Broad-Spectrum Antiviral Agents

With the increased prevalence of new SARS-CoV-2 variants of concern, such as Delta and Omicron, the COVID-19 pandemic has become an ongoing human health disaster, killing millions worldwide. SARS-CoV-2 invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate (HS) on the surface of host cells plays an important role as a co-receptor for this viral pathogen–host cell interaction. Our previous studies demonstrated that many sulfated glycans, such as heparin, fucoidans, and rhamnan sulfate have anti-SARS-CoV-2 activities. In the current study, a small library of sulfated glycans and highly negatively charged compounds, including pentosan polysulfate (PPS), mucopolysaccharide polysulfate (MPS), sulfated lactobionic acid, sulodexide, and defibrotide, was assembled and evaluated for binding to the S-proteins and inhibition of viral infectivity in vitro. These compounds inhibited the interaction of the S-protein receptor-binding domain (RBD) (wild type and different variants) with immobilized heparin, a highly sulfated HS, as determined using surface plasmon resonance (SPR). PPS and MPS showed the strongest inhibition of interaction of heparin and S-protein RBD. The competitive binding studies showed that the IC₅₀ of PPS and MPS against the S-protein RBD binding to immobilized heparin was ~35 nM and ~9 nM, respectively, much lower than the IC₅₀ for soluble heparin (IC₅₀ = 56 nM). Both PPS and MPS showed stronger inhibition than heparin on the S-protein RBD or spike pseudotyped lentiviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, PPS and MPS exhibited strong antiviral activities against pseudotyped viral particles of SARS-CoV-2 containing wild-type or Delta S-proteins. Full article

Enterovirus genus has over one hundred genotypes and could cause several kinds of severe animal and human diseases. Understanding the role of conserved residues in the VP1 capsid protein among the enterovirus genus may lead to anti-enteroviral drug development. The highly conserved residues were found to be located at the loop and ß-barrel intersections. To elucidate the role of these VP1 residues among the enterovirus genus, alanine substitution reverse genetics (rg) variants were generated, and virus properties were investigated for their impact. Six highly conserved residues were identified as located near the inside of the canyon, and four of them were close to the ß-barrel and loop intersection. The variants rgVP1-R86A, rgVP1-P193A, rgVP1-G231A, and rgVP1-K256A were unable to be obtained, which may be due to disruption in the virus replication process. In contrast, rgVP1-E134A and rgVP1-P157A replicated well and rgVP1-P157A showed smaller plaque size, lower viral growth kinetics, and thermal instability at 39.5°C when compared to the rg wild type virus. These findings showed that the conserved residues located at the ß-barrel and loop junction play roles in modulating viral replication, which may provide a pivotal role for pan-enteroviral inhibitor candidate. Full article

As a key element during HBV replication, a nucleocapsid is considered a promising target for the treatment of chronic hepatitis B. The present study aimed to identify small molecules as novel capsid assembly modulators with antiviral activity. Structure-based virtual screening of an integrated compound library led to the identification of several types of HBV inhibitors. Among these inhibitors, N-sulfonylpiperidine-3-carboxamides (SPCs) potently reduced the amount of secreted HBV DNA. Through structure–activity relationship studies, we identified an SPC derivative, namely, C-39, which exhibited the highest antiviral activity without causing cytotoxicity. Mechanism studies showed that C-39 dose-dependently inhibited the formation of HBV capsid, synthesis of cccDNA, e antigen (HBeAg), viral pregenomic RNA (pgRNA), and HBV DNA levels, thereby restraining HBV replication. In summary, SPCs represent a new class of capsid assembly modulators. Further optimization of SPCs is expected to obtain new antiviral drugs against HBV infection. Full article

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. Full article

Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC₅₀ ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability. Full article

