently no effective antivirals to prevent or treat non-polio enterovirus infection. In this study, we report our progress in developing potent and broad-spectrum antivirals against these non-polio enteroviruses. Starting from our previously developed lead compounds that had potent antiviral activity against EV-D68, we synthesized 43 analogs and profiled their broad-spectrum antiviral activity against additional EV-D68, EV-A71, and CVB3 viruses. Promising candidates were also selected for mouse microsomal stability test to prioritize lead compounds for future in vivo mouse model studies. Collectively, this multi-parameter optimization process revealed a promising lead compound 6aw that showed single-digit to submicromolar EC 50 values against two EV-D68 strains (US/KY and US/MO), two EV-A71 strains (Tainan and US/AK), and one CVB3 strain, with a high selectivity index. Encouragingly, 6aw was stable in mouse microsomes with a half-life of 114.7 min. Overall, 6aw represents one of the most potent broad-spectrum antiviral against non-polio enteroviruses, rendering it a promising lead candidate for non-polio enteroviruses with translational potential., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown that bacteria promote oral infection with a variety of enteric viruses. However, it is unknown whether antibiotics have microbiota-independent antiviral effects for enteric viruses or whether antibiotics influence extra-intestinal, systemic infection. Here, we examined the effects of antibiotics on systemic enteric virus infection by performing intraperitoneal injections of either coxsackievirus B3 (CVB3) or poliovirus followed by quantification of viral titers. We found that antibiotic treatment reduced systemic infection for both viruses. Interestingly, antibiotics reduced CVB3 titers in germ-free mice, suggesting that antibiotic treatment alters CVB3 infection through a microbiota-independent mechanism. Overall, these data provide further evidence that antibiotics can have noncanonical effects on viral infection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV71) is one of the main pathogens causing hand-foot-and-mouth disease (HFMD). The nose and mouth are usually the main infection entries of EV71 virus. However, its dynamic transport pathway from mouth to the whole body remains unknown. The reveal of this physiological mechanism in vivo will help to understand its transport direction, find its key proliferation nodes, and develop new preventive strategies. We trained a new strain of GFP-EV71 virus to be susceptible to mice brain by intracranial injection of mice. The adapted virus was oral-administrated to suckling mice. Then, the dynamic distributions of the virus in vivo were detected by living image system and fluorescence quantitation polymerase chain reaction (qPCR). We figured out the dynamic pathway of EV71 transport in vivo from intestine to peripheral tissue, then to the other organs. Small intestine was identified as a gateway for EV71 infection in vivo. Ileum was proved to be the main part of proliferation and transport of EV71 in small intestine of mice. EV71 was verified to enter small intestinal villus of mice through the infection of small intestinal epithelial cell. Acarbose displayed a good preventive effect on EV71 infection both in vivo and in vitro. Acarbose possibly decreased the intestinal infection of EV71 by blocking the receptor-binding sites on the surface of EV71 virion or by inhibiting various glycolic receptors on the cell surface. Thus, acarbose and its analogue may be the potential medicines to prevent EV71 infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The existence of multiple serotypes renders vaccine development challenging for most viruses in the Enterovirus genus. An alternative and potentially more viable strategy for control of these viruses is to develop broad-spectrum antivirals by targeting highly conserved proteins that are indispensable for the virus life cycle, such as the 3C protease. Previously, two single-chain antibody fragments, YDF and GGVV, were reported to effectively inhibit human rhinovirus 14 proliferation. Here, we found that both single-chain antibody fragments target sites on the 3C protease that are distinct from its known drug site (peptidase active site) and possess different mechanisms of inhibition. YDF does not block the active site but instead noncompetitively inhibits 3C peptidase activity through an allosteric effect that is rarely seen for antibody protease inhibitors. Meanwhile, GGVV antagonizes the less-explored regulatory function of 3C in genome replication. The interaction between 3C and the viral genome 5' noncoding region has been reported to be important for enterovirus genome replication. Here, the interface between human rhinovirus 14 3C and its 5' noncoding region was probed by hydrogen-deuterium exchange coupled mass spectrometry and found to partially overlap with the interface between GGVV and 3C. Consistently, prebinding of GGVV completely abolishes interaction between human rhinovirus 14 3C and its 5' noncoding region. The epitopes of YDF and GGVV, therefore, represent two additional sites of therapeutic vulnerability in rhinovirus. Importantly, the GGVV epitope appears to be conserved across many enteroviruses, suggesting that it is a promising target for pan-enterovirus inhibitor screening and design., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families, including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (coxsackievirus, rhinovirus), flavirivuses (Zika virus), and coronaviruses (human coronavirus 229E [HCoV-229E] and Middle East respiratory syndrome CoV [MERS-CoV]). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication., (Copyright © 2020 American Society for Microbiology.)

