Inhibit Enterovirus 71 Infection Research Articles

AIMP2 restricts EV71 replication by recruiting SMURF2 to promote the degradation of 3D polymerase

Hand, foot and mouth disease (HFMD), mainly caused by enterovirus 71 (EV71), has frequently occurred in the Asia-Pacific region, posing a significant threat to the health of infants and young children. Therefore, research on the infection mechanism and pathogenicity of enteroviruses is increasingly becoming important. The 3D polymerase, as the most critical RNA-dependent RNA polymerase (RdRp) for EV71 replication, is widely targeted to inhibit EV71 infection. In this study, we identified a novel host protein, AIMP2, capable of binding to 3D polymerase and inhibiting EV71 infection. Subsequent investigations revealed that AIMP2 recruits the E3 ligase SMURF2, which mediates the polyubiquitination and degradation of 3D polymerase. Furthermore, the antiviral effect of AIMP2 extended to the CVA16 and CVB1 serotypes. Our research has uncovered the dynamic regulatory function of AIMP2 during EV71 infection, revealing a novel antiviral mechanism and providing new insights for the development of antienteroviral therapeutic strategies.

Cathelicidin peptide analogues inhibit EV71 infection through blocking viral entry and uncoating.

Given the serious neurological complications and deaths associated with enterovirus 71 (EV71) infection, there is an urgent need to develop effective antivirals against this viral infection. In this study, we demonstrated that two Cathelicidin-derived peptides, LL-18 and FF-18 were more potent against EV71 infection than the parent peptide LL-37, which is the mature and processed form of Cathelicidin. These peptides could directly bind to the EV71 virus particles, but not to coxsackievirus, indicative of their high specificity. The binding of peptides with the virus surface occupied the viral canyon region in a way that could block virus-receptor interactions and inhibit viral uncoating. In addition, these peptide analogues could also relieve the deleterious effect of EV71 infection in vivo. Therefore, Cathelicidin-derived peptides might be excellent candidates for further development of antivirals to treat EV71 infection.

Ubiquitin-specific protease 24 promotes EV71 infection by restricting K63-linked polyubiquitination of TBK1

TANK-binding kinase 1 (TBK1) is an essential protein kinase for activation of interferon regulatory factor 3 (IRF3) and induction of the type I interferons (IFN-I). Although the biochemical regulation of TBK1 activation has been studied, little is known about how enterovirus 71 (EV71) employs the deubiquitinases (DUBs) to regulate TBK1 activation for viral immune evasion. Here, we found that EV71 infection upregulated the expression of ubiquitin-specific protease 24 (USP24). Further studies revealed that USP24 physically interacted with TBK1, and can reduce K63-linked polyubiquitination of TBK1. Knockdown of USP24 upregulated TBK1 K63-linked polyubiquitination, promoted the phosphorylation and nuclear translocation of IRF3, and in turn improved IFN-I production during EV71 infection. As a consequence, USP24 knockdown dramatically inhibited EV71 infection. This study revealed USP24 as a novel regulator of TBK1 activation, which promotes the understanding of immune evasion mechanisms of EV71 and could provide a potential strategy for treatment of EV71 infection.

A sulfated glucuronorhamnan from the green seaweed Monostroma nitidum: Characteristics of its structure and antiviral activity

A water-soluble polysaccharide from Monostroma nitidum, designated MWS, was isolated using water extraction, anion-exchange and size-exclusion chromatography. MWS was a sulfated glucuronorhamnan consisting of →3)-α-l-Rhap-(1→, →4)-β-d-GlcpA-(1→ and →2)-α-l-Rhap-(1→ units. Sulfate ester groups located at C-4/C-2 of →3)-α-l-Rhap-(1→ and C-4/C-3 of →2)-α-l-Rhap-(1→ units. In in vitro tests, it was proved that MWS possessed broad spectrum against different viruses, especially for enterovirus 71 (EV71) with nearly no toxicity in relation to cell lines used. MWS may largely inhibit EV71 infection before or during viral adsorption through binding to virus particles and block some early steps of virus life cycle by down-regulating host phosphoinositide 3-kinase /protein kinase B signaling pathway. Intramuscular injection of MWS markedly reduced viral titers in EV71-infected mice. The data demonstrated that MWS could have great promising to become an antiviral drug for prevention and therapy of EV71 infection.