Severe Acute Respiratory Syndrome Coronavirus- 2 (SARS-CoV-2), including the recently reported severe variant B.1.617.2, has been reported to attack the respiratory tract with symptoms that may ultimately lead to death. While studies have been conducted to evaluate therapeutic interventions against the virus, this study evaluated the inhibitory potential of virtually screened novel derivatives and structurally similar compounds towards SARS-CoV-2 via a computational approach. A molecular docking simulation of the inhibitory potentials of the compounds against the SARS-CoV-2 drug targets—main protease (Mpro), spike protein (Spro), and RNA-dependent RNA polymerase (RdRp)—were evaluated and achieved utilizing AutoDock Vina in PyRx workspace. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of these compounds were assessed using SwissADME and ADMETLab servers. All the compounds displayed high binding affinities for the SARS-CoV-2 drug targets. However, the C13 exhibited the highest binding affinity for the drug targets, Spro and RdRp, while C15 exhibited the highest binding affinity for Mpro. The compounds interacted with the LEU A:271, LEU A:287, ASP A:289, and LEU A:272 of Mpro and the HIS A:540, PRO A:415, PHE A:486, and LEU A:370 of the Spro receptor binding motif and some active site amino acids of RdRp. The compounds also possess a favourable ADMET profile and showed no tendency towards hERG inhibition, hepatotoxicity, carcinogenicity, mutagenicity, or drug-liver injury. These novel compounds could offer therapeutic benefits against SARS-CoV-2, and wet laboratory experiments are necessary to further validate the results of this computational study. Full article

Influenza is an acute respiratory infection caused by the influenza virus, but few drugs are available for its treatment. Consequently, researchers have been engaged in efforts to discover new antiviral mechanisms that can lay the foundation for novel anti-influenza drugs. The viral RNA-dependent RNA polymerase (RdRp) is an enzyme that plays an indispensable role in the viral infection process, which is directly linked to the survival of the virus. Methods of inhibiting PB1–PB2 (basic polymerase 1–basic polymerase 2) interactions, which are a key part of RdRp enzyme activity, are integral in the design of novel antiviral drugs, a specific PB1–PB2 interactions inhibitor has not been reported. We have screened Enamine’s database and conducted a parallel screening of multiple docking schemes, followed by simulations of molecular dynamics to determine the structure of a stable ligand—PB1 complex. We also calculated the free energy of binding between the screened compounds and PB1 protein. Ultimately, we screened and identified a potential PB1–PB2 inhibitor using the ADMET prediction model. Full article

Viruses are the most common causative agents of inflammation in the oral cavity and throat region. Most respiratory tract infections are self-limiting and require no specific treatment. However, patients often use different self-medication therapies that can treat both the symptoms and the cause. Throat lozenges with a fixed combination of benzydamine hydrochloride and cetypiridinium chloride (BH/CPC) have been shown to provide effective symptomatic relief for sore throat, but their effect on viruses has not been investigated to date. The antiseptic, cetylpyridinium chloride (CPC), has already been described as a successful bactericide. In addition, there are some studies suggesting its efficacy against certain enveloped viruses. Thus, the aim of our study was to examine the virucidal activity of CPC and a combination of BH/CPC as a free active substance or as lozenge on SARS-CoV-2 in vitro. Under in-laboratory simulated conditions of lozenge administration, we incubated SARS-CoV-2 with three different concentrations of each of the active substances, CPC, free BH/CPC or BH/CPC, as a lozenge suspension for 1 min, 5 min and 15 min of contact time. Infective viral particles were detected in cell cultures and the viral titre was calculated accordingly. Our results show that all active substances in high-concentration suspensions, as well as a medium concentration of the BH/CPC combination, exhibited a 4-log reduction in viral titre. Additionally, the highest concentration of BH/CPC as a lozenge had a faster virucidal effect compared to CPC as a free active substance alone, since a contact time as short as 1 min reduced the initial virus concentration by more than 4-log. This study demonstrates the effective strong virucidal effect of the lozenge, with the possibility of viral load reduction in the oral cavity and, consequently, reduced risk of viral transmission. Full article