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC 50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

Glucosamine (GlcN), a dietary supplement widely utilized to promote joint health and effective in the treatment of osteoarthritis, is an effective macroautophagy/autophagy activator in vitro and in vivo . Previous studies have shown that autophagy is required for hepatitis B virus (HBV) replication and envelopment. The objective of this study was to determine whether and how GlcN affects HBV replication, using in vitro and in vivo experiments. Our data demonstrated that HBsAg production and HBV replication were significantly increased by GlcN treatment. Confocal microscopy and western blot analysis showed that the amount of autophagosomes and the levels of autophagic markers MAP1LC3/LC3-II and SQSTM1 were clearly elevated by GlcN treatment. GlcN strongly blocked autophagic degradation of HBV virions and proteins by inhibiting lysosomal acidification through its amino group. Moreover, GlcN further promoted HBV replication by inducing autophagosome formation via feedback inhibition of mechanistic target of rapamycin kinase complex 1 (MTORC1) signaling in an RRAGA (Ras related GTP binding A) GTPase-dependent manner. In vivo , GlcN application promoted HBV replication and blocked autophagic degradation in an HBV hydrodynamic injection mouse model. In addition, GlcN promoted influenza A virus, enterovirus 71, and vesicular stomatitis virus replication in vitro . In conclusion, GlcN efficiently promotes virus replication by inducing autophagic stress through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling. Thus, there is a potential risk of enhanced viral replication by oral GlcN intake in chronically virally infected patients. Abbreviations : ACTB: actin beta; ATG: autophagy-related; CMIA: chemiluminescence immunoassay; ConA: concanavalin A; CQ: chloroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; EV71: enterovirus 71; GalN: galactosamine; GFP: green fluorescence protein; GlcN: glucosamine; GNPNAT1: glucosamine-phosphate N-acetyltransferase 1; HBP: hexosamine biosynthesis pathway; HBV: hepatitis B virus; HBcAg: hepatitis B core antigen; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen; HBV RI: hepatitis B replicative intermediate; IAV: influenza A virus; LAMP1: lysosomal associated membrane protein 1; LAMTOR: late endosomal/lysosomal adaptor, MAPK and MTOR activator; ManN: mannosamine; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PHH: primary human hepatocyte; RAB7: RAB7A, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; RRAGA: Ras related GTP binding A; RT-PCR: reverse transcriptase polymerase chain reaction; SEM: standard error of the mean; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; UAP1: UDP-N-acetylglucosamine pyrophosphorylase 1; VSV: vesicular stomatitis virus.

Viruses in species Parechovirus A ( Picornaviridae ) are associated with a wide variety of clinical manifestations. Parechovirus A3 (PeV-A3) is known to cause sepsis-like illness, meningitis, and encephalitis in infants and young children. To date, no specific therapies are available to treat PeV-A3-infected children. We had previously identified two FDA-cleared antifungal drugs, itraconazole (ITC) and posaconazole (POS), with potent and specific antiviral activity against PeV-A3. Time-of-addition and synchronized infection assays revealed that POS targets an early stage of the PeV-A3 life cycle. POS exerts an antiviral effect, evidenced by a reduction in viral titer following the addition of POS to Vero-P cells before infection, coaddition of POS and PeV-A3 to Vero-P cells, incubation of POS and PeV-A3 prior to Vero-P infection, and at attachment. POS exerts less of an effect on virus entry. A PeV-A3 enzyme-linked immunosorbent assay inhibition experiment, using an anti-PeV-A3 monoclonal antibody, suggested that POS binds directly to the PeV-A3 capsid. POS-resistant PeV-A3 strains developed by serial passage in the presence of POS acquired substitutions in multiple regions of the genome, including the capsid. Reverse genetics confirmed substitutions in capsid proteins VP0, VP3, and VP1 and nonstructural proteins 2A and 3A. Single mutants VP0&#95;K66R, VP0&#95;A124T, VP3&#95;N88S, VP1&#95;Y224C, 2A&#95;S78L, and 3A&#95;T1I were 4-, 9-, 12-, 34-, 51-, and 119-fold more resistant to POS, respectively, than the susceptible prototype strain. Our studies demonstrate that POS may be a valuable tool in developing an antiviral therapy for PeV-A3.