Sophocarpine against enterovirus 71 in vitro.

Sophocarpine (SCA) is a bioactive alkaloid present in Sophoraflavescens Ait. The present study demonstrated that SCA inhibited enterovirus 71 (EV71) infection in Vero cells. The results indicated that the 50% cytotoxicity concentration of SCA for Vero cells was 1,346 µg/ml, and the 50% inhibition concentration of SCA against EV71 was 350 µg/ml. SCA produced a marked inhibitory effect against EV71 when the Vero cells were treated with SCA prior to infection with the virus. Additionally, SCA was effective against EV71 when the Vero cells were infected with EV71 (100xTCID50) that had been treated with SCA for 2 h, and was effective when the Vero cells were infected with EV71 (100xTCID50) at 37°C under 5% CO2 for 2 h prior to treatment with SCA for 2 h. SCA was demonstrated to inhibit the attachment and penetration of EV71 and was more effective at inhibiting attachment. The assay additionally verified that SCA suppressed the replication of viral genomic RNA and indicated that SCA may inhibit EV71 infection in vitro.

Antiviral effects of Retro-2cycl and Retro-2.1 against Enterovirus 71 in vitro and in vivo

Enterovirus 71 (EV71) is one of the causative pathogens of hand, foot and mouth disease (HFMD), especially the form associated with fatal neurological disorders. Sustained outbreaks of EV71 infections remain a serious health threat worldwide. However, no antiviral agent against EV71 for clinical therapy has been approved. Retro-2cycl and Retro-2.1 are inhibitors of several pathogens specifically targeting the intracellular vesicle transport, which also participates in the EV71 lifecycle processes including progeny virus release. Here, we reported that Retro-2cycl and Retro-2.1, respectively, could inhibit EV71 infection with 50% effective concentrations of 12.56 μM and 0.05 μM in a cytopathic effect inhibition assay and showed relatively low cytotoxicity with 50% cytotoxicity concentrations of more than 500 μM and 267.80 μM. Preliminary mechanism studies revealed that Retro-2cycl and Retro-2.1 did not inhibit EV71 protein synthesis or RNA replication but could block progeny EV71 release specifically. Furthermore, administration of Retro-2cycl at the dose of 10 mg/kg significantly protected 90% of newborn mice from lethal EV71 challenge. Consequently, our results for the first time identified Retro-2cycl and Retro-2.1 as effective inhibitors of EV71 as well as lead compounds, which would contribute to anti-EV71 drug development. We also identified progeny virus release and the intracellular vesicle transport as antiviral targets for EV71.

The binding of a monoclonal antibody to the apical region of SCARB2 blocks EV71 infection

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77–113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.

MiR-127-5p negatively regulates enterovirus 71 replication by directly targeting SCARB2.

Enterovirus 71 (EV71) is the major causative agent of hand‐foot‐and‐mouth disease in young children and can cause severe cerebral and pulmonary complications and even fatality. This study aimed at elucidating whether and how EV71 infection is regulated by a cellular microRNA, miR‐127‐5p. We found that miR‐127‐5p can downregulate the expression of SCARB2, a main receptor of EV71, by targeting two potential sites in its 3′ UTR region and inhibit EV71 infection. Meanwhile, miR‐127‐5p expression was upregulated during EV71 infection. Notably, transfecting cells with miR‐127‐5p mimics led to a significant decrease in viral replication, while inhibition of endogenous miR‐127‐5p facilitated viral replication. Furthermore, our evidence showed that miR‐127‐5p did not affect postentry viral replication. Taken together, these results indicated that miR‐127‐5p inhibited EV71 replication by targeting the SCARB2 mRNA.