Interferons (IFNs) are cytokines involved in the immune response that act on innate and adaptive immunity. These proteins are natural cell-signaling glycoproteins expressed in response to viral infections, tumors, and biological inducers and constitute the first line of defense of vertebrates against infectious agents. They have been marketed for more than 30 years with considerable impact on the global therapeutic protein market thanks to their diversity in terms of biological activities. They have been used as single agents or with combination treatment regimens, demonstrating promising clinical results, resulting in 22 different formulations approved by regulatory agencies. The 163 clinical trials with currently active IFNs reinforce their importance as therapeutics for human health. However, their application has presented difficulties due to the molecules’ size, sensitivity to degradation, and rapid elimination from the bloodstream. For some years now, work has been underway to obtain new drug delivery systems to provide adequate therapeutic concentrations for these cytokines, decrease their toxicity and prolong their half-life in the circulation. Although different research groups have presented various formulations that encapsulate IFNs, to date, there is no formulation approved for use in humans. The current review exhibits an updated summary of all encapsulation forms presented in the scientific literature for IFN-α, IFN-ß, and IFN-γ, from the year 1996 to the year 2021, considering parameters such as: encapsulating matrix, route of administration, target, advantages, and disadvantages of each formulation. Full article

This retrospective study included COPD patients who attended our medical center between January and October 2018, and analyzed the outcomes of their influenza vaccination, including medical visits, hospitalization, medical expenses, and the incidence of respiratory failure. Airflow limitation was stratified according to GOLD guidelines. Overall, 543 COPD patients were enrolled, including 197, 113, 126, and 107 mild, moderate, severe, and very severe patients, respectively. Of all the participants, 238 received an influenza vaccination (43.8%), which significantly reduced hospital utilization for moderate (odds ratio [OR] 0.22, 95%CI 0.09–0.51), severe (OR 0.19, 95%CI 0.08–0.44), and very severe patients (OR 0.15, 95%CI 0.05–0.5) compared to mild patients (OR 0.51, 95%CI 0.2–1.26); reduced emergency department utilization for moderate (OR 0.33, 95%CI 0.14–0.77), severe (OR 0.22, 95%CI 0.10–0.52), and very severe patients (OR 0.30, 95%CI 0.10–0.88) compared to mild patients (OR 0.64, 95%CI 0.30–1.37); and reduced the occurrence of respiratory failure for moderate (OR 0.20, 95%CI 0.06–0.68), severe (OR 0.40, 95%CI 0.16–0.98), and very severe patients (OR 0.36, 95%CI 0.15–0.82) compared to mild patients (OR 0% CI 0.14–3.20). Influenza vaccination is more effective in COPD patients with moderate, severe, and very severe airflow obstruction than in those with mild obstruction with respect to hospital utilization, emergency department utilization, and respiratory failure. Full article

The Michael addition reaction is a spontaneous and quick chemical reaction that is widely applied in various fields. This reaction is performed by conjugating an addition of nucleophiles with α, β-unsaturated carbonyl compounds, resulting in the bond formation of C-N, C-S, C-O, and so on. In the development of molecular materials, the Michael addition is not only used to synthesize chemical compounds but is also involved in the mechanism of drug action. Several covalent drugs that bond via Michael addition are regarded as anticarcinogens and anti-inflammatory drugs. Although drug development is mainly focused on pharmaceutical drug discovery, target-based discovery can provide a different perspective for drug usage. However, considerable time and labor are required to define a molecular target through molecular biological experiments. In this review, we systematically examine the chemical structures of current FDA-approved antiviral drugs for potential Michael addition moieties with α, β-unsaturated carbonyl groups, which may exert an unidentified broad-spectrum inhibitory mechanism to target viral or host factors. We thus propose that profiling the targets of antiviral agents, such as Michael addition products, can be achieved by employing a high-throughput LC-MS approach to comprehensively analyze the interaction between drugs and targets, and the subsequent drug responses in the cellular environment to facilitate drug repurposing and/or identify potential adverse effects, with a particular emphasis on the pros and cons of this shotgun proteomic approach. Full article