Enterovirus 71 (EV71) is the causative pathogen of hand, foot, and mouth disease (HFMD). However, no effective antiviral therapy is currently available. Some viruses could escape the host's innate immunity by upregulating suppressor of cytokine signaling (SOCS) proteins. Until now, whether EV71 evades the host immune system by regulating the expression of SOCS proteins remains unknown. In this study, we found that EV71 infection promoted SOCS expression at both mRNA and protein levels in vitro and in vivo. Consistently, the infectivity of EV71 was decreased significantly in the SOCS3 or SOCS1 knockdown cells, suggesting that SOCS1 and especially SOCS3 are crucial for EV71 infection. Further investigation showed that SOCS3 promoted virus infection by inhibiting interferon-induced STAT3 phosphorylation. SOCS1 and SOCS3 mRNA expressions were independent on virus-induced type I interferon expression but were blocked by the inhibitor of NF- κ B. Therefore, EV71 infection stimulates the expression of SOCS proteins in an interferon-independent way and negatively regulates the JAK/STAT signaling pathway, thus escaping host immunity. All these results may add new information to the mechanism of EV71 in fighting against type I interferon responses., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Wenying Gao et al.)

Today, there are no medicines to treat enterovirus and rhinovirus infections. In the present study, a series of novel pleconaril derivatives with substitutions in the isoxazole and phenyl rings was synthesized and evaluated for their antiviral activity against a panel of pleconaril-sensitive and -resistant enteroviruses. Studies of the structure-activity relationship demonstrate the crucial role of the N,N-dimethylcarbamoyl group in the isoxazole ring for antiviral activity against pleconaril-resistant viruses. In addition, one or two substituents in the phenyl ring directly impact on the spectrum of antienteroviral activity. The 3-(3-methyl-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 10g was among the compounds exhibiting the strongest activity against pleconaril-resistant as well as pleconaril-susceptible enteroviruses with IC 50 values from 0.02 to 5.25 μM in this series. Compound 10g demonstrated markedly less CYP3A4 induction than pleconaril, was non-mutagenic, and was bioavailable after intragastric administration in mice. These results highlight compound 10g as a promising potential candidate as a broad spectrum enterovirus and rhinovirus inhibitor for further preclinical investigations., Competing Interests: Declaration of competing interest Sean Ekins is CEO of Collaborations Pharmaceuticals, Inc. Vadim Makarov, Michaela Schmidtke and Sean Ekins are co-inventors on a US provisional patent relating to this work., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Coxsackievirus B3 (CVB3), a member of the Picornaviridae family, is considered to be one of the most important infectious agents to cause virus-induced myocarditis. Despite improvements in studying viral pathology, structure and molecular biology, as well as diagnosis of this disease, there is still no virus-specific drug in clinical use. Structural and nonstructural proteins produced during the coxsackievirus life cycle have been identified as potential targets for blocking viral replication at the step of attachment, entry, uncoating, RNA and protein synthesis by synthetic or natural compounds. Moreover, WIN (for Winthrop) compounds and application of nucleic-acid based strategies were shown to target viral capsid, entry and viral proteases, but have not reached to the clinical trials as a successful antiviral agent. There is an urgent need for diverse molecular libraries for phenotype-selective and high-throughput screening., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

IRES-driven translation plays an essential role in picornavirus infection. However, there are seldom reports of compounds targeting this pathway with effective protection in animal models. Here, we identified emetine, an antiprotozoal drug, which inhibits EV-A71 with an EC 50 value of 0.04 μM and a CC 50 value of 10 μM in RD cell culture. Interestingly, emetine exhibits activities against a series of human enteroviruses, including CV-A16, CV-B1, EV-D68, Echov-6, etc., at the nanomolar level. When orally administered at 0.20 mg/kg twice a day in an EV-A71 mouse model, emetine reduced viral loads in various organs and completely prevented diseases and death. A mechanistic study demonstrated that emetine suppressed EV-A71 by inhibiting viral IRES-driven translation. Taken together, these data indicate emetine as a promising candidate to treat picornavirus infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The main objective of this work to explore new safe antiviral agents against hepatitis A virus (HAV), norovirus (NoV) and Coxsackievirus B4 (CoxB4) infections. In this context, we have successfully prepared new polyquaternary phosphonium oligochitosans (PQPOC 1,2 ) to use them as natural synergistic in-situ bioreductants of silver ions into nanosilver and stabilizing agent for these nanosilver to fabricate PQPOCs-AgNPs nano-biocomposites (NBC1,2). The antiviral performance of the PQPOCs and NBCs against FCV, HAV, and CoxB4 reflects great virucidal activities for NBCs as compared with PQPOCs with maximum viral reduction% (41.42, 80.62, and 84.04%) for NBC1 against FCV, HAV, and CoxB4, respectively. Furthermore, the antiviral activity of NBC1 is concentration- / pH-dependent where NBC1 acquired its maximum antiviral at [NBC1] = 200 μL/mL and pH 4. Based upon these facts, we could attribute the enhanced virucidal efficacy of NBC1: (i) binding of AgNPs to the virions active sites. (ii) Electrostatic interaction between the positive brushes of PQPOC and negative targets of viruses. (iii) Inducing ribonuclease catalyzed by CS to degrade the viral RNA and consequently prevents its transcription and translation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here, we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, as seen with poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. The quantity and composition of bacterial communities were altered by treatment with the five-antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more-subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the standard concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 are more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ with respect to their reliance on microbiota. IMPORTANCE Recent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here, we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that closely related viruses may differ with respect to their reliance on microbiota., (Copyright © 2019 American Society for Microbiology.)