Berberine inhibits enterovirus 71 replication by downregulating the MEK/ERK signaling pathway and autophagy

BackgroundThe MEK-ERK signaling pathway and autophagy play an important role for enterovirus71(EV71) replication. Inhibition of MEK-ERK signaling pathway and autophagy is shown to impair EV71 replication. Berberine (BBR), an isoquinoline alkaloid isolated from Berberis vulgaris L., has been reported to have ability to regulate this signaling pathway and autophagy. Herein, we want to determine whether berberine can inhibit EV71 infection by downregulating the MEK/ERK signaling pathway and autophagy.MethodsThe antiviral effect of berberine was determined by cytopathic effect (CPE) assay, western blotting assay and qRT-PCR assay. The mechanism of BBR anti-virus was determined by western blotting assay and immunofluorescence assay.ResultsWe showed that berberine does-dependently reduced EV71 RNA and protein synthesis, which was, at least in part, the result of inhibition of activation of MEK/ERK signaling pathway. Furthermore, we found that berberine suppressed the EV71-induced autophagy by activating AKT protein and inhibiting the phosphorylation of JNK and PI3KIII.ConclusionsBBR inhibited EV71 replication by downregulating autophagy and MEK/ERK signaling pathway. These findings suggest that BBR may be a potential agent or supplement against EV71 infection.

Flos Farfarae Inhibits Enterovirus 71-Induced Cell Injury by Preventing Viral Replication and Structural Protein Expression.

Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.

Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry.

Antibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre- and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs. Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during the viral entry process and interfere with EV71 binding to heparan sulfate, SCARB2, and PSGL-1 molecules, which are key receptors involved in different steps of EV71 entry. Our findings greatly enhance the understanding of the interplays among EV71, neutralizing antibodies, and host receptors, which in turn should facilitate the development of an MAb-based anti-EV71 therapy.

Yakammaoto inhibits enterovirus 71 infection by reducing viral attachment, internalization, replication, and translation

Enterovirus 71 (EV71) can cause central nervous system infections with mortality and neurologic sequelae. At present, there is no effective therapeutic modality for EV71 infection. The infection is more common in families with poor socioeconomic status. Therefore, finding a readily available, cost-effective therapeutic modality would be very helpful to these socioeconomically disadvantaged families. Yakammaoto is a cheap and readily available traditional prescription that is proven to have antiviral activity against coxsackievirus B4 (CVB4). CVB4 and EV71 are enteroviruses. In this study, we evaluated the antiviral activity of hot water extract of yakammaoto against EV71. The results of plaque reduction assay and flow cytometry demonstrated that yakammaoto dose dependently inhibited EV71 infection. In addition, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR results showed that yakammaoto reduced viral replication. Western blotting analysis showed that yakammaoto can inhibit viral protein production. Thus, our results suggest that yakammaoto should be considered to manage EV71 infection in the future.

Saururus chinensis (Lour.) Baill blocks enterovirus 71 infection by hijacking MEK1–ERK signaling pathway

The aerial parts of Saururus chinensis (Lour.) Baill are a Chinese herbal medicine used for the treatment of edema and inflammatory diseases. However, the effect of this medicine on enterovirus 71 (EV71) infection has not been explored. Previous studies showed that MEK1–ERK signal pathway was required for efficient replication of EV71 infection and inhibition of this signal pathway has been shown to suppress virus infection. Here we show that the water extract of S. chinensis (Lour.) Baill (SCB) significantly blocks EV71 infection by inhibiting the activation of MEK1–ERK signal pathway with an IC50 of 8.9μg/mL. SCB at 30 and 60μg/mL blocked EV71-induced cytopathic effect (CPE) and production of infectious virion by 1.9 and 5.1logs, respectively. Virucidal assay suggested that SCB had no virucidal activity against EV71 and probably exerted its effect by targeting multiple steps in EV71 infection. Knockdown of MEK1 but not MEK2 blocked EV71 replication. And SCB treatment inhibited the activation of MEK1–ERK signal during EV71 infection. Furthermore, we found that rutin at 200μM, one of the major components of SCB, significantly suppressed EV71 induced CPE and inhibited viral replication in a dose dependent manner. Taken together, SCB inhibited EV71 infection by hijacking MEK1–ERK signal pathway and rutin was the responsible antiviral component of SCB.