Large-scale efforts have been persistently undertaken for medical prophylaxis and treatment of COVID-19 disasters worldwide. A variety of novel viral spike protein-targeted vaccines have been extensively distributed for global inoculation based on accelerated approval. With concerns of emerging spike protein mutations, we revisited the early but inconclusive clinical interest in the repurposed combination of azithromycin (AZT) and zinc supplements with safety advantages. The aim of this study is to provide in vitro proof of concept for IκBα associated rapid and synergistic suppression of angiotensin-converting enzymes 2 (ACE2) following combination treatments with AZT plus zinc sulfate in two human airway cells with ACE2 expression, Calu-3 and H322M, representative cells of the human upper and lower airway origin respectively. Clinical timing of AZT combined with zinc is indicated based on suppression of the key cellular entry molecule, ACE2, of SARS-CoV-2. Full article

Therapeutic modalities designed specifically to inhibit COVID-19 infection and replication would limit progressive COVID-19-associated pulmonary disease in infected patients and prevent or limit systemic disease. If effective, antivirals could reduce viral transmission rates by reducing viral burden and allow time for immune clearance. For individuals infected with acute-stage disease, antivirals in support of the existing vaccines could reduce COVID-19 hospitalizations and deaths. Here, we evaluate MRCV-19, a phosphorodiamidate morpholino oligo with delivery dendrimer (Vivo-Morpholino), to prevent coronavirus infection in a cell culture model. This is a novel antiviral that effectively inhibits SARS-CoV-2 replication in vitro. By design, MRCV-19 targets the SARS-CoV-2 5’UTR and overlaps the pp1a start site of translation in order to block access of the translation initiation complex to the start. MRCV-19 testing is conducted in a high-throughput, 384-well plate format with a 10-point dose-response curve (common ratio of 2) assayed in duplicate with parallel cytotoxicity evaluations. MRCV-19 was shown to be more effective than hydroxychloroquine and remdesivir in our CPE reduction assay with low toxicity. The clinical translational impact of this study is providing the basis for evaluating MRCV-19 on a large scale in an appropriate infection model for toxicity and systemic high-level inhibition of SARS-CoV-2, which could lead in time to phase I testing in humans. Full article

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections. Full article

Influenza A viruses are serious zoonotic pathogens that continuously cause pandemics in several animal hosts, including birds, pigs, and humans. Indole derivatives containing an indole core framework have been extensively studied and developed to prevent and/or treat viral infection. This study evaluated the anti-influenza activity of several indole derivatives, including 3-indoleacetonitrile, indole-3-carboxaldehyde, 3-carboxyindole, and gramine, in A549 and MDCK cells. Among these compounds, 3-indoleacetonitrile exerts profound antiviral activity against a broad spectrum of influenza A viruses, as tested in A549 cells. Importantly, in a mouse model, 3-indoleacetonitrile with a non-toxic concentration of 20 mg/kg effectively reduced the mortality and weight loss, diminished lung virus titers, and alleviated lung lesions of mice lethally challenged with A/duck/Hubei/WH18/2015 H5N6 and A/Puerto Rico/8/1934 H1N1 influenza A viruses. The antiviral properties enable the potential use of 3-indoleacetonitrile for the treatment of IAV infection. Full article

Amino acids have been implicated with virus infection and replication. Here, we demonstrate the effects of two basic amino acids, arginine and lysine, and their ester derivatives on infection of two enveloped viruses, SARS-CoV-2, and influenza A virus. We found that lysine and its ester derivative can efficiently block infection of both viruses in vitro. Furthermore, the arginine ester derivative caused a significant boost in virus infection. Studies on their mechanism of action revealed that the compounds potentially disturb virus uncoating rather than virus attachment and endosomal acidification. Our findings suggest that lysine supplementation and the reduction of arginine-rich food intake can be considered as prophylactic and therapeutic regimens against these viruses while also providing a paradigm for the development of broad-spectrum antivirals. Full article