Oxysterol-binding Protein (OSBP) is a human lipid-transport protein required for the cellular replication of many types of viruses, including several human pathogens. The structurally-diverse small molecule compounds OSW-1, itraconazole (ITZ), T-00127-HEV2 (THEV) and TTP-8307 (TTP) inhibit viral replication through interaction with the OSBP protein. The OSW-1 compound reduces intracellular OSBP, and the reduction of OSBP protein levels persists multiple days after the OSW-1-compound treatment is stopped. The OSW-1-induced reduction of OSBP levels inhibited Enterovirus replication prophylactically in cells. In this report, the OSBP-interacting compounds ITZ, THEV, and TTP are shown not to reduce OSBP levels in cells, unlike the OSW-1-compound, and the OSW-1 compound is determined to be the only compound capable of providing prophylactic antiviral activity in cells. Furthermore, OSW-1 and THEV inhibit the binding of 25-hydroxycholesterol (25-OHC) to OSBP indicating that these compounds bind at the conserved sterol ligand binding site. The ITZ and TTP compounds do not inhibit 25-hydroxycholesterol binding to OSBP, and therefore ITZ and TTP interact with OSBP through other, unidentified binding sites. Co-administration of the THEV compound partially blocks the cellular activity of OSW-1, including the reduction of cellular OSBP protein levels; co-administration of the ITZ and TTP compounds have minimal effect on OSW-1 cellular activity further supporting different modes of interaction with these compounds to OSBP. OSW-1, ITZ, THEV, and TTP treatment alter OSBP cellular localization and levels, but in four distinct ways. Co-administration of OSW-1 and ITZ induced OSBP cellular localization patterns with features similar to the effects of ITZ and OSW-1 treatment alone. Based on these results, OSBP is capable of interacting with multiple structural classes of antiviral small molecule compounds at different binding sites, and the different compounds have distinct effects on OSBP cellular activity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It is demonstrated that carbon quantum dots derived from curcumin (Cur-CQDs) through one-step dry heating are effective antiviral agents against enterovirus 71 (EV71). The surface properties of Cur-CQDs, as well as their antiviral activity, are highly dependent on the heating temperature during synthesis. The one-step heating of curcumin at 180 °C preserves many of the moieties of polymeric curcumin on the surfaces of the as-synthesized Cur-CQDs, resulting in superior antiviral characteristics. It is proposed that curcumin undergoes a series of structural changes through dehydration, polymerization, and carbonization to form core-shell CQDs whose surfaces remain a pyrolytic curcumin-like polymer, boosting the antiviral activity. The results reveal that curcumin possesses insignificant inhibitory activity against EV71 infection in RD cells [half-maximal effective concentration (EC 50 ) >200 µg mL -1 ] but exhibits high cytotoxicity toward RD cells (half-maximal cytotoxic concentration (CC 50 ) <13 µg mL -1 ). The EC 50 (0.2 µg mL -1 ) and CC 50 (452.2 µg mL -1 ) of Cur-CQDs are >1000-fold lower and >34-fold higher, respectively, than those of curcumin, demonstrating their far superior antiviral capabilities and high biocompatibility. In vivo, intraperitoneal administration of Cur-CQDs significantly decreases mortality and provides protection against virus-induced hind-limb paralysis in new-born mice challenged with a lethal dose of EV71., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The hydrophobic pocket within viral capsid protein 1 is a target to combat the rhino- and enteroviruses (RV and EV) using small molecules. The highly conserved amino acids lining this pocket enable the development of antivirals with broad-spectrum of activity against numerous RVs and EVs. Inhibitor binding blocks: the attachment of the virion to the host cell membrane, viral uncoating, and/or production of infectious virus particles. Syntheses and biological studies of the most well-known antipicornaviral capsid binders have been reviewed and we propose next steps in this research., (Copyright © 2019. Published by Elsevier Masson SAS.)