Formononetin inhibits enterovirus 71 replication by regulating COX- 2/PGE2 expression

BackgroundThe activation of ERK, p38 and JNK signal cascade in host cells has been demonstrated to up-regulate of enterovirus 71 (EV71)-induced cyclooxygenase-2 (COX-2)/ prostaglandins E2 (PGE2) expression which is essential for viral replication. So, we want to know whether a compound can inhibit EV71 infection by suppressing COX-2/PGE2 expression.MethodsThe antiviral effect of formononetin was determined by cytopathic effect (CPE) assay and the time course assays. The influence of formononetin for EV71 replication was determined by immunofluorescence assay, western blotting assay and qRT-PCR assay. The mechanism of the antiviral activity of formononetin was determined by western blotting assay and ELISA assay.ResultsFormononetin could reduce EV71 RNA and protein synthesis in a dose-dependent manner. The time course assays showed that formononetin displayed significant antiviral activity both before (24 or 12 h) and after (0–6 h) EV71 inoculation in SK-N-SH cells. Formononetin was also able to prevent EV71-induced cytopathic effect (CPE) and suppress the activation of ERK, p38 and JNK signal pathways. Furthermore, formononetin could suppress the EV71-induced COX-2/PGE2 expression. Also, formononetin exhibited similar antiviral activities against other members of Picornaviridae including coxsackievirus B2 (CVB2), coxsackievirus B3 (CVB3) and coxsackievirus B6 (CVB6).ConclusionsFormononetin could inhibit EV71-induced COX-2 expression and PGE2 production via MAPKs pathway including ERK, p38 and JNK. Formononetin exhibited antiviral activities against some members of Picornaviridae. These findings suggest that formononetin could be a potential lead or supplement for the development of new anti-EV71 agents in the future.

Antiviral activity of Paulownia tomentosa against enterovirus 71 of hand, foot, and mouth disease.

The bark, leaves, and flowers of Paulownia trees have been used in traditional Chinese medicine to treat infectious and inflammatory diseases. We investigated the antiviral effects of Paulownia tomentosa flowers, an herbal medicine used in some provinces of P. R. China for the treatment of skin rashes and blisters. Dried flowers of P. tomentosa were extracted with methanol and tested for antiviral activity against enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), the predominant etiologic agents of hand, foot, and mouth disease in P. R. China. The extract inhibited EV71 infection, although no effect was detected against CAV16 infection. Bioactivity-guided fractionation was performed to identify apigenin as an active component of the flowers. The EC50 value for apigenin to block EV71 infection was 11.0 µM, with a selectivity index of approximately 9.3. Although it is a common dietary flavonoid, only apigenin, and not similar compounds like naringenin and quercetin, were active against EV71 infection. As an RNA virus, the genome of EV71 has an internal ribosome entry site that interacts with heterogeneous nuclear ribonucleoproteins (hnRNPs) and regulates viral translation. Cross-linking followed by immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that EV71 RNA was associated with hnRNPs A1 and A2. Apigenin treatment disrupted this association, indicating that apigenin suppressed EV71 replication through a novel mechanism by targeting the trans-acting factors. This study therefore validates the effects of Paulownia against EV71 infection. It also yielded mechanistic insights on apigenin as an active compound for the antiviral activity of P. tomentosa against EV71 infection.