In late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic emerged to severely impact the global population, creating an unprecedented need for effective treatments. This study aims to investigate the potential of Scutellaria barbata D. Don (SB) as a treatment for SARS-CoV-2 infection through the inhibition of the proteases playing important functions in the infection by SARS-CoV-2. FRET assay was applied to investigate the inhibitory effects of SB on the two proteases involved in SARS-CoV-2 infection, M^pro and TMPRSS2. Additionally, to measure the potential effectiveness of SB treatment on infection inhibition, cellular models based on the Calu3 and VeroE6 cells and their TMPRSS2- expressing derivatives were assessed by viral pseudoparticles (Vpp) infection assays. The experimental approaches were conjugated with LC/MS analyses of the aqueous extracts of SB to identify the major constituent compounds, followed by a literature review to determine the potential active components of the inhibitory effects on protease activities. Our results showed that SB extracts inhibited the enzyme activities of M^pro and TMPRSS2. Furthermore, SB extracts effectively inhibited SARS-CoV-2 Vpp infection through a TMPRSS2-dependent mechanism. The aqueous extract analysis identified six major constituent compounds present in SB. Some of them have been known associated with inhibitory activities of TMPRSS2 or M^pro. Thus, SB may effectively prevent SARS-CoV-2 infection and replication through inhibiting M^pro and TMPRSS2 protease activities. Full article

Dengue virus (DENV) infection, which causes dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, is a severe global health problem in tropical and subtropical areas. There is no effective vaccine or drug against DENV infection. Thus, the development of anti-DENV agents is imperative. This study aimed to assess the anti-DENV activity of (E)-guggulsterone using a DENV infectious system. A specific inhibitor targeting signal molecules was used to evaluate the molecular mechanisms of action. Western blotting and qRT-PCR were used to determine DENV protein expression and RNA replication, respectively. Finally, an ICR suckling mouse model was used to examine the anti-DENV activity of (E)-guggulsterone in vivo. A dose-dependent inhibitory effect of (E)-guggulsterone on DENV protein synthesis and RNA replication without cytotoxicity was observed. The mechanistic studied revealed that (E)-guggulsterone stimulates Nrf2-mediated heme oxygenase-1 (HO-1) expression, which increases the antiviral interferon responses and downstream antiviral gene expression by blocking DENV NS2B/3B protease activity. Moreover, (E)-guggulsterone protected ICR suckling mice from life-threatening DENV infection. These results suggest that (E)-guggulsterone can be a potential supplement for controlling DENV replication. Full article

The emergence or re-emergence of viruses with epidemic and/or pandemic potential, such as Ebola, Zika, Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus 1 and 2 (SARS and SARS-CoV-2) viruses, or new strains of influenza represents significant human health threats due to the absence of available treatments. Vaccines represent a key answer to control these viruses. However, in the case of a public health emergency, vaccine development, safety, and partial efficacy concerns may hinder their prompt deployment. Thus, developing broad-spectrum antiviral molecules for a fast response is essential to face an outbreak crisis as well as for bioweapon countermeasures. So far, broad-spectrum antivirals include two main categories: the family of drugs targeting the host-cell machinery essential for virus infection and replication, and the family of drugs directly targeting viruses. Among the molecules directly targeting viruses, nucleoside analogues form an essential class of broad-spectrum antiviral drugs. In this review, we will discuss the interest for broad-spectrum antiviral strategies and their limitations, with an emphasis on virus-targeted, broad-spectrum, antiviral nucleoside analogues and their mechanisms of action. Full article

The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses. Full article

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication. Full article