Hand, foot, and mouth disease (HFMD) is a global health concern, especially in the Asia-Pacific region. HFMD caused by Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection is usually self-limited but occasionally leads to severe pulmonary edema, neurological complications, and even death. Unfortunately, no effective drugs are currently available in clinical practice for the prevention and treatment of HFMD. Thus, anti-HFMD drugs must be urgently developed. A previous study had reported that lycorine could inhibit EV71 replication. In the present study, we found that LY-55, a lycorine derivative, inhibited the replication of EV71 and CVA16 in vitro and provided partial protection to mice from EV71 infection, as indicated by the decreased viral load and protein expression levels in muscles, clinical scores, and increased survival rates of infected mice. Mechanistically, LY-55 was not directly viricidal. Instead, the LY-55-mediated inhibition of EV71 and CVA16 was found to be mechanistically possible, at least in part, through downregulating autophagy, which plays an important role for EV71 and CVA16 replication. These findings suggest that LY-55 could be a potential lead or supplement for the development of anti-HFMD agents in the future.

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are "capsid binders" that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo-electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens., Competing Interests: The authors have declared that no competing interests exist.

Three new megastigmane glucosides (1-3) and two new monoterpenes (4-5), together with 14 related known compounds (6-19) were isolated from the twigs and leaves of Lyonia ovalifolia. The structures of the new compounds were determined by extensive MS, NMR, CD experiments and chemical methods. Compounds 2, 6, and 18 displayed potent antiviral activity against Coxsackie B3, with IC 50 values between 6.4 and 14.6 µM. Additionally, compounds 6, 10, and 11 exhibited noteworthy anti-inflammatory activities, with inhibition rates ranging from 54.55% to 83.33% under the concentration of 10 -5 M.

Coxsackievirus A16 (CVA16) is one of the most prevalent enteroviral pathogens associated with hand, foot, and mouth disease. In the present study, we have investigated (1) whether the bioactive compound acetylshikonin (AS) inhibits CVA16 infection in vitro and in vivo and (2) the potential antiviral mechanism(s). The results suggest that AS is nontoxic at concentrations of up to 5 μmol/L and could directly inactivate virus particles at relatively low concentrations (0.08 μmol/L), thereby rendering CVA16 incapable of cellular entry. Correspondingly, the expression of viral RNA in vitro was also reduced 100-fold ( P < 0.05) when compared to infected, untreated controls. Results from a CVA16-infected neonatal mouse model indicate that, in comparison to the virus-infected, untreated group, body weights of the mice in the virus-infected, compound-treated group increased more steadily with less severe clinical symptoms. In addition, viral loads in internal organs significantly decreased in treated animals, concomitantly with both reduced pathology and diminished expression of the proinflammatory cytokines IFN-γ and IL-6. In conclusion, AS exerted an inhibitory effect on CVA16 infection in vitro and in vivo. Our study provides a basis for further investigations of AS-type compounds to develop therapeutics to mitigate CVA-associated disease in children.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells., Competing Interests: The authors have declared that no competing interests exist.

Enterovirus 71 (EV71), a single-stranded positive-sense RNA virus, is the causative agent of hand, foot, and mouth disease (HFMD), for which no effective antiviral therapy is currently available. Apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) is a cytidine deaminase that inhibits the replication of several viruses, such as human immunodeficiency virus-1, hepatitis B virus and hepatitis C virus. In our efforts toward understanding the antiviral spectrum and mechanism of A3G, we found that ectopic expression of A3G inhibited EV71 replication, whereas knockdown of endogenous A3G expression promoted EV71 replication. Moreover, inhibition of EV71 replication by IMB-Z, a N-phenylbenzamide derivative, is associated with increased levels of intracellular A3G, but reducing the level of A3G by RNA interference diminished the antiviral activity of IMB-Z. Mechanistically, we obtained evidence suggesting that the cytidine deaminase activity is not required for A3G inhibition of EV71 replication. Instead, we demonstrated that A3G can interact with viral 3D RNA-dependent RNA polymerase (RdRp) and viral RNA and be packaged into progeny virions to reduce its infectivity. Taken together, our results indicate that A3G is a cellular restriction factor of EV71 and mediator of the antiviral activity of IMB-Z. Pharmacological induction and/or stabilization of A3G is a potential therapeutic approach to treat diseases caused by EV71 infection, such as HFMD., (Copyright © 2019 Elsevier B.V. All rights reserved.)