A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the causative agents of hand, foot, and mouth disease (HFMD). During recent epidemics of HFMD in China, medicinal herbals and preparations containing herbal extracts have demonstrated therapeutic efficacy with relative safety profiles. There have been no microbiological studies to validate their usefulness for HFMD. We selected 12 commonly used herbs for HFMD from government recommended guidelines as well as published reports and tested for their antiviral activity and anti-inflammatory activity. A water extract of Houttuynia cordata Thunb. (HCT) inhibited EV71 infection significantly and was marginally active against CVA16 infection. The IC50 (concentration to have 50% inhibitory effect) values of HCT against a Fuyang strain and a BrCr strain of EV71 were determined at 8.9 μg/mL and 20.6 μg/mL, respectively. Mentha haplocalyx Briq. (MHB) water extract was active against CVA16, with an IC50 value of 70.3 μg/mL. The extract did not exhibit activity against EV71 infection. Although the majority of the extracts showed no activity against viral infection, several extracts demonstrated activity in blocking proinflammatory response by viral infection. This study therefore validates the effectiveness of Chinese herbs for HFMD since some formulations containing the correct combination of the herbs can block viral replication as well as proinflammatory response of HFMD.

In vitro evaluation of the antiviral activity of heparan sulfate mimetic compounds against Enterovirus 71

Enterovirus 71 (EV71) is the causative agent of hand foot and mouth disease (HFMD) and can also cause fatal neurological complications for which currently there is no vaccine or approved antiviral drug. Despite suggestions that heparan sulfate (HS)-like compounds are effective antivirals against various viruses, no research has been undertaken to examine their effects upon EV71. Therefore, this study aimed to investigate the in vitro anti-EV71 effect of HS mimetics (heparin, heparan sulfate, and pentosan polysulfate). The results revealed that all of the compounds exhibited significant antiviral actions (p<0.05) against EV71 at concentrations less than 250μg/mL, compared to virus control and positive control, ribavirin. Among the compounds, heparin exhibited the most potent antiviral activity, as 7.81μg/mL of it prevented the infection by more than 90% (p<0.05). Assays to reveal the mode of action revealed that all of the compounds were capable of exerting antiviral activity through hindrance of viral attachment to the cells. In addition, some of the compounds could inhibit viral replication when added to cells 1h before infection, but none significantly reduced viral penetration. Overall, this research revealed that HS mimetic compounds could inhibit EV71 infection, and that HS may be involved in EV71–host cell interactions, as the virus binding to the host cells was significantly hindered by the HS-like compounds but not by ribavirin. Thus, further investigations to discover the molecular mechanisms underlying the anti-EV71 action of HS-like compounds are warranted.

Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells

BackgroundMany viruses recognize specific sugar residues, particularly sulfated or sialylated glycans, as the infection receptors. A change of sialic acid (2-6)-linked galactose (SA-α2,6Gal) to SA-α2,3Gal determines the receptor for avian flu infection. The receptor for enterovirus 71 (EV71) infection that frequently causes fatal encephalitis in Asian children remains unclear. Currently, there is no effective vaccine or anti-virus agent for EV71 infection. Using DLD-1 intestinal cells, this study investigated whether SA-linked glycan on DLD-1 intestinal cells was a receptor for EV71, and whether natural SA-linked sugars from human milk could block EV71 infection.ResultsEV71 specifically infected DLD-1 intestinal cells but not K562 myeloid cells. Depletion of O-linked glycans or glycolipids, but not N-linked glycans, significantly decreased EV71 infection of DLD-1 cells. Pretreatment of DLD-1 cells with sialidase (10 mU, 2 hours) significantly reduced 20-fold EV71 replication (p < 0.01). Taken together, these results suggest that SA-linked O-glycans and glycolipids, but not N-glycans, on DLD-1 cells were responsible for EV71 infection. Purified SA-α2,3Gal and SA-α2,6Gal from human milk significantly inhibited EV71 infection of DLD-1 cells, indicating terminal SA-linked glycans could be receptors and inhibitors of EV71 infection.ConclusionThis is the first in the literature to demonstrate that EV71 uses SA-linked glycans as receptors for infection, and natural SA-linked glycans from human milk can protect intestinal cells from EV71 infection. Further studies will test how a SA-containing glycan can prevent EV71 in the future.

Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells

The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30 min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-α expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.